(19)
(11)EP 3 494 139 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
12.01.2022 Bulletin 2022/02

(21)Application number: 17754895.5

(22)Date of filing:  04.08.2017
(51)International Patent Classification (IPC): 
C07K 16/28(2006.01)
A61P 11/00(2006.01)
A61P 13/10(2006.01)
A61P 17/00(2006.01)
A61P 13/12(2006.01)
A61K 39/00(2006.01)
A61P 13/02(2006.01)
A61P 15/00(2006.01)
A61P 35/00(2006.01)
(52)Cooperative Patent Classification (CPC):
C07K 2299/00; A61P 15/00; A61P 35/00; A61P 11/00; A61P 13/02; A61P 17/00; C07K 16/2818; C07K 16/2878; A61P 13/10; C07K 2317/92; A61P 13/12; C07K 2317/55; C07K 2317/75; A61K 2039/505; C07K 2317/34; C07K 2317/24; C07K 2317/90; C07K 2317/35; C07K 2317/71
(86)International application number:
PCT/US2017/045642
(87)International publication number:
WO 2018/027204 (08.02.2018 Gazette  2018/06)

(54)

MULTIVALENT AND MULTIEPITOPIC ANITIBODIES HAVING AGONISTIC ACTIVITY AND METHODS OF USE

MULTIVALENTE UND MULTIEPITOPISCHE ANITIKÖRPER MIT AGONISTISCHER WIRKUNG UND VERFAHREN ZUR VERWENDUNG

ANTICORPS MULTIVALENTS ET MULTIÉPITOPIQUES AYANT UNE ACTIVITÉ AGONISTE ET PROCÉDÉS D'UTILISATION


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 05.08.2016 US 201662371671 P

(43)Date of publication of application:
12.06.2019 Bulletin 2019/24

(73)Proprietor: F. Hoffmann-La Roche AG
4070 Basel (CH)

(72)Inventors:
  • HARRIS, Seth
    South San Francisco, California 94080 (US)
  • LAZAR, Greg
    South San Francisco, California 94080 (US)
  • YANG, Yanli
    South San Francisco, California 94080 (US)
  • CHRISTENSEN, Erin H.
    South San Francisco, California 94080 (US)
  • HANG, Julie
    San Jose, California 95129 (US)
  • KIM, Jeong
    South San Francisco, California 94080 (US)

(74)Representative: Klostermeyer-Rauber, Dörte 
F. Hoffmann-La Roche AG Corporate Law Patents (CLP) Grenzacherstrasse 124
4070 Basel
4070 Basel (CH)


(56)References cited: : 
WO-A1-2015/017822
US-A1- 2015 307 620
WO-A2-2017/123673
  
      
    Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


    Description

    FIELD OF THE INVENTION



    [0001] The present invention relates to multivalent and multiepitopic antigen binding complexes having agonistic activity and methods of using the same.

    BACKGROUND



    [0002] Functional antibodies and antigen binding complexes are an important therapeutic option for treatment of a wide variety of diseases. There is a need in the art for better means for identifying functional antibodies and antigen binding complexes, particularly those having agonistic activity, from pools of candidate molecules. There is a further need in the art for antibodies and antigen binding complexes with more potent agonistic activity. The present invention is directed to these and other needs.

    [0003] WO 2017/123673 discloses multivalent and multispecific TNF receptor superfamily binding fusion polypeptides comprising OX40 single domain antibodies (VHH). WO 2015/017822 describes antibodies or fusion proteins that are multimerized via a homomultimerizing peptide and US 2015/0307620 A1 discloses modified immunotherapeutic agent wherein an OX40 monoclonal antibody is covalently linked to a 4-1BB monoclonal antibody.

    SUMMARY



    [0004] The scope of the invention is defined by the appended set of claims. The invention provides antigen binding complexes (e.g., multivalent and multiepitopic antibodies/antigen binding complexes) having agonistic activity and methods of using the same. Provided herein is a tetravalent antigen binding complex having agonist activity for OX40, the complex comprising four antigen binding domains that bind OX40, wherein each of the four antigen binding domains comprises an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, wherein (a) the complex comprises one or more antigen binding domains that bind a first epitope of OX40 and one or more antigen binding domains that bind a second epitope of OX40, and wherein the first and second epitopes of OX40 are different and/or (b) the antigen binding domains that bind the first epitope do not cross-compete for binding OX40 with the antigen binding domains that bind the second epitope; wherein each of the antibody heavy chain polypeptides comprises:

            VH1-L1- VH2-L2-CH1-hinge-CH2-CH3     [I];

    wherein each of the antibody light chain polypeptides comprises:

            VL1-L3-VL2-L4-CL     [II];

    wherein each of the antibody heavy chain polypeptides associates with one antibody light chain polypeptide such that VH1 and VL1 form an antigen binding domain that binds the first epitope of OX40 and VH2 and VL2 form an antigen binding domain that binds the second epitope of OX40; and
    wherein VH1 is a first antibody heavy chain variable domain, VH2 is a second antibody heavy chain variable domain, VL1 is a first antibody light chain variable domain, VL2 is a second antibody light chain variable domain, CL is an antibody light chain constant domain, CH1 is an antibody first heavy chain constant domain, hinge is an antibody hinge region, CH2 is an antibody second heavy chain constant domain, CH3 is an antibody third heavy chain constant domain, and L1, L2, L3, and L4 are amino acid linkers.

    [0005] In some aspects, the first epitope comprises one or more amino acid residues selected from the group consisting of: 114-119, 124, 126, 127, 129, 130, 132, 140, and 142 of SEQ ID NO:281. In some aspects, the first epitope comprises amino acid residues 114-119, 124, 126, 127, 129, 130, 132, 140, and 142 of SEQ ID NO:281. In some aspects, the antigen binding domain that binds the first epitope of OX40 comprises: (a) a VH domain comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; and (b) a VL domain comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In some aspects, the VH domain of the antigen binding domain that binds the first epitope of OX40 comprises the amino acid sequence of SEQ ID NO:56, and the VL domain of the antigen binding domain that binds the first epitope of OX40 comprises the amino acid sequence of SEQ ID NO:57. In some aspects, the second epitope comprises one or more amino acid residues selected from the group consisting of: 68-71, 83-90, 95, and 98 of SEQ ID NO:281.

    [0006] In some aspects, the second epitope comprises amino acid residues 68-71, 83-90, 95, and 98 of SEQ ID NO:281. In some aspects, the antigen binding domain that binds the second epitope of OX40 comprises: (a) a VH domain comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; and (b) a VL domain comprising: an HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:42. In some aspects, the VH domain of the antigen binding domain that binds the second epitope of OX40 comprises the amino acid sequence of SEQ ID NO: 128, and the VL domain of the antigen binding domain that binds the second epitope of OX40 comprises the amino acid sequence of SEQ ID NO: 129. In some aspects, the antigen binding domain that binds the first epitope of OX40 comprises: (a) a VH domain comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; and (b) a VL domain comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and the antigen binding domain that binds the second epitope of OX40 comprises: (c) a VH domain comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; and (d) a VL domain comprising: an HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:42. In some aspects, the VH domain of the antigen binding domain that binds the first epitope of OX40 comprises the amino acid sequence of SEQ ID NO:56, the VL domain of the antigen binding domain that binds the first epitope of OX40 comprises the amino acid sequence of SEQ ID NO:57; the VH domain of the antigen binding domain that binds the second epitope of OX40 comprises the amino acid sequence of SEQ ID NO: 128, and the VL domain of the antigen binding domain that binds the second epitope of OX40 comprises the amino acid sequence of SEQ ID NO:129.

    [0007] In some aspects, L2 and L4 are 0 amino acids in length. In some aspects, L1 is between 0 and 20 amino acids in length. In some aspects, at least 90% of the amino acids of L1 are glycine and/or serine amino acids. In some aspects, L1 comprises an amino acid sequence selected from the group consisting of GGGGSG (SEQ ID NO:270), GGGGSGGGGS (SEQ ID NO:272), and GGGGSGGGGSGGGG (SEQ ID NO:273). In some aspects, L3 comprises an amino acid sequence selected from the group consisting of GGSGG (SEQ ID NO:271), GGGGSGGGGS (SEQ ID NO:272), and GGSGGGGSGGGGS (SEQ ID NO:274). In some aspects, L1 comprises the amino acid sequence GGGGSG (SEQ ID NO:270), and L3 comprises the amino acid sequence GGSGG (SEQ ID NO:271). In some aspects, L1 and L3 both comprise the amino acid sequence GGGGSGGGGS (SEQ ID NO:272). In some aspects, L1 comprises the amino acid sequence GGGGSGGGGSGGGG (SEQ ID NO:273), and L3 comprises the amino acid sequence GGSGGGGSGGGGS (SEQ ID NO:274). In some aspects, L1 comprises an amino acid sequence found within a human antibody constant domain sequence. In some aspects, L1 comprises the amino acid sequence ASTKGP (SEQ ID NO:275) or ASTKGPSVFPLAP (SEQ ID NO:277). In some aspects, L3 comprises the amino acid sequence RTVAAP (SEQ ID NO:276) or RTVAAPSVFIFPP (SEQ ID NO:278). In some aspects, L1 comprises the amino acid sequence ASTKGP (SEQ ID NO:275), and L3 comprises the amino acid sequence RTVAAP (SEQ ID NO:276). In some aspects, L1 comprises the amino acid sequence ASTKGPSVFPLAP (SEQ ID NO:277), and L3 comprises the amino acid sequence RTVAAPSVFIFPP (SEQ ID NO:278). In some aspects of any of the above aspects, VH1 comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:29 or 288, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:30 or 289, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; VL1 comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:42; VH2 comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2 or 282, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4 or 283; and VL2 comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6 or 286, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In some aspects, VH2 comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:29 or 288, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:30 or 289, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; VL2 comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:42; VH1 comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2 or 282, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4 or 283; and VL1 comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6 or 286, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In some aspects, VH1 comprises the amino acid sequence of SEQ ID NO:128, VL1 comprises the amino acid sequence of SEQ ID NO: 129, VH2 comprises the amino acid sequence of SEQ ID NO:56, and VL2 comprises the amino acid sequence of SEQ ID NO:57. In some aspects, VH2 comprises the amino acid sequence of SEQ ID NO: 128 or 290, VL2 comprises the amino acid sequence of SEQ ID NO: 129, VH1 comprises the amino acid sequence of SEQ ID NO:56, 284, or 285, and VL1 comprises the amino acid sequence of SEQ ID NO:57 or 287. In any of the complexes described above, a VH1, VH2, VL1, and/or VL2 comprises an HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and/or HVR-L3 that comprise one or more of the sequences listed in Tables 4 and 5. In some aspects, both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:240, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:241; both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:242, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:243; both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:250, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:251; both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:252, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:253; both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:254, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:255; both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:256, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:257; both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:258, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:259; both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:260, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:261; both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:262, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:263; both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:264, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:265; or both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:266, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:267. In some aspects of any of the above aspects, the complex comprises an antibody Fc region that comprises a modification for attenuating effector function. In some aspects, the complex comprises an antibody Fc region that comprises an amino acid substitution at one or more amino acid positions (EU numbering) selected from the group consisting of:
    1. (a) 297 in the Fc region of human IgG1,
    2. (b) 234 and 235 in the Fc region of human IgG1,
    3. (c) 234, 235 and 329 in the Fc region of human IgG1,
    4. (d) 234 and 237 in the Fc region of human IgG2,
    5. (e) 235, 237 and 318 in the Fc region of human IgG4,
    6. (f) 228 and 236 in the Fc region of human IgG4,
    7. (g) 268, 309, 330 and 331 in the Fc region of human IgG2,
    8. (h) 220, 226, 229 and 238 in the Fc region of human IgG1,
    9. (i) 226, 229, 233, 234 and 235 in the Fc region of human IgG1,
    10. (j) 234, 235 and 331 in the Fc region of human IgG1,
    11. (k) 226 and 230 in the Fc region of human IgG1, and
    12. (l) 267 and 328 in the Fc region of human IgG1.


    [0008] In some aspects, the complex comprises an antibody Fc region that comprises one or more amino acid substitutions (EU numbering) selected from the group consisting of:
    1. (a) N297A in the Fc region of human IgG1,
    2. (b) L234A and L235A in the Fc region of human IgG1,
    3. (c) L234A, L235A and P329G in the Fc region of human IgG1,
    4. (d) V234A and G237A in the Fc region of human IgG2,
    5. (e) L235A, G237A and E318A in the Fc region of human IgG4,
    6. (f) S228P and L236E in the Fc region of human IgG4,
    7. (g) one or more substitutions in the region spanning amino acid residues 118 to 260 in the Fc region of human IgG2 or in the region spanning amino acids 261 to 447 in the Fc region of human IgG4,
    8. (h) H268Q, V309L, A330S and A331S in the Fc region of human IgG2,
    9. (i) C220S, C226S, C229S and P238S in the Fc region of human IgG1,
    10. (j) C226S, C229S, E233P, L234V and L235A in the Fc region of human IgG1,
    11. (k) L234F, L235E and P331S in the Fc region of human IgG1,
    12. (l) C226S and P230S in the Fc region of human IgG1, and
    13. (m) S267E and L328F in the Fc region of human IgG1.


    [0009] In some aspects, the complex comprises an antibody Fc region that comprises a modification for attenuating effector function that results in an aglycosylated Fc region. In some aspects, the complex comprises an antibody Fc region that comprises a modification for attenuating effector function that does not eliminate glycosylation of the Fc region.

    [0010] Further provided herein are one or more polynucleotides encoding one or more polypeptides of the complex of any of the above aspects. Further provided herein are one or more vectors comprising the one or more polynucleotides of any of the above aspects. Further provided herein are one or more host cells comprising the one or more polynucleotides of any of the above aspects or the one or more vectors of any of the above aspects. Further provided herein is a pharmaceutical formulation comprising the complex of any of the above aspects and a pharmaceutically acceptable carrier.

    [0011] Further provided herein is the complex of any of the above aspects for use in a method of treating an individual having cancer comprising administering to the individual an effective amount of the complex of any of the above aspects. In some aspects, the complex of any of the above aspects is for use in a method that further comprises administering to the individual an additional therapeutic agent. In some aspects, the additional therapeutic agent comprises a chemotherapeutic agent. In some aspects, the additional therapeutic agent comprises a PD-1 axis binding antagonist. In some aspects, the cancer is epithelial ovarian cancer, fallopian tube cancer, primary peritoneal cancer, squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer (including platinum sensitive and platinum resistant ovarian cancer), liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, fallopian tube, peritoneal, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; post-transplant lymphoproliferative disorder (PTLD), or abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome, including metastatic forms of those cancers. In some aspects, the cancer is Urothelial carcinoma (uBC), melanoma, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), renal, or bladder cancer.

    BRIEF DESCRIPTION OF THE FIGURES



    [0012] 

    FIG. 1 shows Analytical Size Exclusion Chromatography (aSEC) chromatograms demonstrating that biepitopic 1A7/3C8 anti-OX40 antibody is able to promote higher order complexes in the presence of target antigen relative to monoepitopic antibody.

    FIG. 2 illustrates the elements of an antibody that may be used to construct the novel antibody formats described herein.

    FIGS. 3A & 3B show exemplary antibody formats of the present disclosure. Schematic illustrations are provided for a coupled IgG-IgG (c:IgG-IgG) wherein two full length IgGs are coupled (e.g., chemically coupled), a coupled Fab-IgG (c:Fab-IgG) wherein two Fabs are coupled (e.g., chemically coupled) to a full length IgG, a recombinant Fab-IgG (r:Fab-IgG) wherein an extra set of Fab arms is fused recombinantly to an IgG, and a recombinant Fv-IgG (r:Fv-IgG) wherein an extra set of Fv regions is fused recombinantly to an IgG. For each format are illustrated a monoepitopic version (top row), wherein all four variable regions bind the same epitope on the target antigen, and a biepitopic version (bottom row), wherein two variable regions bind one epitope and the other variable regions bind a different epitope on the target antigen. Lines between antibodies, or between antibodies and Fab fragments, represent linkers, and stars represent attachment points where the linker is chemically coupled (e.g., at an engineered cysteine of the antibody or Fab fragment).

    FIG. 4 depicts a first version of a biepitopic c:IgG-IgG format.

    FIG. 5 depicts a second version of a biepitopic c:IgG-IgG format.

    FIG. 6 depicts a biepitopic c:Fab-IgG format.

    FIG. 7 depicts a biepitopic r:Fv-IgG format.

    FIG. 8 depicts a first version of a biepitopic r:Fab-IgG format.

    FIG. 9 depicts a second version of a biepitopic r:Fab-IgG format.

    FIG. 10 shows co-stimulation of CD4+ memory T cell proliferation by anti-OX40 or control antibodies in the presence of anti-CD3 antibody and CD80+ FcγRIIa+ L cells. T cell proliferation was monitored by CellTiter-Glo® (Promega). All groups included anti-CD3. Coupled IgGs include monoepitopic 1A7_hIgG1(N297G)-BM(PEG)2-1A7_hIgG1(N297G) and biepitopic 1A7/3C8_hIgG1(N297G)-BM(PEG)3-1A7/3C8_hIgG1(N297G).

    FIG. 11 shows co-stimulation of CD4+ memory T cell proliferation by anti-OX40 or control antibodies in the presence of anti-CD3 antibody and FcγRIIa+ L cells. T cell proliferation was monitored by CellTiter-Glo® (Promega). Coupled IgGs include monoepitopic 1A7_hIgG1(N297G)-BM(PEG)3-1A7_hIgG1(N297G), monoepitopic 3C8_hIgG1(N297G)-BM(PEG)3-3C8_hIgG1(N297G), and biepitopic 1A7/3C8_hIgG1(N297G)-BM(PEG)3-1A7/3C8_hIgG1(N297G). L cells in this assay are FcγRII+ but lack CD80. The four controls are L cells, L cells + anti-CD3, L cells + T cells, and L cells + T cells + anti-CD3. All antibodies tested included anti-CD3 unless noted ("-αCD3).

    FIG. 12 shows SEC-MALS data on coupled Fab-IgGs 1A7/3C8 and 1A7(N297G)/3C8.

    FIG. 13 shows co-stimulation of CD4+ memory T cell proliferation by anti-OX40 or control antibodies in the presence of anti-CD3 antibody and FcγRIIa+ L cells. T cell proliferation was monitored by CellTiter-Glo® (Promega). L cells in this assay are FcγRII+ but lack CD80. The four controls are L cells, L cells + anti-CD3, L cells + T cells, and L cells + T cells + anti-CD3. All antibodies tested included anti-CD3. Coupled Fab-IgG format is two 3C8 Fabs coupled with 1A7_hIgG1(K149C) via BM(PEG)3 linker.

    FIG. 14 shows co-stimulation of CD4+ memory T cell proliferation by anti-OX40 or control antibodies in the presence of anti-CD3 antibody and FcγRIIa+ L cells. T cell proliferation was monitored by CellTiter-Glo® (Promega). L cells in this assay are FcγRII+ but lack CD80. The four controls are L cells, L cells + anti-CD3, L cells + T cells, and L cells + T cells + anti-CD3. All antibodies tested included anti-CD3. Coupled Fab-IgG format is two 3C8 Fabs coupled with 1A7_hIgG1(K149C/N297G) via BM(PEG)3 linker.

    FIG. 15 shows agonist activity of anti-OX40 native (1A7) and recombinant Fv-IgG monoepitopic (1A7-1A7 and 3C8-3C8) and biepitopic (1A7-3C8 and 3C8-1A7) antibodies using OX40 expressing Jurkat cells with a luciferase reporter.

    FIG. 16 shows co-stimulation of CD4+ memory T cell proliferation by anti-OX40 native (1A7) or r:Fv-IgG antibodies in the presence of anti-CD3 antibody and FcγRIIa+ L cells. T cell proliferation was monitored by CellTiter-Glo® (Promega). L cells in this assay are FcγRII+ but lack CD80. The four controls are L cells, L cells + anti-CD3, L cells + T cells, and L cells + T cells + anti-CD3. All antibodies tested included anti-CD3.

    FIG. 17 shows SEC-MALS data on r:Fab-IgG 3C8-1A7 anti-OX40 antibody compared to a native IgG1 antibody, as labeled.

    FIG. 18 shows SEC-MALS data on r:Fab-IgG 1A7-3C8 anti-OX40 antibody compared to a native IgG1 antibody, as labeled.

    FIG. 19 shows agonist activity of anti-OX40 native recombinant Fab-IgG monoepitopic (1A7-1A7 and 3C8-3C8) and biepitopic (1A7-3C8 and 3C8-1A7) antibodies using OX40 expressing Jurkat cells with a luciferase reporter.

    FIG. 20 shows co-stimulation of CD4+ memory T cell proliferation by anti-OX40 native (1A7) or r:Fab-IgG antibodies in the presence of anti-CD3 antibody and FcγRIIa+ L cells. T cell proliferation was monitored by CellTiter-Glo® (Promega). L cells in this assay are FcγRII+ but lack CD80. The four controls are L cells, L cells + anti-CD3, L cells + T cells, and L cells + T cells + anti-CD3. All antibodies tested included anti-CD3.

    FIG. 21 shows co-stimulation of CD4+ memory T cell proliferation by anti-OX40 antibodies in the presence of anti-CD3 antibody and FcγRIIa+ L cells. The left graph shows results from IgG1 versions, while the right graph shows results from IgG1 LALAPG versions of the r:Fv-IgGs. r:Fv-IgG's included were 3C8-1A7, 1A7-3C8, 3C8-3C8, and 1A7-1A7. IgG1 1A7 refers to the human IgG1 version of the 1A7 antibody on both left and right panels of the figure.

    FIG. 22 shows agonist activity of anti-OX40 control and r:Fab-IgG and r:Fv-IgG antibodies in the OX40 expressing Jurkat cells luciferase reporter assay. r:Fab-IgG (left panel) and r:Fv-IgG (right panel) include both biepitopic (1A7-3C8 and 3C8-1A7) formats as well as the monoepitopic 1A7-1A7 format. Bivalent IgG1 controls include 1A7, 3C8, and a bivalent biepitopic antibody 1A7/3C8. All heavy chain constant regions were human IgG1 in these assays.

    FIG. 23 shows agonist activity of anti-OX40 control and cIgG-IgG and c:Fab-IgG antibodies in the OX40 expressing Jurkat cells luciferase reporter assay. c:IgG-IgG (left panel) and c:Fab-IgG (right panel) include both biepitopic formats (c:IgG-IgG 1A7/3C8-1A7/3C8 and c:Fab-IgG 1A7-3C8) as well as the monoepitopic 1A7-1A7 format. Bivalent IgG1 controls include 1A7, 3C8, and a bivalent biepitopic antibody 1A7/3C8. All heavy chain constant regions were human IgG1 in these assays.

    FIG. 24 shows agonist activity of control and tetravalent biepitopic anti-OX40 antibodies in the OX40 expressing Jurkat cells luciferase reporter assay. All heavy chain constant regions were human IgG1 in these assays.

    FIG. 25 shows Size Exclusion Chromatography (SEC) chromatograms for biepitopic r:Fv-IgG 1A7-3C8 (black) and monoepitopic r:Fv-IgG 1A7-1A7 (gray) alone (solid lines) and in complex with target OX40 (dashed lines). Earlier elution time is proportional to greater protein size.

    FIG. 26 shows results from Multiple Angle Light Scattering (MALS) data upon SEC column elution. The Y axis reflects size of antibody/OX40 complex in KDa. Monoepitopic formats are shown as gray bars, biepitopic formats are shown as open white bars.

    FIG. 27 shows pharmacokinetics (PK) of anti-OX40 r:Fv-IgG and r:Fab-IgG antibody formats in C.B-17 SCID mice. IgG1 1A7 and IgG1 anti-gD were included as controls.

    FIG. 28 shows PK of anti-OX40 c:Fab-IgG and c:IgG-IgG antibody formats in C.B-17 SCID mice. IgG1 1A7 and IgG1 anti-gD were included as controls.

    FIG. 29 shows PK of anti-OX40 r:Fv-IgG, r:Fab-IgG, c:Fab-IgG, and c:IgG-IgG antibody formats in C57BL-6 mice. IgG1 1A7 was included as a control.

    FIG. 30 shows results from the KLH immunization PD experiment in human OX40 knockin (hOX40ki) mice comparing r:Fv-IgG 1A7-3C8 to IgG1 versions of 1A7 and 3C8. The left panel shows level of detected anti-KLH IgG, and the right panel shows percent expansion of CD4 T cells. p values comparing r:Fv-IgG to IgG1 1A7 are shown.

    FIG. 31 shows results from the KLH immunization PD experiment in hOX40ki mice comparing r:Fv-IgG 1A7-3C8 to IgG1 versions of 1A7 and 3C8. The left panel shows total number of CD8+ T cells, and the right panel shows total number of cells in the draining lymph node. p values comparing r:Fv-IgG to IgG1 1A7 are shown.

    FIG. 32 shows results from the KLH immunization PD experiment in hOX40ki mice comparing r:Fv-IgG 1A7-3C8 to IgG1 versions of 1A7 and 3C8. The graph shows release of interferon gamma (IFN-γ) after ex vivo restimulation of purified CD4+ T cells with KLH. p values comparing r:Fv-IgG to IgG1 1A7 are shown.

    FIG. 33 shows results from the KLH immunization PD experiment in hOX40ki mice comparing anti-OX40 biepitopic r:Fv-IgG, r:Fab-IgG, and c:Fab-IgG antibody formats, along with IgG1 versions of 1A7 and 3C8. The left panel shows total number of CD4 T cells, and the right panel shows total number of CD8 T cells in the draining lymph node. Three p values are shown comparing tetravalent biepitopic formats to IgG1 3C8 ("vs 3C8"), IgG1 1A7 ("vs 1A7"), and control IgG1 anti-gD ("vs ctl"), as indicated. Number of asterisks represents p value as follows: * < 0.05, ** < 0.005, *** < 0.0005, * < 0.00005.

    FIG. 34 shows anti-tumor activity of antibodies in a syngeneic E.G7-OVA (EL4 expressing chicken ovalbumin (OVA)) lymphoma tumor model in female hOX40ki C57BL/6 mice. Test articles included tetravalent biepitopic r:Fv-IgG 3C8-1A7, IgG1 1A7, and isotype control IgG1 anti-gD.

    FIG. 35 shows the crystallographic structure of the ternary complex between OX40 and the 1A7 and 3C8 Fabs. An N-linked glycosylation is highlighted at position 160 of the OX40 receptor.

    FIG. 36 shows the sequence of human OX40 highlighting the binding epitopes of 1A7 (grey boxes) and 3C8 (black outlined boxes) based on the ternary complex structure. Shown are residues 29-214 of SEQ ID NO:281.

    FIG. 37 shows agonist activity of IgG and r:Fv-IgG antibodies tested in the OX40+ Jurkat cell luciferase reporter assay. All heavy chain constant regions were human IgG1 in these assays.

    FIG. 38 shows OX40 agonist activity of engineered linker variants of r:Fv-IgGs and 1A7 human IgG1 (1A7) control antibody in both the primary human T cell assay (left panel) and OX40+ Jurkat cell luciferase reporter assay (right panel). The primary cell assay included CD4+ memory T cells, anti-CD3 antibody, and FcγRIIa+ L cells. GS(Long), GS(Medium), GS(Short), Elbow(Long), and Elbow(Short) refers to linker variants of r:Fv-IgG 3C8-1A7 as described in Example 16.

    FIG. 39 shows a plot of the affinities of high and low affinity variant versions of IgG1 and r:Fv-IgG formats. H corresponds to High (lower KD, stronger affinity) and L corresponds to Low (higher KD, weaker affinity) variants. Parentheses designate whether the 3C8 Fv and/or 1A7 Fv were increased or reduced in affinity. For example, 3C8(H)/1A7(L) corresponds to an r:Fv-IgG 3C8-1A7 in which the 3C8 Fv has stronger affinity (N31I/K64L variant) and in which the 1A7 Fv has weaker affinity (P96A variant) relative to the r:Fv-IgG 3C8-1A7 parent.

    FIG. 40 shows OX40 agonist activity of engineered affinity variants of r:Fv-IgGs and 1A7 human IgG1 (1A7) and 3C8 human IgG1 (3C8) control antibodies in both the primary human T cell assay (left panel) and OX40+ Jurkat cell luciferase reporter assay (right panel). The primary cell assay included CD4+ memory T cells, anti-CD3 antibody, and FcyRIIa+ L cells. High corresponds to stronger affinity (lower KD) and Low corresponds to weaker affinity (higher KD) variants relative to the r:Fv-IgG 3C8-1A7 parent. Parentheses designate whether the 3C8 Fv and/or 1A7 Fv were increased or reduced in affinity. For example, 3C8(High)-1A7(Low) corresponds to an r:Fv-IgG 3C8-1A7 in which the 3C8 Fv has stronger affinity (N31I/K64L variant) and in which the 1A7 Fv has weaker affinity (P96A variant) relative to the r:Fv-IgG 3C8-1A7 parent.

    FIG. 41 shows caspase 8 activity for IgG1, biepitopic IgG1, and biepitopic r:Fab-IgG versions of anti-DR5 antibodies against HT-29 cells. Antibodies were tested at 50 nM concentration. Drozitumab is abbreviated to Droz. IgG1 antibodies include Drozitumab, 13E3, 3H3, 4D9, and 11H12. Bivalent biepitopic IgG1 antibodies include IgG1 Droz-4D9, IgG1 13E3-3H3, and IgG1 11H12-4D9. r:Fab-IgGs include tetravalent monoepitopics r:Fab-IgG 4D9-4D9 and r:Fab-IgG Droz-Droz and tetravalent biepitopics r:Fab-IgG Droz-4D9, r:Fab-IgG 13E3-3H3, and r:Fab-IgG 11H12-4D9.

    FIG. 42 shows dose-response caspase 8 activity for IgG1, biepitopic IgG1, and biepitopic r:Fab-IgG versions of anti-DR5 antibodies against HT-29 cells. Drozitumab is abbreviated to Droz. IgG1 antibodies include IgG1 Droz and IgG1 4D9. Bivalent biepitopic IgG1 antibodies include IgG1 Droz-4D9. r:Fab-IgGs include tetravalent monoepitopics r:Fab-IgG 4D9-4D9 and r:Fab-IgG Droz-Droz, and tetravalent biepitopic r:Fab-IgG Droz-4D9.

    FIG. 43 shows dose-response anti-proliferative activity for IgG1, biepitopic IgG1, and biepitopic r:Fab-IgG versions of anti-DR5 antibodies against HT-29 cells. Drozitumab is abbreviated to Droz. IgG1 antibodies include IgG1 Droz and IgG1 4D9. Bivalent biepitopic IgG1 antibodies include IgG1 Droz-4D9. r:Fab-IgGs include tetravalent monoepitopics r:Fab-IgG 4D9-4D9 and r:Fab-IgG Droz-Droz, and tetravalent biepitopic r:Fab-IgG Droz-4D9.

    FIG. 44 shows dose-response caspase 8 activity for IgG1, biepitopic IgG1, and biepitopic r:Fab-IgG versions of anti-DR5 antibodies against Colo205 cells. Drozitumab is abbreviated to Droz. IgG1 antibodies include IgG1 Droz and IgG1 4D9. Bivalent biepitopic IgG1 antibodies include IgG1 Droz-4D9. r:Fab-IgGs include tetravalent monoepitopics r:Fab-IgG 4D9-4D9 and r:Fab-IgG Droz-Droz, and tetravalent biepitopic r:Fab-IgG Droz-4D9.

    FIG. 45 shows dose-response antiproliferative activity for IgG1, biepitopic IgG1, and biepitopic r:Fab-IgG versions of anti-DR5 antibodies against Colo205 cells. Drozitumab is abbreviated to Droz. IgG1 antibodies include IgG1 Droz and IgG1 4D9. Bivalent biepitopic IgG1 antibodies include IgG1 Droz-4D9. r:Fab-IgGs include tetravalent monoepitopics r:Fab-IgG 4D9-4D9 and r:Fab-IgG Droz-Droz, and tetravalent biepitopic r:Fab-IgG Droz-4D9.


    DETAILED DESCRIPTION


    I. Definitions



    [0013] The term "polypeptide" or "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component or toxin. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. The terms "polypeptide" and "protein" as used herein specifically encompass antibodies.

    [0014] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. The term "immunoglobulin" (Ig) is used interchangeable with antibody herein.

    [0015] Antibodies are naturally occurring immunoglobulin molecules which have varying structures, all based upon the immunoglobulin fold. For example, IgG antibodies have two "heavy" chains and two "light" chains that are disulphide-bonded to form a functional antibody. Each heavy and light chain itself comprises a "constant" (C) and a "variable" (V) region. The V regions determine the antigen binding specificity of the antibody, whilst the C regions provide structural support and function in non-antigen- specific interactions with immune effectors. The antigen binding specificity of an antibody or antigen-binding fragment of an antibody is the ability of an antibody to specifically bind to a particular antigen.

    [0016] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β- sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

    [0017] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence ("complementarity determining regions" or "CDRs") and/or form structurally defined loops ("hypervariable loops") and/or contain the antigen-contacting residues ("antigen contacts"). Generally, antibodies comprise six HVRs: three in the VH (HI, H2, H3), and three in the VL (L1, L2, L3). Exemplary HVRs herein include:
    1. (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));
    2. (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));
    3. (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and
    4. (d) combinations of (a), (b), and/or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).


    [0018] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

    [0019] "Antibody fragments" comprise a portion of an intact antibody, preferably comprising the "antigen binding region" thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; tandem diabodies (taDb), linear antibodies (e.g., U.S. Patent No. 5,641,870, Example 2; Zapata et al, Protein Eng. 8(10): 1057-1062 (1995)); one-armed antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., including but not limited to, Db- Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem (di,tri)-scFv); and Bi-specific T-cell engagers (BiTEs).

    [0020] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

    [0021] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

    [0022] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

    [0023] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

    [0024] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, y, and µ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

    [0025] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In some aspects, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pliickthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994).

    [0026] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH - VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et ah, Proc. Natl. Acad. Set USA, 90:6444-6448 (1993).

    [0027] The term "multispecific antibody" is used in the broadest sense and specifically covers an antibody that has polyepitopic specificity. Such multispecific antibodies include, but are not limited to, an antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), where the VHVL unit has polyepitopic specificity, antibodies having two or more VL and VH domains with each VHVL unit binding to a different epitope, antibodies having two or more single variable domains with each single variable domain binding to a different epitope, full length antibodies, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, triabodies, tri-functional antibodies, antibody fragments that have been linked covalently or non-covalently. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). "Monospecific" refers to the ability to bind only one epitope. According to one aspect the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5 µM to 0.001 pM, 3 µM to 0.001 pM, 1 µM to 0.001 pM, 0.5 µM to 0.001 pM, or 0.1 µM to 0.001 pM.

    [0028] The expression "single domain antibodies" (sdAbs) or "single variable domain (SVD) antibodies" generally refers to antibodies in which a single variable domain (VH or VL) can confer antigen binding. In other words, the single variable domain does not need to interact with another variable domain in order to recognize the target antigen. Examples of single domain antibodies include those derived from camelids (lamas and camels) and cartilaginous fish (e.g., nurse sharks) and those derived from recombinant methods from humans and mouse antibodies (Nature (1989) 341:544-546; Dev Comp Immunol (2006) 30:43-56; Trend Biochem Sci (2001) 26:230-235; Trends Biotechnol (2003):21:484-490; WO 2005/035572; WO 03/035694; Febs Lett (1994) 339:285-290; WO00/29004; WO 02/051870).

    [0029] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the methods provided herein may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al, Nature 352:624-628 (1991) and Marks et al, J. Mol. Biol. 222:581-597 (1991), for example.

    [0030] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey, such as baboon, rhesus or cynomolgus monkey) and human constant region sequences (US Pat No. 5,693,780).

    [0031] An "acceptor human framework" for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some aspects, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some aspects, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

    [0032] A "human consensus framework" is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one aspect, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one aspect, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

    [0033] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence, except for FR substitution(s) as noted above. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522- 525 (1986); Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

    [0034] A "human antibody" is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

    [0035] In some aspects, antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity(CDC); Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors.

    [0036] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells in summarized is Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et ah, Proc. Natl. Acad. Set (USA) 95:652-656 (1998).

    [0037] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. In some aspects, the cells express at least FcγRIII and carry out ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred.

    [0038] "Complement dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g. polypeptide (e.g., an antibody)) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996), may be performed.

    [0039] The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the Fc region of an antibody. In some aspects, the FcR is a native sequence human FcR.

    [0040] Moreover, a preferred FcR is one that binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcyRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcyRIIA (an activating receptor) and FcγRIIB (an inhibiting receptor), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, {see Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25- 34 (1994); and de Haas et al, J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al, J. Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)).

    [0041] The term "Fc region", as used herein, generally refers to a dimer complex comprising the C-terminal polypeptide sequences of an immunoglobulin heavy chain, wherein a C-terminal polypeptide sequence is that which is obtainable by papain digestion of an intact antibody. The Fc region may comprise native or variant Fc sequences. Although the boundaries of the Fc sequence of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc sequence is usually defined to stretch from an amino acid residue at about position Cys226, or from about position Pro230, to the carboxyl terminus of the Fc sequence. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The Fc sequence of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. By "Fc polypeptide" herein is meant one of the polypeptides that make up an Fc region, e.g., a monomeric Fc. An Fc region may be obtained from any suitable immunoglobulin, such as IgG1 lgG2, lgG3, or lgG4 subtypes, IgA, IgE, IgD or IgM. The Fc region comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region; this region is also the part recognized by Fc receptors (FcR) found on certain types of cells. In some aspects, an Fc polypeptide comprises part or all of a wild type hinge sequence (generally at its N terminus). In some aspects, an Fc polypeptide does not comprise a functional or wild type hinge sequence.

    [0042] A "modified Fc region" or "Fc variant" comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the modified Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The modified Fc region herein will preferably possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology therewith.

    [0043] The term "agonist", "agonistic", "agonism" or "agonize" as used herein in general refers to a binding molecule (e.g., an antigen binding polypeptide or antigen binding complex) which binds to a receptor on the surface of a cell and is capable of initiating/mimicking/stimulating a reaction or activity that is similar to or the same as that initiated/mimicked/stimulated by the receptor's natural ligand. In exemplary aspects, an agonist as described herein is capable of inducing/augmenting/enhancing/stimulating the activation of a signal transduction pathway associated with the receptor.

    [0044] The term "cell surface receptor," as used herein, refers to any native cell surface receptor from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed cell surface receptor as well as any form of cell surface receptor that results from processing in the cell. The term also encompasses naturally occurring variants of cell surface receptor, e.g., splice variants or allelic variants.

    [0045] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include epithelial ovarian cancer, fallopian tube cancer, primary peritoneal cancer, squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer (including platinum sensitive and platinum resistant ovarian cancer), liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, fallopian tube, peritoneal, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.

    [0046] An "affinity matured" antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

    [0047] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

    [0048] The terms "full length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

    [0049] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

    [0050] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

    [0051] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the individual or subject is a human.

    [0052] An "isolated" antibody is one which has been separated from a component of its natural environment. In some aspects, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

    [0053] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

    [0054] "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

    [0055] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

    [0056] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

    where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

    [0057] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

    [0058] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

    [0059] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

    II Antigen Binding Complexes With Agonist Activity



    [0060] Provided herein are tetravalent antigen binding complexes, having agonist activity. A variety of configurations for the antigen binding complexes of the present disclosure are described and illustrated herein, with different formats including both mono-epitopic and bi-or multi-epitopic forms (see, e.g., FIGS. 3A & 3B). As demonstrated herein, targeting a cell surface receptor with a tetravalent antigen binding complex having agonist activity for the cell surface receptor results in strong receptor activation. Moreover, it is a surprising discovery of the present disclosure that targeting multiple epitopes of a cell surface receptor with an antigen binding complex (e.g., a tetravalent antigen binding complex) having agonist activity for the cell surface receptor results in a synergistic increase in receptor-mediated signaling (e.g., as compared to targeting only a single epitope with an equivalent number of antigen binding moieties). Importantly, the present disclosure demonstrates that antigen binding regions (e.g., antibody CDRs and/or variable domains) of antibodies that already show agonist activity when in monovalent form demonstrate superior agonist activity when used in the multivalent, biepitopic formats described herein. Exemplary antigen binding complex formats are described infra.

    [0061] An exemplary configuration of the antigen binding complex is described and illustrated herein as an "r:Fv-IgG" format (see, e.g., FIGS. 3A & 3B). A variety of configurations, including both mono-epitopic and bi- or multi-epitopic forms, are contemplated. For example, in some aspects, the antibody heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain (VH1), a second heavy chain variable domain (VH2), a heavy chain CH1 domain, a heavy chain CH2 domain, a heavy chain CH3 domain, and, optionally, a heavy chain CH4 domain. In some aspects, the antibody light chain comprises, from N-terminus to C-terminus, a first light chain variable domain (VL1), a second light chain variable domain (VL2), and a light chain constant domain (e.g., VK or VL). In some aspects, the first heavy chain variable domain (VH1) and the first light chain variable domain (VL1) recognize a first epitope of a cell surface receptor, and the second heavy chain variable domain (VH2) and the second light chain variable domain (VL2) recognize the same epitope of the cell surface receptor. In some aspects, the first heavy chain variable domain (VH1) and the first light chain variable domain (VL1) recognize a first epitope of a cell surface receptor, and the second heavy chain variable domain (VH2) and the second light chain variable domain (VL2) recognize a different epitope of the same cell surface receptor.

    [0062] In some aspects, an antigen binding complex (e.g., a tetravalent antigen binding complex) of the present disclosure comprises two or more antibody heavy chains and/or two or more antibody light chains. In some aspects, each heavy chain comprises a heavy chain variable domain (VH) and a set of heavy chain constant domains, e.g., a CH1 domain, a CH2 domain, a CH3 domain, and, optionally (e.g., for IgM or IgE antibodies), a CH4 domain. In some aspects, each light chain comprises a light chain variable domain (VL) and a constant light (CL) domain (e.g., kappa or lambda). In some aspects, the heavy chains of each subunit comprise one or more modifications that reduce effector function, e.g., as described herein.

    [0063] In certain aspects of any of the above formats, the VH1 comprises: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; and/or the VL1 comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and (iii) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:7. In some aspects, the VH1 comprises one, two, or three antibody HVR sequences selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; and/or the VL1 comprises one, two, or three antibody HVR sequences selected from: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and (vi) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:7. In some aspects, the VH1 comprises the amino acid sequence of SEQ ID NO:56 and/or the VL1 comprises the amino acid sequence of SEQ ID NO:57. In certain aspects of any of the above formats, the VH2 comprises: (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; and/or the VL2 comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and (iii) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:42. In some aspects, the VH2 comprises one, two, or three antibody HVR sequences selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; and/or the VL2 comprises one, two, or three antibody HVR sequences selected from: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and (vi) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:42. In some aspects, the VH2 comprises the amino acid sequence of SEQ ID NO: 126 and/or the VL2 comprises the amino acid sequence of SEQ ID NO:129. In certain aspects of any of the above formats, the VH1 comprises: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; the VL1 comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and (iii) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:7; the VH2 comprises: (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; and the VL2 comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and (iii) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:42. In certain aspects, the VH1 comprises the amino acid sequence of SEQ ID NO:56, the VL1 comprises the amino acid sequence of SEQ ID NO:57, the VH2 comprises the amino acid sequence of SEQ ID NO:126, and the VL2 comprises the amino acid sequence of SEQ ID NO:129.

    [0064] In certain aspects of any of the above formats, the VH2 comprises: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; and/or the VL2 comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and (iii) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:7. In some aspects, the VH2 comprises one, two, or three antibody HVR sequences selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; and/or the VL2 comprises one, two, or three antibody HVR sequences selected from: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and (vi) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:7. In some aspects, the VH2 comprises the amino acid sequence of SEQ ID NO:56 and/or the VL2 comprises the amino acid sequence of SEQ ID NO:57. In certain aspects of any of the above formats, the VH1 comprises: (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; and/or the VL1 comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and (iii) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:42. In some aspects, the VH1 comprises one, two, or three antibody HVR sequences selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; and/or the VL1 comprises one, two, or three antibody HVR sequences selected from: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and (vi) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:42. In some aspects, the VH1 comprises the amino acid sequence of SEQ ID NO: 126 and/or the VL1 comprises the amino acid sequence of SEQ ID NO:129. In certain aspects of any of the above formats, the VH2 comprises: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; the VL2 comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and (iii) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:7; the VH1 comprises: (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; and the VL1 comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and (iii) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:42. In certain aspects, the VH1 comprises the amino acid sequence of SEQ ID NO:126, the VL1 comprises the amino acid sequence of SEQ ID NO: 129, the VH2 comprises the amino acid sequence of SEQ ID NO:56, and the VL2 comprises the amino acid sequence of SEQ ID NO:57.

    [0065] In some aspects, a complex comprises two antibody heavy chain polypeptides and two antibody light chain polypeptides; wherein each of the antibody heavy chain polypeptides comprises: VH-L1-VH2-L2-CH1-hinge-CH2-CH3 [I]; wherein each of the antibody light chain polypeptides comprises: VL1-L3-VL2-L4-CL [II]; wherein each of the antibody heavy chain polypeptides associates with one antibody light chain polypeptide such that VH1 and VL1 form an antigen binding domain and VH2 and VL2 form an antigen binding domain; wherein VH1 is a first antibody heavy chain variable domain, VH2 is a second antibody heavy chain variable domain, VL1 is a first antibody light chain variable domain, VL2 is a second antibody light chain variable domain, CL is an antibody light chain constant domain, CH1 is an antibody first heavy chain constant domain, hinge is an antibody hinge region, CH2 is an antibody second heavy chain constant domain, CH3 is an antibody third heavy chain constant domain, and L1, L2, L3, and L4 are amino acid linkers.

    [0066] In some aspects, VH1 comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; wherein VL1 comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:42; wherein VH2 comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; and wherein VL2 comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In some aspects, VH2 comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; wherein VL2 comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:42; wherein VH1 comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; and wherein VL1 comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In some aspects, VH1 comprises the amino acid sequence of SEQ ID NO: 128, wherein VL1 comprises the amino acid sequence of SEQ ID NO: 129, wherein VH2 comprises the amino acid sequence of SEQ ID NO:56, and wherein VL2 comprises the amino acid sequence of SEQ ID NO:57. In some aspects, VH2 comprises the amino acid sequence of SEQ ID NO:128, wherein VL2 comprises the amino acid sequence of SEQ ID NO:129, wherein VH1 comprises the amino acid sequence of SEQ ID NO:56, and wherein VL1 comprises the amino acid sequence of SEQ ID NO:57. In some aspects, a VH domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs:56 or 128. In some aspects, a VL domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs:57 or 129. In some aspects, an antibody heavy chain polypeptide according to formula [I] comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 240, 242, 250, 252, 254, 256, 258, 260, 262, 264, or 266. In some aspects, an antibody heavy chain polypeptide according to formula [I] comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 240 or 242. In some aspects, an antibody heavy chain polypeptide according to formula [I] comprises the amino acid sequence of SEQ ID NO:242 or 291.

    [0067] In any of the complexes described above, a VH1, VH2, VL1, and/or VL2 comprises an HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and/or HVR-L3 that comprise one or more of the sequences listed in Tables 4 and 5.

    [0068] A complex of the present disclosure can have one or more of the agonist activities described below.

    [0069] In some aspects, the OX40 agonist complex binds human OX40 with an affinity of less than or equal to about 0.45 nM. In some aspects, the OX40 agonist complex binds human OX40 with an affinity of less than or equal to about 1 nM. In some aspects, the OX40 complex binds human OX40 with an affinity of less than or equal to about 0.4 nM. In some aspects, the OX40 complex binds human OX40 with an affinity of less than or equal to about 0.5nM. In some aspects, the binding affinity is determined using radioimmunoassay.

    [0070] In some aspects, the OX40 agonist complex binds human OX40 and cynomolgus OX40. In some aspects, binding is determined using a FACS assay. In some aspects, binding to human OX40 has an EC50 of about 0.2 ug/ml. In some aspects, binding to human OX40 has an EC50 of about 0.3 ug/ml or lower. In some aspects, binding to cynomolgus OX40 has an EC50 of about 1.5 ug/ml. In some aspects, binding to cynomolgus OX40 has an EC50 of about 1.4 ug/ml.

    [0071] In some aspects, the OX40 agonist complex does not bind to rat OX40 or mouse OX40.

    [0072] In some aspects, the OX40 agonist complex does not induce apoptosis in OX40-expressing cells (e.g., Treg). In some aspects, apoptosis is assayed using an antibody concentration of 30ug/ml, e.g., by determining whether apoptosis has occurred using annexin V and proprodium iodide stained Treg.

    [0073] In some aspects, the OX40 agonist complex increases memory T cell proliferation and/or increasing cytokine production by the memory cell. In some aspects, the cytokine is IFN-γ. In some aspects, the OX40 agonist complex enhances memory T cell function, for example by increasing memory T cell proliferation and/or increasing cytokine production by the memory cell. In some aspects, the cytokine is gamma interferon.

    [0074] In some aspects, the OX40 agonist complex increases CD4+ effector T cell proliferation and/or increases cytokine production by the CD4+ effector T cell as compared to proliferation and/or cytokine production prior to treatment with the OX40 agonist complex. In some aspects, the cytokine is IFN-γ.

    [0075] In some aspects, the anti-human OX40 agonist complex enhances CD4+ effector T cell function, for example, by increasing CD4+ effector T cell proliferation and/or increasing gamma interferon production by the CD4+ effector T cell (for example, as compared to proliferation and/or cytokine production prior to treatment with anti-human OX40 agonist complex). In some aspects, the cytokine is gamma interferon. In some aspects, the anti-human OX40 agonist complex increases number of intratumoral (infiltrating) CD4+ effector T cells (e.g., total number of CD4+ effector T cells, or e.g., percentage of CD4+ cells in CD45+ cells), e.g., as compared to number of intratumoral (infiltrating) CD4+ T cells prior to treatment with anti-human OX40 agonist complex. In some aspects, the anti-human OX40 agonist complex increases number of intratumoral (infiltrating) CD4+ effector T cells that express gamma interferon (e.g., total gamma interferon expressing CD4+ cells, or e.g., percentage of gamma interferon expressing CD4+ cells in total CD4+ cells), e.g., as compared to number of intratumoral (infiltrating) CD4+ T cells that express gamma interferon prior to treatment with anti-human OX40 agonist complex.

    [0076] In some aspects, the number of CD4+ effector T cells is elevated relative to prior to administration of the OX40 agonist complex. In some aspects, CD4+ effector T cell cytokine secretion is elevated relative to prior to administration of the OX40 agonist complex. In some aspects of any of the methods, the CD8+ effector T cells in the individual have enhanced proliferation, cytokine secretion and/or cytolytic activity relative to prior to the administration of the OX40 agonist complex. In some aspects, the number of CD8+ effector T cells is elevated relative to prior to administration of the OX40 agonist complex. In some aspects, CD8+ effector T cell cytokine secretion is elevated relative to prior to administration of the OX40 agonist complex.

    [0077] In some aspects, the anti-human OX40 agonist complex increases number of intratumoral (infiltrating) CD8+ effector T cells (e.g., total number of CD8+ effector T cells, or e.g., percentage of CD8+ in CD45+ cells), e.g., as compared to number of intratumoral (infiltrating) CD8+ T effector cells prior to treatment with anti-human OX40 agonist complex. In some aspects, the anti-human OX40 agonist complex increases number of intratumoral (infiltrating) CD8+ effector T cells that express gamma interferon (e.g., percentage of CD8+ cells that express gamma interferon in total CD8+ cells), e.g., compared to number of intratumoral (infiltrating) CD8+ T cells that express gamma interferon prior to treatment with anti-human OX40 agonist complex.

    [0078] In some aspects, the number of intratumoral (infiltrating) CD8+ effector T cells (e.g., total number of CD8+ effector T cells, or e.g., percentage of CD8+ in CD45+ cells) is elevated relative to prior to administration of the OX40 agonist complex. In some aspects of any of the methods of the invention, number of intratumoral (infiltrating) CD8+ effector T cells that express gamma interferon (e.g., percentage of CD8+ cells that express gamma interferon in total CD8+ cells) is increased relative to prior to administration of the OX40 agonist complex.

    [0079] In some aspects, the memory T cells in the individual have enhanced proliferation and/or cytokine secretion relative to prior to the administration of the OX40 agonist complex. In some aspects, the number of memory T cells is elevated relative to prior to administration of the OX40 agonist complex. In some aspects, memory T cell cytokine secretion (level) is elevated relative to prior to administration of the OX40 agonist complex. In some aspects of any of the methods, the Treg in the individual have decreased inhibition of effector T cell function (e.g., proliferation and/or cytokine secretion) relative to prior to the administration of the OX40 agonist complex. In some aspects, the number of effector T cells is elevated relative to prior to administration of the OX40 agonist complex. In some aspects, effector T cell cytokine secretion (level) is elevated relative to prior to administration of the OX40 agonist complex.

    [0080] In some aspects, the OX40 agonist complex inhibits Treg suppression of effector T cell function. In some aspects, effector T cell function is effector T cell proliferation and/or cytokine production. In some aspects, the effector T cell is a CD4+ effector T cell.

    [0081] In some aspects, the OX40 agonist complex inhibits Treg function, for example, by decreasing Treg suppression of effector T cell function (e.g., effector T cell proliferation and/or effector T cell cytokine secretion). In some aspects, the effector T cell is a CD4+ effector T cell. In some aspects, the OX40 agonist complex reduces the number of intratumoral (infiltrating) Treg (e.g., total number of Treg or e.g., percentage of Fox3p+ cells in CD4+ cells).

    [0082] In some aspects, the number of intratumoral (infiltrating) Treg (e.g., total number of Treg or e.g., percentage of Fox3p+ cells in CD4+ cells) is reduced relative to prior to administration of the OX40 agonist complex.

    [0083] In some aspects, the number of intratumoral (infiltrating) CD4+ effector T cells (e.g., total number of CD4+ effector T cells, or e.g., percentage of CD4+ cells in CD45+ cells) is elevated relative to prior to administration of the OX40 agonist complex. In some aspects of any of the methods of the invention, number of intratumoral (infiltrating) CD4+ effector T cells that express gamma interferon (e.g., total gamma interferon expressing CD4+ cells, or e.g., percentage of gamma interferon expressing CD4+ cells in total CD4+ cells) is elevated relative to prior to administration of the OX40 agonist complex.

    [0084] In some aspects, the OX40 agonist complex increases OX40 signal transduction in a target cell that expresses OX40. In some aspects, OX40 signal transduction is detected by monitoring NFkB downstream signaling.

    [0085] In some aspects, the OX40 agonist complex is stable after treatment at 40°C for two weeks.

    [0086] In some aspects, the OX40 agonist complex competes for binding to human OX40 with OX40L. In some aspects, addition of OX40L does not enhance OX40 complex function in an in vitro assay.

    [0087] According to another aspect, the OX40 agonist complexes include any one, any combination, or all of the following properties: (1) binds human OX40 with an affinity of less than or equal to about 0.45 nM, in some aspects, binds human OX40 with an affinity of less than or equal to about 0.4 nM, in some aspects, binds human OX40 with an affinity of less than or equal to about 0.5nM, in some aspects, the binding affinity is determined using radioimmunoassay; (2) binds human OX40 and cynomolgus OX40, in some aspects, binding is determined using a FACS assay, (3) binds human OX40 with an EC50 of about 0.2 ug/ml, in some aspects, binds to human OX40 has an EC50 of about 0.3 ug/ml or lower, in some aspects, binds to cynomolgus OX40 with an EC50 of about 1.5 ug/ml, in some aspects, binds to cynomolgus OX40 has an EC50 of about 1.4 ug/ml, (4) does not substantially bind to rat OX40 or mouse OX40, (5) enhances CD4+ effector T cell function, for example, by increasing CD4+ effector T cell proliferation and/or increasing gamma interferon production by the CD4+ effector T cell (for example, as compared to proliferation and/or cytokine production prior to treatment with anti-human OX40 agonist complex), (6) enhances memory T cell function, for example by increasing memory T cell proliferation and/or increasing cytokine production by the memory cell, (7) inhibits Treg function, for example, by decreasing Treg suppression of effector T cell function (e.g., effector T cell proliferation and/or effector T cell cytokine secretion). In some aspects, the effector T cell is a CD4+ effector T cell, (8) increases OX40 signal transduction in a target cell that expresses OX40 (in some aspects, OX40 signal transduction is detected by monitoring NFkB downstream signaling), and (9) is stable after treatment at 40°C for two weeks.

    [0088] Further contemplated herein are bispecific or multispecific antibodies, wherein each arm of a bispecific or multispecific antibody binds to the same cell surface receptor, e.g., OX40. In some aspects, each arm of a bispecific or multispecific antibody binds to a different epitope of the same cell surface receptor, e.g., OX40. For example, in certain aspects, a bispecific antibody may comprise two arms, wherein each arm binds a different epitope of OX40. It is to be understood that any of the exemplary antibodies, antigen binding domains, and/or antibody fragments that bind OX40 (e.g., HVRs, VH, and/or VL domains of any of the OX40 agonist antibodies described herein) may be combined in a bispecific or multispecific antibody in any combination.

    [0089] Any of the other antibody HVR sequences, VH/VL sequences, and/or binding specificities described herein may be used in addition to or in place of one or more of the sequences described above.

    Antigen Binding Polypeptides



    [0090] Antigen binding polypeptides or subunits suitable for forming the complexes described herein comprise at least one antigen binding region for a cell surface receptor. The antigen binding polypeptides described herein may comprise an antibody, an antigen binding region of an antibody (e.g., an antibody fragment) fused to an Fc region, or a non-antibody antigen binding region protein fused to an Fc region. In exemplary aspects, the antigen binding polypeptide is an antibody which binds to a cell surface receptor and has a modified Fc region.

    [0091] In some aspects, an antibody or subunit described herein contains at least two polypeptides. In some aspects, the two polypeptides are half-antibodies, e.g., a single heavy chain (comprising a heavy chain variable region, a CH1 domain, and an Fc domain) and a single light chain (comprising a light chain variable region and CL domain). In particular, the Fc region for each antibody is a dimer formed between two polypeptides (either a homodimer or a heterodimer as described further below). Similarly, the antigen binding region attached to one of the polypeptides of the Fc region may contain two polypeptides, e.g., when the antigen binding region is an antibody fragment it may contain heavy and light chain variable regions.

    Antibodies



    [0092] In certain aspects, an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) of the present disclosure, or a subunit thereof, provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody.

    [0093] In certain aspects, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some aspects, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

    [0094] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" approach to FR shuffling).

    [0095] Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

    [0096] In certain aspects, an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) of the present disclosure, or a subunit thereof, provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

    [0097] Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007/0061900, describing VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

    [0098] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).

    [0099] Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

    [0100] Antibodies suitable as antigen binding polypeptides or subunits as described herein may be isolated by screening combinatorial libraries for polypeptides with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

    [0101] In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360. Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

    Antigen Binding Region of an Antibody



    [0102] In certain aspects, the antigen binding complexes (e.g., a tetravalent antigen binding complex having agonist activity) of the present disclosure or subunit(s) thereof described herein comprise an antigen binding region that binds to a cell surface receptor and an Fc region. In an exemplary aspect, the antigen binding polypeptides, antibodies, or subunits described herein comprise an antigen binding region of an antibody that binds to a cell surface receptor fused to an Fc region. In some aspects, an antigen binding region of the present disclosure refers to an antigen binding domain comprising a heavy chain variable domain (VH) and light chain variable domain (VL). In exemplary aspects, an antigen binding region of an antibody refers to an antibody fragment, such as, for example, a Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

    [0103] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

    [0104] Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain aspects, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

    [0105] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein. Antibody fragments can be made from any of the antibodies described herein, including for example, monoclonal, chimeric, humanized, human, bispecific, multispecific, DAF, etc. antibody formats.

    [0106] The antigen binding complexes (e.g., a tetravalent antigen binding complex having agonist activity) of the present disclosure or subunit(s) thereof described herein may comprise one or more polypeptides. In certain aspects, the antigen binding regions comprises one polypeptide, such as, for example a single chain Fv (scFv) wherein the heavy and light chain variable regions of an antibody are attached via a linker. In other aspects, the antigen binding region comprises two polypeptide, such as, for example a Fab antibody fragment wherein the heavy and light chain variable regions are separate polypeptide chains that naturally associate to form an antigen binding region having 6 CDRs.

    [0107] In some aspects, the complex comprises one or more amino acid substitutions in VH1, VL1, (CH1)x, or (CL)x that promote VH1 and VL1 forming an antigen binding domain; and/or one or more amino acid substitutions in VH2, VL2, (CH1)y, or (CL)y that promote VH2 and VL2 forming an antigen binding domain. See, e.g., International Pub. No. WO2016172485 and Example 6. For example, exemplary substitutions include without limitation VH-Q39K / VL-Q38E or VH Q39E / VL Q38K (Kabat numbering), CH1-S183E/ CL-V133K or CH1-S183K / CL-V133E (EU numbering); CH1 A141I, F170S, S181M, S183A, V185A; and CL F116A, L135V, S174A, S176F, and T178V (EU numbering).

    [0108] In certain aspects a multispecific antigen-binding protein comprises a CH1 domain of H1 comprising (including consisting of and consisting essentially of) A141I, F170S, S181M, S183A, and V185A mutations (EU numbering) and a CL domain of L1 comprising (including consisting of or consisting essentially of) F116A, L135V, S174A, S176F, and T178V mutations (EU numbering).

    [0109] In certain aspects, the VH domain of H1 of the multispecific antigen-binding protein comprises a Q39E substitution mutation (Kabat numbering), the VL domain of L1 the multispecific antigen-binding protein comprises a Q38K substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen-binding protein comprises (such as consists of or consists essentially of) an S 183E substitution mutation (EU numbering), and the CL domain of L2 of the multispecific antigen-binding protein comprises (such as consists of or consists essentially of) a V133K substitution mutation (EU numbering

    [0110] In certain aspects, the VH domain of H1 of the multispecific antigen-binding protein comprises a Q39K substitution mutation (Kabat numbering), the VL domain of L1 the multispecific antigen-binding protein comprises a Q38E substitution mutation (Kabat numbering), the CH1 domain of H2 of the multispecific antigen-binding protein comprises (such as consists of or consists essentially of) an S183E substitution mutation (EU numbering), and the CL domain of L2 of the multispecific antigen-binding protein comprises (such as consists of or consists essentially of) a V133K substitution mutation (EU numbering).

    Non-Antibody Antigen Binding Regions



    [0111] In certain aspects, the antigen binding polypeptides described herein comprise an antigen binding region that binds to a cell surface receptor and an Fc region. In an exemplary aspect, the antigen binding polypeptides described herein comprise a non-antibody antigen binding region that binds to a cell surface receptor fused to an Fc region. Examples of non-antibody antigen binding regions include, for example, ligands, ligand fragments, or multimers thereof, that bind to a cell surface receptor. Examples of non-antibody antigen binding regions that bind to OX40 are described below. Examples of non-antibody binding regions that bind to Tie2 are described in WO 2008/049227.

    Attachment of an Antigen Binding Region to an Fc region



    [0112] The antigen binding regions described herein (both antibody derived antigen binding regions and non-antibody antigen binding regions) may be fused to a variant Fc region as described herein. Any method for covalently attaching two polypeptides may be used to fuse together the antigen binding region with the Fc domain, including for example, expression as a single polypeptide (with or without an intervening polypeptide linker), chemical linkage or linkage via a polymeric group (such as, for example, a single or branched polyethylene glycol (PEG) linker). In certain aspects, the linker may be a cleavable linker.

    [0113] In certain aspects, a linker may be a polypeptide linker. In one aspect, the polypeptide linker is a hinge sequence from an antibody, or a variant thereof. For example, the hinge sequence may comprise amino acid residues 216-238 (EU numbering) of an antibody, such as, for example, an IgG1, IgG2, IgG3 or IgG4 antibody, or fragments or derivatives thereof. In an exemplary aspect, a hinge based linker comprises the sequence CDKTHTCPPCPAPELLGGP (SEQ ID NO:219), or fragments or derivatives thereof. In certain aspects, the polypeptide linker may be a flexible linker of varying length (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids). Suitable linkers are known in the art, see for example, Protein Engineering, 9(3), 299-305, 1996. Exemplary peptide linkers include, for example:

    Ser

    Gly-Ser

    Gly-Gly-Ser

    Ser-Gly-Gly

    Gly-Gly-Gly-Ser (SEQ ID NO:220)

    Ser-Gly-Gly-Gly (SEQ ID NO:221)

    Gly-Gly-Gly-Gly-Ser (SEQ ID NO:222)

    Ser-Gly-Gly-Gly-Gly (SEQ ID NO:223)

    Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:224)

    Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:225)

    Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:226)

    Ser-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:227)

    (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO:228)and

    (Ser-Gly-Gly-Gly-Gly)n (SEQ ID NO:229)

    wherein n is an integer not less than one. In certain aspects n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20.



    [0114] In some aspects, a linker is between 0 and 20 amino acids in length (inclusive), e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

    [0115] In some aspects, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acids of a linker are glycine and/or serine amino acids. For example, in some aspects, a linker of the present disclosure comprises the sequence GGGGSG (SEQ ID NO:270), GGGGSGGGGS (SEQ ID NO:272), GGGGSGGGGSGGGG (SEQ ID NO:273), GGSGG (SEQ ID NO:271), GGGGSGGGGS (SEQ ID NO:272), or GGSGGGGSGGGGS (SEQ ID NO:274).

    [0116] In some aspects, a linker comprises an amino acid sequence found within a human antibody constant domain sequence (e.g., a CH1 domain or hinge region sequence). In some aspects, a linker of the present disclosure comprises the sequence ASTKGP (SEQ ID NO:275), ASTKGPSVFPLAP (SEQ ID NO:277), RTVAAP (SEQ ID NO:276), or RTVAAPSVFIFPP (SEQ ID NO:278). In some aspects, a linker comprises both an amino acid sequence found within a human antibody constant domain sequence (e.g., a CH1 domain or hinge region sequence) and a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% glycine and/or serine amino acids.

    [0117] In certain aspects, a linker may be a chemical linker. Suitable chemical linkers are known in the art and commercially available. Exemplary chemical linkers include, for example, N-hydroxy succinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycolbis(succinimidyl succinate) (EGS), ethylene glycolbis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimido oxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimido oxycarbonyloxy) ethyl]sulfone (sulfo-BSOCOES).

    [0118] In certain aspects, an antigen binding region of an antibody is expressed as a single polypeptide with the Fc domain. As the Fc domain is a dimer, each polypeptide contained in the Fc dimer may be fused to an antigen binding region of an antibody or only of the polypeptides contained in the Fc dimer may be fused to an antigen binding region of an antibody.

    [0119] In certain aspects, a non-antibody antigen binding region is expressed as a single polypeptide with the Fc domain. As the Fc domain is a dimer, each polypeptide contained in the Fc dimer may be fused to a non-antibody antigen binding region or only of the polypeptides contained in the Fc dimer may be fused to a non-antibody antigen binding region.

    Antigen Binding Regions Comprising OX40 Agonists



    [0120] In one aspect, the antigen binding polypeptide or complex described herein comprises an antigen binding region that binds to and agonizes human OX40. In certain aspects, the antigen binding polypeptide comprises an antigen binding region of an anti-human OX40 agonist antibody. In certain aspects, the antigen binding polypeptide comprises an antigen binding region that is a non-antibody OX40 agonist. Any of the variable domains of any of the exemplary OX40 agonist antibodies described infra can be used in a complex of the present disclosure.

    OX40 Agonist Antibodies



    [0121] Certain aspects of the present disclosure relate to antigen binding complexes that bind OX40. In some aspects, the complex is a tetravalent antigen binding complex having agonist activity for OX40 (e.g., human OX40). Exemplary antibody subunits and exemplary features thereof are described infra. Without wishing to be bound to theory, it is thought that agonist antigen binding complexes (e.g., tetravalent agonist antigen binding complexes) may be particularly advantageous for scenarios in which antibody cross-linking by effector cells may be important for agonist activity (e.g., by inducing clustering of, and subsequent signaling by, the target), but effector cells may not be plentiful at the site of action (e.g., in a tumor with low levels of effector cells). In these scenarios, an agonist antigen binding complex such as a tetravalent agonist antigen binding complex may allow for and/or enhance agonist activity in the absence of plentiful effector cells.

    [0122] In some aspects, the antigen binding complex comprises an anti-human OX40 agonist antibody or Fab fragment that comprises at least one, two, three, four, five, or all six HVRs for the same antibody as listed in Table A. For example, in certain aspects, the OX40 antibody or Fab fragment included in the complex contains all six of the HVRs from the same antibody as listed in Table A. In other aspects, the complex comprises an anti-human OX40 agonist antibody or Fab fragment that comprises a heavy chain variable region (VH) and/or a light chain variable region (VL) as for the same antibody as listed in Table A below. It will be appreciated, however, that the the HVR, VH, and/or VL sequences as listed in Table A with reference to particular antibodies are not limited to these particular antibodies; instead, these sequences can be suitably combined in a variety of configurations not explicitly listed in Table A by one of skill in the art.

    [0123] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:7.

    [0124] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4. In one aspect, the antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:4. In another aspect, the antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:4 and HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In a further aspect, the antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:4, HVR-L3 comprising the amino acid sequence of SEQ ID NO:7, and HVR-H2 comprising the amino acid sequence of SEQ ID NO:3. In a further aspect, the antibody or Fab fragment comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4.

    [0125] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In one aspect, the antibody or Fab fragment comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.

    [0126] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:4; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.

    [0127] In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:7.

    [0128] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:26.

    [0129] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:4 and HVR-L3 comprising the amino acid sequence of SEQ ID NO:26. In a further aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:4, HVR-L3 comprising the amino acid sequence of SEQ ID NO:26, and HVR-H2 comprising the amino acid sequence of SEQ ID NO:3.

    [0130] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:4; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:26.

    [0131] In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:26.

    [0132] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:27.

    [0133] In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:4 and HVR-L3 comprising the amino acid sequence of SEQ ID NO:27. In a further aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:4, HVR-L3 comprising the amino acid sequence of SEQ ID NO:27, and HVR-H2 comprising the amino acid sequence of SEQ ID NO:3.

    [0134] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:4; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:27.

    [0135] In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:27.

    [0136] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, 8 or 9; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, 10, 11, 12, 13 or 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4, 15, or 19; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7, 22, 23, 24, 25, 26, 27, or 28.

    [0137] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2, 8 or 9; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3, 10, 11, 12, 13 or 14; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4, 15, or 19. In one aspect, the antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4, 15, or 19. In another aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:4, 15, or 19 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 7, 22, 23, 24, 25, 26, 27, or 28. In a further aspect, the antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4, 15, or 19, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 7, 22, 23, 24, 25, 26, 27, or 28, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3, 10, 11, 12, 13 or 14. In a further aspect, the antibody or Fab fragment comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2, 8 or 9; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3, 10, 11, 12, 13 or 14; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4, 15, or 19.

    [0138] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 7, 22, 23, 24, 25, 26, 27, or 28. In one aspect, the antibody or Fab fragment comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 7, 22, 23, 24, 25, 26, 27, or 28.

    [0139] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2, 8 or 9, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3, 10, 11, 12, 13 or 14, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 4, 15, or 19; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 7, 22, 23, 24, 25, 26, 27, or 28.

    [0140] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2, 8 or 9; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3, 10, 11, 12, 13 or 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4, 15, or 19; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 7, 22, 23, 24, 25, 26, 27, or 28.

    [0141] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:172; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:173; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:174; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:175. In some aspect, HVR-H2 is not DMYPDAAAASYNQKFRE (SEQ ID NO: 216).In some aspects, HVR-H3 is not APRWAAAA (SEQ ID NO: 217). In some aspects, HVR-L3 is not QAAAAAAAT (SEQ ID NO: 218).

    [0142] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:172; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:173; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:174. In one aspect, the antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:174. In another aspect, the antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:174 and HVR-L3 comprising the amino acid sequence of SEQ ID NO:175. In a further aspect, the antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:174, HVR-L3 comprising the amino acid sequence of SEQ ID NO:175, and HVR-H2 comprising the amino acid sequence of SEQ ID NO:173. In a further aspect, the antibody or Fab fragment comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:172; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:173; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:174. In some aspect, HVR-H2 is not DMYPDAAAASYNQKFRE (SEQ ID NO: 216).In some aspects, HVR-H3 is not APRWAAAA (SEQ ID NO: 217). In some aspects, HVR-L3 is not QAAAAAAAT (SEQ ID NO: 218).

    [0143] In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:175. In some aspects, HVR-L3 is not QAAAAAAAT (SEQ ID NO: 218).

    [0144] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:172, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:173, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 174; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:175.

    [0145] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:172; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:173; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:174; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:175. In some aspect, HVR-H2 is not DMYPDAAAASYNQKFRE (SEQ ID NO: 216). In some aspects, HVR-H3 is not APRWAAAA (SEQ ID NO: 217). In some aspects, HVR-L3 is not QAAAAAAAT (SEQ ID NO: 218).

    [0146] All possible combinations of the above substitutions are encompassed by the consensus sequences of SEQ ID NO: 172, 173, 174 and 175.

    [0147] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.

    [0148] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33. In one aspect, the antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:33. In another aspect, the antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:33 and HVR-L3 comprising the amino acid sequence of SEQ ID NO:42. In a further aspect, the antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:33, HVR-L3 comprising the amino acid sequence of SEQ ID NO:42, and HVR-H2 comprising the amino acid sequence of SEQ ID NO:30. In a further aspect, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33.

    [0149] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42. In one aspect, the antibody or Fab fragment comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42. In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33. In one aspect, the antibody or Fab fragment comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33.

    [0150] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:33; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:38, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.

    [0151] In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:42.

    [0152] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42. In one aspect, the antibody or Fab fragment comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42. In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33. In one aspect, the antibody or Fab fragment comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33.

    [0153] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:33; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.

    [0154] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:40; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.

    [0155] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:40; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42. In one aspect, the antibody or Fab fragment comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:40; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.

    [0156] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:33; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:40, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.

    [0157] In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:40; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:42.

    [0158] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, 31, or 32; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, 40 or 41; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42, 43, or 44.

    [0159] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30, 31, or 32; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33. In another aspect, the antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:33 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42, 43, or 44. In a further aspect, the antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:33, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42, 43, or 44, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 39, 40 or 41. In a further aspect, the antibody or Fab fragment comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, 31, or 32; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33.

    [0160] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39, 40 or 41; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42, 43, or 44. In one aspect, the antibody or Fab fragment comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39, 40 or 41; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42, 43, or 44.

    [0161] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30, 31, or 32, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:33; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39, 40 or 41, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42, 43, or 44.

    [0162] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30, 31, or 32; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39, 40 or 41; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 42, 43, or 44.

    [0163] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:175; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33. In another aspect, the antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:33 and HVR-L3 comprising the amino acid sequence of SEQ ID NO:177. In a further aspect, the antibody or Fab fragment comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:33, HVR-L3 comprising the amino acid sequence of SEQ ID NO:178, and HVR-H2 comprising the amino acid sequence of SEQ ID NO:176. In a further aspect, the antibody or Fab fragment comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 176; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33.

    [0164] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:177; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:177. In one aspect, the antibody or Fab fragment comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:177; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:178.

    [0165] In some aspects, the anti-human OX40 agonist antibody or Fab fragment comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:176, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:33; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:177, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:178.

    [0166] In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:176; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:177; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:178.

    [0167] In any of the above aspects, an anti-OX40 agonist antibody or Fab fragment is humanized. In one aspect, an anti-OX40 antibody or Fab fragment comprises HVRs as in any of the above aspects and further comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework.

    [0168] In another aspect, the anti-human OX40 agonist antibody or Fab fragment comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 108, 114, 116, 183 or 184. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody or Fab fragment comprising that sequence retains the ability to bind to OX40. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 108, 114, 116, 183 or 184. In certain aspects, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonist antibody or Fab fragment comprises the VH sequence in SEQ ID NO: SEQ ID NO:56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 108, 114, 116, 183 or 184, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4.

    [0169] In another aspect, the anti-human OX40 agonist antibody or Fab fragment comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 109, 115 or 117. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody or Fab fragment comprising that sequence retains the ability to bind to OX40. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 109, 115 or 117. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonist antibody or Fab fragment comprises the VL sequence in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 109, 115 or 117, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.

    [0170] In another aspect, the anti-human OX40 agonist antibody or Fab fragment comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:56. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody or Fab fragment comprising that sequence retains the ability to bind to OX40. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:56. In certain aspects, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonist antibody or Fab fragment comprises the VH sequence in SEQ ID NO:56, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4.

    [0171] In another aspect, the anti-human OX40 agonist antibody or Fab fragment comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:57. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody or Fab fragment comprising that sequence retains the ability to bind to OX40. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 57. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonist antibody or Fab fragment comprises the VL sequence in SEQ ID NO: 57, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.

    [0172] In another aspect, the anti-human OX40 agonist antibody or Fab fragment comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 180. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody or Fab fragment comprising that sequence retains the ability to bind to OX40. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:180. In certain aspects, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonist antibody or Fab fragment comprises the VH sequence in SEQ ID NO: 180, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4.

    [0173] In another aspect, the anti-human OX40 agonist antibody or Fab fragment comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 179. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody or Fab fragment comprising that sequence retains the ability to bind to OX40. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 179. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonist antibody or Fab fragment comprises the VL sequence in SEQ ID NO: 179, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.

    [0174] In another aspect, the anti-human OX40 agonist antibody or Fab fragment comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:94. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody or Fab fragment comprising that sequence retains the ability to bind to OX40. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:94. In certain aspects, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonist antibody or Fab fragment comprises the VH sequence in SEQ ID NO:94, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4.

    [0175] In another aspect, the anti-human OX40 agonist antibody or Fab fragment comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:95. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody or Fab fragment comprising that sequence retains the ability to bind to OX40. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:95. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonist antibody or Fab fragment comprises the VL sequence in SEQ ID NO:95, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:26.

    [0176] In another aspect, the anti-human OX40 agonist antibody or Fab fragment comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:96. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody or Fab fragment comprising that sequence retains the ability to bind to OX40. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:96. In certain aspects, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonist antibody or Fab fragment comprises the VH sequence in SEQ ID NO:96, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4.

    [0177] In another aspect, the anti-human OX40 agonist antibody or Fab fragment comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:97. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody or Fab fragment comprising that sequence retains the ability to bind to OX40. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:97. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonist antibody or Fab fragment comprises the VL sequence in SEQ ID NO:97, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:27.

    [0178] In another aspect, the anti-human OX40 agonist antibody or Fab fragment comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, or 148. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody or Fab fragment comprising that sequence retains the ability to bind to OX40. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, or 148. In certain aspects, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonist antibody or Fab fragment comprises the VH sequence in SEQ ID NO: SEQ ID NO: 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, or 148, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 29, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33.

    [0179] In another aspect, the anti-human OX40 agonist antibody or Fab fragment comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, or 149. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody or Fab fragment comprising that sequence retains the ability to bind to OX40. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, or 149. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-human OX40 agonist antibody or Fab fragment comprises the VL sequence in SEQ ID NO: 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, or 149, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.

    [0180] In certain aspects, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:56 and SEQ ID NO:57, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:58 and SEQ ID NO:59, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:60 and SEQ ID NO:61, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:62 and SEQ ID NO:63, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:64 and SEQ ID NO:65, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:66 and SEQ ID NO:67, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:68 and SEQ ID NO:69, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:70 and SEQ ID NO:71, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:72 and SEQ ID NO:73, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:74 and SEQ ID NO:75, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:76 and SEQ ID NO:77, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:78 and SEQ ID NO:79, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:80 and SEQ ID NO:81, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:82 and SEQ ID NO:83, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:84 and SEQ ID NO:85, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:86 and SEQ ID NO:87, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:88 and SEQ ID NO:89, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:90 and SEQ ID NO:91, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:92 and SEQ ID NO:93, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:94 and SEQ ID NO:95, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:96 and SEQ ID NO:97, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:98 and SEQ ID NO:99, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:100 and SEQ ID NO:101, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:108 and SEQ ID NO:109, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:114 and SEQ ID NO:115, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:116 and SEQ ID NO:117, respectively, including post-translational modifications of those sequences. In one aspect, the antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:183 and SEQ ID NO:65, respectively, including post-translational modifications of those sequences. In one aspect, the antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:184 and SEQ ID NO:69, respectively, including post-translational modifications of those sequences.

    [0181] In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:118 and SEQ ID NO:119, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:120 and SEQ ID NO:121, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:122 and SEQ ID NO:123, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:124 and SEQ ID NO:125, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:126 and SEQ ID NO:127, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:128 and SEQ ID NO:129, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:130 and SEQ ID NO:131, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:132 and SEQ ID NO:133, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:134 and SEQ ID NO:135, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:136 and SEQ ID NO:137, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:138 and SEQ ID NO:139, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:140 and SEQ ID NO:141, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:142 and SEQ ID NO:143, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:144 and SEQ ID NO: 145, respectively, including post-translational modifications of those sequences. In one aspect, the anti-human OX40 agonist antibody or Fab fragment comprises the VH and VL sequences in SEQ ID NO:146 and SEQ ID NO:147, respectively, including post-translational modifications of those sequences.

    [0182] As described above, certain aspects of the present disclosure relate to complexes. It is to be understood that any of the exemplary antibodies, antigen binding domains, and/or antibody Fab fragments that bind OX40 (e.g., HVRs, VH, and/or VL domains of any of the OX40 agonist antibodies described herein) may be combined in a complex of the present disclosure in any combination or configuration. In some aspects, a complex may comprise two or more different OX40 agonist antibodies or antigen binding polypeptides that bind to the same epitope of OX40. In some aspects, a complex may comprise two or more different OX40 agonist antibodies or antigen binding polypeptides that bind to different epitopes of OX40 (e.g., partially non-overlapping or completely non-overlapping epitopes of an OX40 polypeptide, such as human OX40).

    [0183] In some aspects, a complex of the present disclosure comprises four antigen binding domains that bind OX40, wherein each of the four antigen binding domains comprises an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, wherein the complex comprises one or more antigen binding domains that bind a first epitope of OX40 and one or more antigen binding domains that bind a second epitope of OX40, and wherein the first and second epitopes of OX40 are different. In some aspects, the antigen binding domains that bind the first epitope do not cross-compete for binding OX40 with the antigen binding domains that bind the second epitope.

    [0184] In some aspects, a complex of the present disclosure comprises four antigen binding domains that bind OX40, each of the four antigen binding domains comprising an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain. In some aspects, the complex comprises one or more antigen binding domains that bind a first epitope of OX40 and one or more antigen binding domains that bind a second epitope of OX40, where the antigen binding domains that bind the first epitope do not cross-compete for binding OX40 with the antigen binding domains that bind the second epitope. In some aspects, competition between two antigen binding domains is performed by testing each antigen binding domain as part of a separate antibody or fragment thereof, rather than testing the two domains as part of a shared complex. Exemplary competition assays are provided infra. In some aspects, a first antigen binding domains that does not cross-compete for binding with OX40 with a second antigen binding domain blocks binding of the second antigen binding domain to OX40 in a competition assay by 50% or less.

    [0185] In some aspects, an antigen binding domain of the present disclosure binds an epitope of OX40 (e.g., human OX40) comprising one or more amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 residues) selected from: residues 114-119, 124, 126, 127, 129, 130, 132, 140, and 142 of SEQ ID NO:281. In some aspects, an antigen binding domain of the present disclosure binds an epitope of OX40 (e.g., human OX40) comprising one or more amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 residues) selected from: residues 68-71, 83-90, 95, and 98 of SEQ ID NO:281.

    [0186] Full sequence of human OX40 with signal peptide (signal peptide underlined):



    [0187] In some aspects, the OX40 agonist antibody binds human OX40 with an affinity of less than or equal to about 0.45 nM. In some aspects, the OX40 agonist antibody binds human OX40 with an affinity of less than or equal to about 1 nM. In some aspects, the OX40 antibody binds human OX40 with an affinity of less than or equal to about 0.4 nM. In some aspects, the OX40 antibody binds human OX40 with an affinity of less than or equal to about 0.5nM. In some aspects, the binding affinity is determined using radioimmunoassay.

    [0188] In some aspects, the OX40 agonist antibody binds human OX40 and cynomolgus OX40. In some aspects, binding is determined using a FACS assay. In some aspects, binding to human OX40 has an EC50 of about 0.2 ug/ml. In some aspects, binding to human OX40 has an EC50 of about 0.3 ug/ml or lower. In some aspects, binding to cynomolgus OX40 has an EC50 of about 1.5 ug/ml. In some aspects, binding to cynomolgus OX40 has an EC50 of about 1.4 ug/ml.

    [0189] In some aspects, the OX40 agonist antibody does not bind to rat OX40 or mouse OX40.

    [0190] In some aspects, the OX40 agonist antibody does not induce apoptosis in OX40-expressing cells (e.g., Treg). In some aspects, apoptosis is assayed using an antibody concentration of 30ug/ml, e.g., by determining whether apoptosis has occurred using annexin V and proprodium iodide stained Treg.

    [0191] In some aspects, the OX40 agonist antibody increases memory T cell proliferation and/or increasing cytokine production by the memory cell. In some aspects, the cytokine is IFN-γ. In some aspects, the OX40 agonist antibody enhances memory T cell function, for example by increasing memory T cell proliferation and/or increasing cytokine production by the memory cell. In some aspects, the cytokine is gamma interferon.

    [0192] In some aspects, the OX40 agonist antibody increases CD4+ effector T cell proliferation and/or increases cytokine production by the CD4+ effector T cell as compared to proliferation and/or cytokine production prior to treatment with the OX40 agonist antibody. In some aspects, the cytokine is IFN-γ.

    [0193] In some aspects, the anti-human OX40 agonist antibody enhances CD4+ effector T cell function, for example, by increasing CD4+ effector T cell proliferation and/or increasing gamma interferon production by the CD4+ effector T cell (for example, as compared to proliferation and/or cytokine production prior to treatment with anti-human OX40 agonist antibody). In some aspects, the cytokine is gamma interferon. In some aspects, the anti-human OX40 agonist antibody increases number of intratumoral (infiltrating) CD4+ effector T cells (e.g., total number of CD4+ effector T cells, or e.g., percentage of CD4+ cells in CD45+ cells), e.g., as compared to number of intratumoral (infiltrating) CD4+ T cells prior to treatment with anti-human OX40 agonist antibody. In some aspects, the anti-human OX40 agonist antibody increases number of intratumoral (infiltrating) CD4+ effector T cells that express gamma interferon (e.g., total gamma interferon expressing CD4+ cells, or e.g., percentage of gamma interferon expressing CD4+ cells in total CD4+ cells), e.g., as compared to number of intratumoral (infiltrating) CD4+ T cells that express gamma interferon prior to treatment with anti-human OX40 agonist antibody.

    [0194] In some aspects, the number of CD4+ effector T cells is elevated relative to prior to administration of the OX40 agonist antibody. In some aspects, CD4+ effector T cell cytokine secretion is elevated relative to prior to administration of the OX40 agonist antibody. In some aspects of any of the methods, the CD8+ effector T cells in the individual have enhanced proliferation, cytokine secretion and/or cytolytic activity relative to prior to the administration of the OX40 agonist antibody. In some aspects, the number of CD8+ effector T cells is elevated relative to prior to administration of the OX40 agonist antibody. In some aspects, CD8+ effector T cell cytokine secretion is elevated relative to prior to administration of the OX40 agonist antibody.

    [0195] In some aspects, the anti-human OX40 agonist antibody increases number of intratumoral (infiltrating) CD8+ effector T cells (e.g., total number of CD8+ effector T cells, or e.g., percentage of CD8+ in CD45+ cells), e.g., as compared to number of intratumoral (infiltrating) CD8+ T effector cells prior to treatment with anti-human OX40 agonist antibody. In some aspects, the anti-human OX40 agonist antibody increases number of intratumoral (infiltrating) CD8+ effector T cells that express gamma interferon (e.g., percentage of CD8+ cells that express gamma interferon in total CD8+ cells), e.g., compared to number of intratumoral (infiltrating) CD8+ T cells that express gamma interferon prior to treatment with anti-human OX40 agonist antibody.

    [0196] In some aspects, the number of intratumoral (infiltrating) CD8+ effector T cells (e.g., total number of CD8+ effector T cells, or e.g., percentage of CD8+ in CD45+ cells) is elevated relative to prior to administration of the OX40 agonist antibody. In some aspects of any of the methods of the invention, number of intratumoral (infiltrating) CD8+ effector T cells that express gamma interferon (e.g., percentage of CD8+ cells that express gamma interferon in total CD8+ cells) is increased relative to prior to administration of the OX40 agonist antibody.

    [0197] In some aspects, the memory T cells in the individual have enhanced proliferation and/or cytokine secretion relative to prior to the administration of the OX40 agonist antibody. In some aspects, the number of memory T cells is elevated relative to prior to administration of the OX40 agonist antibody. In some aspects, memory T cell cytokine secretion (level) is elevated relative to prior to administration of the OX40 agonist antibody. In some aspects of any of the methods, the Treg in the individual have decreased inhibition of effector T cell function (e.g., proliferation and/or cytokine secretion) relative to prior to the administration of the OX40 agonist antibody. In some aspects, the number of effector T cells is elevated relative to prior to administration of the OX40 agonist antibody. In some aspects, effector T cell cytokine secretion (level) is elevated relative to prior to administration of the OX40 agonist antibody.

    [0198] In some aspects, the OX40 agonist antibody inhibits Treg suppression of effector T cell function. In some aspects, effector T cell function is effector T cell proliferation and/or cytokine production. In some aspects, the effector T cell is a CD4+ effector T cell.

    [0199] In some aspects, the OX40 agonist antibody inhibits Treg function, for example, by decreasing Treg suppression of effector T cell function (e.g., effector T cell proliferation and/or effector T cell cytokine secretion). In some aspects, the effector T cell is a CD4+ effector T cell. In some aspects, the OX40 agonist antibody reduces the number of intratumoral (infiltrating) Treg (e.g., total number of Treg or e.g., percentage of Fox3p+ cells in CD4+ cells).

    [0200] In some aspects, the number of intratumoral (infiltrating) Treg (e.g., total number of Treg or e.g., percentage of Fox3p+ cells in CD4+ cells) is reduced relative to prior to administration of the OX40 agonist antibody.

    [0201] In some aspects, the number of intratumoral (infiltrating) CD4+ effector T cells (e.g., total number of CD4+ effector T cells, or e.g., percentage of CD4+ cells in CD45+ cells) is elevated relative to prior to administration of the OX40 agonist antibody. In some aspects of any of the methods of the invention, number of intratumoral (infiltrating) CD4+ effector T cells that express gamma interferon (e.g., total gamma interferon expressing CD4+ cells, or e.g., percentage of gamma interferon expressing CD4+ cells in total CD4+ cells) is elevated relative to prior to administration of the OX40 agonist antibody.

    [0202] In some aspects, the OX40 agonist antibody increases OX40 signal transduction in a target cell that expresses OX40. In some aspects, OX40 signal transduction is detected by monitoring NFkB downstream signaling.

    [0203] In some aspects, the OX40 agonist antibody is stable after treatment at 40°C for two weeks.

    [0204] In some aspects, the OX40 agonist antibody competes for binding to human OX40 with OX40L. In some aspects, addition of OX40L does not enhance OX40 antibody function in an in vitro assay.

    [0205] According to another aspect, the OX40 agonist antibodies include any one, any combination, or all of the following properties: (1) binds human OX40 with an affinity of less than or equal to about 0.45 nM, in some aspects, binds human OX40 with an affinity of less than or equal to about 0.4 nM, in some aspects, binds human OX40 with an affinity of less than or equal to about 0.5nM, in some aspects, the binding affinity is determined using radioimmunoassay; (2) binds human OX40 and cynomolgus OX40, in some aspects, binding is determined using a FACS assay, (3) binds human OX40 with an EC50 of about 0.2 ug/ml, in some aspects, binds to human OX40 has an EC50 of about 0.3 ug/ml or lower, in some aspects, binds to cynomolgus OX40 with an EC50 of about 1.5 ug/ml, in some aspects, binds to cynomolgus OX40 has an EC50 of about 1.4 ug/ml, (4) does not substantially bind to rat OX40 or mouse OX40, (5) enhances CD4+ effector T cell function, for example, by increasing CD4+ effector T cell proliferation and/or increasing gamma interferon production by the CD4+ effector T cell (for example, as compared to proliferation and/or cytokine production prior to treatment with anti-human OX40 agonist antibody), (6) enhances memory T cell function, for example by increasing memory T cell proliferation and/or increasing cytokine production by the memory cell, (7) inhibits Treg function, for example, by decreasing Treg suppression of effector T cell function (e.g., effector T cell proliferation and/or effector T cell cytokine secretion). In some aspects, the effector T cell is a CD4+ effector T cell, (8) increases OX40 signal transduction in a target cell that expresses OX40 (in some aspects, OX40 signal transduction is detected by monitoring NFkB downstream signaling), and (9) is stable after treatment at 40°C for two weeks.

    [0206] Exemplary anti-OX40 HVR, VH, and VL sequences suitable for use in any of the antibodies and/or Fab fragments described herein in any suitable combination of the present disclosure are provided in Table A.
    Table A. OX40 Antibody Sequences
    Name SEQUENCE SEQ ID NO:
    Human OX40 (lacking the signal peptide)

     
    1
    HVR-H1-   2
    1A7.gr. 1    
    1A7.gr.2    
    1A7.gr.3    
    1A7.gr.4    
    1A7.gr.5    
    IA7.gr.5'    
    1A7.gr.6    
    1A7.gr.7    
    1A7.gr.7' DSYMS  
    1A7.gr.NADS    
    1A7.gr.NADA    
    1A7.gr.NGDA    
    1A7.gr.SGDS    
    1A7.gr.NGSS    
    1A7.Ala.1    
    1A7.Ala.2    
    1A7.Ala.3    
    1A7.Ala.4    
    1A7.Ala.5    
    1A7.Ala.6    
    1A7.Ala.7    
    1A7.Ala.8    
    1A7.Ala.9    
    1A7.Ala.10    
    1A7.Ala.11    
    1A7.Ala.12    
    1A7.Ala.13    
    1A7.Ala.14    
    1A7.Ala.15    
    1A7.Ala.16    
    HVR-H2-   3
    1A7.gr. 1    
    1A7.gr.2    
    1A7.gr.3    
    1A7.gr.4    
    1A7.gr.5    
    1A7.gr.5'    
    1A7.gr.6    
    1A7.gr.7    
    1A7.gr.7'    
    1A7.gr.DA    
    1A7.gr.ES    
    1A7.Ala.1    
    1A7.Ala.2    
    1A7.Ala.3    
    1A7.Ala.4    
    1A7.Ala.5    
    1A7.Ala.6    
    1A7.Ala.7    
    1A7.Ala.8    
    1A7.Ala.9    
    1A7.Ala.10    
    1A7.Ala.11    
    1A7.Ala.12    
    1A7.Ala.13    
    1A7.Ala.14    
    1A7.Ala.15    
    1A7.Ala.16 DMYPDNGDSSYNQKFRE  
    HVR-H3-   4
    1A7.gr. 1    
    1A7.gr.2    
    1A7.gr.3    
    1A7.gr.4    
    1A7.gr.5    
    1A7.gr.5'    
    1A7.gr.6    
    1A7.gr.7    
    1A7.gr.7'    
    1A7.gr.DA    
    1A7.gr.ES    
    1A7.gr.NADS    
    1A7.gr.NADA    
    1A7.gr.NGDA    
    1A7.gr.SGDS    
    1A7.gr.NGSS    
    1A7.gr.DANAD    
    A    
    1A7.Ala.1    
    1A7.Ala.2    
    1A7.Ala.3    
    1A7.Ala.4    
    1A7.Ala.5    
    1A7.Ala.6    
    1A7.Ala.7    
    1A7-Ala.15    
    1A7.Ala.16 APRWYFSV  
    HVR-L1-   5
    1A7.gr. 1    
    1A7.gr.2    
    1A7.gr.3    
    1A7.gr.4    
    1A7.gr.5    
    1A7.gr.5'    
    1A7.gr.6    
    1A7.gr.7    
    1A7.gr.7'    
    1A7.gr.DA    
    1A7.gr.ES    
    1A7.gr.NADS    
    1A7.gr.NADA    
    1A7.gr.NGDA    
    1A7.gr.SGDS    
    1A7.gr.NGSS    
    1A7.gr.DANAD    
    A    
    1A7.Ala.1    
    1A7.Ala.2 RASQDISNYLN  
    1A7.Ala.3    
    1A7.Ala.4    
    1A7.Ala.5    
    1A7.Ala.6    
    1A7.Ala.7    
    1A7.Ala.8    
    1A7.Ala.9    
    1A7.Ala.10    
    1A7.Ala.11    
    1A7.Ala.12    
    1A7.Ala.13    
    1A7.Ala.14    
    1A7.Ala.15    
    1A7.Ala.16    
    HVR-L2-   6
    1A7.gr. 1    
    1A7.gr.2    
    1A7.gr.3    
    1A7.gr.4    
    1A7.gr.5    
    1A7.gr.5'    
    1A7.gr.6    
    1A7.gr.7    
    1A7.gr.7'    
    1A7.gr.DA    
    1A7.gr.ES    
    1A7.gr.NADS    
    1A7.gr.NADA    
    1A7.gr.NGDA    
    1A7.gr.SGDS    
    1A7.gr.NGSS    
    1A7.gr.DANAD    
    A    
    1A7.Ala.1    
    1A7.Ala.2    
    1A7.Ala.3    
    1A7.Ala.4    
    1A7.Ala.5    
    1A7.Ala.6    
    1A7.Ala.7    
    1A7.Ala.8    
    1A7.Ala.9    
    1A7.Ala.10    
    1A7.Ala.11    
    1A7.Ala.12    
    1A7.Ala.13    
    1A7.Ala.14    
    1A7.Ala.15    
    1A7.Ala.16 YTSRLRS  
    HVR-L3-   7
    1A7.gr. 1    
    1A7.gr.2    
    1A7.gr.3    
    1A7.gr.4    
    1A7.gr.5    
    1A7.gr.5'    
    1A7.gr.6    
    1A7.gr.7    
    1A7.gr.7'    
    1A7.gr.DA    
    1A7.gr.ES    
    1A7.gr.NADS    
    1A7.gr.NADA    
    1A7.gr.NGDA    
    1A7.gr.SGDS    
    1A7.gr.NGSS    
    1A7.gr.DANAD    
    A    
    1A7.Ala.8    
    1A7.Ala.9    
    1A7.Ala.10    
    1A7.Ala.11    
    1A7.Ala.12    
    1A7.Ala.13    
    1A7.Ala.14    
    1A7.Ala.15    
    1A7.Ala.16 QQGHTLPPT  
    HVR-H1-   8
    1A7.gr.DA DAYMS  
    HVR-H1-   9
    1A7.gr.ES    
    1A7.gr.DANAD    
    A ESYMS  
    HVR-H2-   10
    1A7.gr.NADS DMYPDNADSSYNQKFRE  
    HVR-H2-   11
    1A7.gr.NADA    
    1A7.gr.DANAD    
    A DMYPDNADASYNQKFRE  
    HVR-H2-   12
    1A7.gr.NGDA DMYPDNGDASYNQKFRE  
    HVR-H2-   13
    1A7.gr.SGDS DMYPDSGDSSYNQKFRE  
    HVR-H2- DMYPDNGS S SYNQKFRE 14
    1A7.gr.NGSS    
    HVR-H3- 1A7.Ala.8 APRWYFSA 15
    HVR-H3- 1A7.Ala.9 APRWYASV 16
    HVR-H3- 1A7.Ala.10 APRWAFSV 17
    HVR-H3- 1A7.Ala.11 APAWYFSV 18
    HVR-H3- 1A7.Ala.12 APRWYFAV 19
    HVR-H3- 1A7.Ala.13 APRAYFSV 20
    HVR-H3- 1A7.Ala.14 AARWYFSV 21
    HVR-L3- 1A7.Ala.1 QQGHTLPAT 22
    HVR-L3- 1A7.Ala.2 QQGHTAPPT 23
    HVR-L3- 1A7.Ala.3 QQGATLPPT 24
    HVR-L3- 1A7.Ala.4 QQGHALPPT 25
    HVR-L3- 1A7.Ala.5 QQAHTLPPT 26
    HVR-L3- 1A7.Ala.6 QQGHTLAPT 27
    HVR-L3- 1A7.Ala.7 QAGHTLPPT 28
    HVR-H1- 3C8.gr.1 NYLIE 29
    3C8.gr.2    
    3C8.gr.3    
    3C8.gr.4    
    3C8.gr.5    
    3C8.gr.5.SG    
    3C8.gr.5.EG    
    3C8.gr.5.QG    
    3C9.gr.5.DQ    
    3C8.gr.5.DA    
    3C8.gr.6    
    3C8.gr.7    
    3C8.gr.8    
    3C8.gr.9    
    3C8.gr.10    
    3C8.gr.11    
    3C8.A.1    
    3C8.A.2    
    3C8.A.3    
    3C8.A.4    
    3C8.A.5    
    3C8.A.6    
    3C8.A.7    
    3C8.A.8    
    3C8.A.9    
    3C8.A.10    
    HVR-H2-   30
    3C8.gr.1    
    3C8.gr.2    
    3C8.gr.3    
    3C8.gr.4    
    3C8.gr.5    
    3C8.gr.5.SG    
    3C8.gr.5.EG    
    3C8.gr.5.QG    
    3C8.gr.6    
    3C8.gr.7    
    3C8.gr.8    
    3C8.gr.9    
    3C8.gr.10    
    3C8.gr.11    
    3C8.A.1    
    3C8.A.2    
    3C8.A.3    
    3C8.A.4    
    3C8.A.5    
    3C8.A.6    
    3C8.A.7    
    3C8.A.8    
    3C8.A.9 VINPGSGDTYYSEKFKG  
    3C8.A.10    
    HVR-H2- 3C8.gr.5.DA VINPGSGDAYYSEKFKG 31
    HVR-H2-   32
    3C8.gr.5.DQ VINPGSGDQYYSEKFKG  
    HVR-H3-   33
    3C8.gr.1    
    3C8.gr.2    
    3C8.gr.3    
    3C8.gr.4    
    3C8.gr.5    
    3C8.gr.5.SG    
    3C8.gr.5.EG    
    3C8.gr.5.QG    
    3C8.gr.5.DA    
    3C8.gr.5.DQ    
    3C8.gr.6    
    3C8.gr.7    
    3C8.gr.8    
    3C8.gr.9    
    3C8.gr.10    
    3C8.gr.11    
    3C8.A.1    
    3C8.A.2    
    3C8.A.3    
    3C8.A.4    
    3C8.A.5    
    3C8.A.6    
    3C8.A.7 DRLDY  
    HVR-H3- 3C8.A.8 ARLDY 34
    HVR-H3- 3C8.A.9 DALDY 35
    HVR-H3- 3C8.A.10 DRADY 36
    HVR-L1-   37
    3C8.gr.1    
    3C8.gr.2    
    3C8.gr.3    
    3C8.gr.4    
    3C8.gr.5    
    3C8.gr.5.SG    
    3C8.gr.5.EG    
    3C8.gr.5.QG    
    3C8.gr.5.DA    
    3C8.gr.5.DQ HASQDISSYIV  
    3C8.gr.6    
    3C8.gr.7    
    3C8.gr.8    
    3C8.gr.9    
    3C8.gr.10    
    3C8.gr.11    
    3C8.A.1    
    3C8.A.2    
    3C8.A.3    
    3C8.A.4    
    3C8.A.5    
    3C8.A.6    
    3C8.A.7    
    3C8.A.8    
    3C8.A.9    
    3C8.A.10    
    HVR-L2-   38
    3C8.gr.1    
    3C8.gr.2    
    3C8.gr.3    
    3C8.gr.4    
    3C8.gr.5    
    3C8.gr.5.DA    
    3C8.gr.5.DQ    
    3C8.gr.6    
    3C8.gr.7    
    3C8.gr.8    
    3C8.gr.9    
    3C8.gr.10    
    3C8.gr.11    
    3C8.A.1    
    3C8.A.2    
    3C8.A.3    
    3C8.A.4    
    3C8.A.5    
    3C8.A.6    
    3C8.A.7    
    3C8.A.8    
    3C8.A.9    
    3C8.A.10 HGTNLED  
    HVR-L2- 3C8.gr5.SG HGTNLES 39
    HVR-L2- 3C8.gr.5.EG HGTNLEE 40
    HVR-L2- 3C8.gr.5.QG HGTNLEQ 41
    HVR-L3   42
    3C8.gr.1    
    3C8.gr.2    
    3C8.gr.3    
    3C8.gr.4    
    3C8.gr.5    
    3C8.gr.5.SG    
    3C8.gr.5.EG    
    3C8.gr.5.QG    
    3C8.gr.5.DA    
    3C8.gr.5.DQ    
    3C8.gr.6    
    3C8.gr.7    
    3C8.gr.8    
    3C8.gr.9    
    3C8.gr.10    
    3C8.gr.11    
    3C8.A.8    
    3C8.A.9    
    3C8.A.10 VHYAQFPYT  
    HVR-L3- 3C8.A.1 AHYAQFPYT 43
    HVR-L3- 3C8.A.2 VAYAQFPYT 44
    HVR-L3- 3C8.A.3 VHAAQFPYT 45
    HVR-L3- 3C8.A.4 VHYAAFPYT 46
    HVR-L3- 3C8.A.5 VHYAQAPYT 47
    HVR-L3- 3C8.A.6 VHYAQFAYT 48
    HVR-L3- 3C8.A.7 VHYAQFPAT 49
    HVR-H1-   50
    1D2.gr.1    
    1D2.gr.2    
    1D2.gr.3 DYGVL  
    HVR-H2-   51
    1D2.gr.1    
    1D2.gr.2 MIWSGGTTDYNAAFIS  
    1D2.gr.3    
    HVR-H3-   52
    1D2.gr.1    
    1D2.gr.2    
    1D2.gr.3 EEMDY  
    HVR-L1- 1D2.gr.1   53
    1D2.gr.2    
    1D2.gr.3 RASQDISNFLN  
    HVR-L2-   54
    1D2.gr.1    
    1D2.gr.2    
    1D2.gr.3 YTSRLHS  
    HVR-L3-   55
    1D2.gr.1    
    1D2.gr.2    
    1D2.gr.3 QQGNTLPWT  
    1A7.gr. 1 VH

     
    56
    1A7.gr. 1 VL

     
    57
    1A7.gr.2 VH

     
    58
    1A7.gr.2 VL

     
    59
    1A7.gr.3 VH

     
    60
    1A7.gr.3 VL

     
    61
    1A7.gr.4 VH

     
    62
    1A7.gr.4 VL

     
    63
    1A7.gr.5 VH

     
    64
    1A7.gr.5 VL

     
    65
     

     
     
    1A7.gr.6 VH

     
    66
    1A7.gr.6 VL

     
    67
    1A7.gr.7 VH

     
    68
    1A7.gr.7 VL

     
    69
    1A7.gr.DA VH

     
    70
    1A7.gr.DA VL

     
    71
    1A7.gr.ES VH

     
    72
    1A7.gr.ES VL

     
    73
    1A7.gr.NADS VH

     
    74
    1A7.gr.NADS VL

     
    75
    1A7.gr.NADA VH

     
    76
    1A7.gr.NADA VL

     
    77
    1A7.gr.NGDA VH

     
    78
    1A7.gr.NGDA VL

     
    79
     

     
     
    1A7.gr.SGDS VH

     
    80
    1A7.gr.SGDS VL

     
    81
    1A7.gr.NGSS VH

     
    82
    1A7.gr.NGSS VL

     
    83
    1A7.gr.DANAD A VH

     
    84
    1A7.gr.DANAD A VL

     
    85
    1A7.Ala.1 VH

     
    86
    1A7.Ala.1 VL

     
    87
    1A7.Ala.2 VH

     
    88
    1A7.Ala.2 VL

     
    89
    1A7.Ala.3 VH

     
    90
    1A7.Ala.3 VL

     
    91
    1A7.Ala.4 VH

     
    92
    1A7.Ala.4 VL

     
    93
    1A7.Ala.5 VH

     
    94
    1A7.Ala.5 VL

     
    95
    1A7.Ala.6 VH

     
    96
    1A7.Ala.6 VL

     
    97
    1A7.Ala.7 VH

     
    98
    1A7.Ala.7 VL

     
    99
    1A7.Ala.8 VH

     
    100
    1A7.Ala.8 VL

     
    101
    1A7.Ala.9 VH

     
    102
    1A7.Ala.9 VL

     
    103
    1A7.Ala.10 VH

     
    104
    1A7.Ala.10 VL

     
    105
    1A7.Ala.11 VH

     
    106
    1A7.Ala.11 VL

     
    107
    1A7.Ala.12 VH

     
    108
     

     
     
    1A7.Ala.12 VL

     
    109
    1A7.Ala.13 VH

     
    110
    1A7.Ala.13 VL

     
    111
    1A7.Ala.14 VH

     
    112
    1A7.Ala.14 VL

     
    113
    1A7.Ala.15 VH

     
    114
    1A7.Ala.15 VL

     
    115
    1A7.Ala.16 VH

     
    116
    1A7.Ala.16 VL

     
    117
    3C8.gr.1 VH

     
    118
    3C8.gr.1 VL

     
    119
    3C8.gr.2 VH

     
    120
    3C8.gr.2 VL

     
    121
    3C8.gr.3

     
    122
    VH

     
     
    3C8.gr.3 VL

     
    123
    3C8.gr.4 VH

     
    124
    3C8.gr.4 VL

     
    125
    3C8.gr.5 VH

     
    126
    3C8.gr.5 VL

     
    127
    3C8.gr.5.SG VH

     
    128
    3C8.gr.5.SG VL

     
    129
    3C8.gr.5.EG VH

     
    130
    3C8.gr.5.EG VL

     
    131
    3C8.gr.5.QG VH

     
    132
    3C8.gr.5.QG VL

     
    133
    3C8.gr.6 VH

     
    134
    3C8.gr.6 VL

     
    135
    3C8.gr.7 VH

     
    136
     

     
     
    3C8.gr.7 VL

     
    137
    3C8.gr.8 VH

     
    138
    3C8.gr.8 VL

     
    139
    3C8.gr.9 VH

     
    140
    3C8.gr.9 VL

     
    141
    3C8.gr.10 VH

     
    142
    3C8.gr.10 VL

     
    143
    3C8.gr.11 VH

     
    144
    3C8.gr.11 VL

     
    145
    3C8.A.1 VH

     
    146
    3C8.A.1 VL

     
    147
    3C8.A.2 VH

     
    148
    3C8.A.2 VL

     
    149
    3C8.A.3 VH

     
    150
     

     
     
    3C8.A.3 VL

     
    151
    3C8.A.4 VH

     
    152
    3C8.A.4 VL

     
    153
    3C8.A.5 VH

     
    154
    3C8.A.5 VL

     
    155
    3C8.A.6 VH

     
    156
    3C8.A.6 VL

     
    157
    3C8.A.7 VH

     
    158
    3C8.A.7 VL

     
    159
    3C8.A.8 VH

     
    160
    3C8.A.8 VL

     
    161
    3C8.A.9 VH

     
    162
    3C8.A.9 VL

     
    163
    3C8.A.10 VH

     
    164
     

     
     
    3C8.A.10 VL

     
    165
    1D2.gr.1 VH

     
    166
    1D2.gr.1 VL

     
    167
    1D2.gr.2 VH

     
    168
    1D2.gr.2 VL

     
    169
    1D2.gr.3 VH

     
    170
    1D2.gr.3 VL

     
    171
    CON1 (1A7)HVR-H1 X1X2YMS, wherein X1 is D or E, and X2 is S or A 172
    CON1 (1A7) HVR-H2 DMYPDX1X2X3X4SYNQKFRE, wherein X1 is N or S, X1 is A or G, X3 is D or S, and X4 is A or S 173
    CON1 (1A7) HVR-H3 APRWX1X2X3X4, wherein X1 is Y or A, X2 is A or F, X3 is S or A, and X4 is A or V. 174
    CON1 (1A7) HVR-L3 QX1X2X3X4X5X6X7T, wherein X1 is A or Q, X2 is A or G, X3 is A or H, X4 is A or T, X5 is A or L, X6 is A or P, and X7 is A or P. 175
    CON2 (3C8) HVR-H2 VINPGSGDX1YYSEKFKG, wherein X1 is T, A or Q. 176
    CON2 (3C8) HVR-L2 HGTNLEX1, wherein X1 is S, E, or Q. 177
    CON2 (3C8) HVR-L3 X1X2YAQFPYX3, wherein X1 is V or A, X2 is H or A, and X3 is Y or A. 178
    1A7 VL

     
    179
    1A7 VH

     
    180
    3C8 VL

     
    181
     

     
     
    3C8 VH

     
    182
    1A7.gr.5' VH

     
    183
    1A7.gr.7' VH

     
    184
    1A7 HVR-H1 M34I variant DSYIS 282
    1A7 HVR-H3 P96A variant AARWYFSV 283
    1A7 VHM34I variant

     
    284
    1A7 VHP96A variant

     
    285
    1A7 HVR-L2 R53Y variant YTSYLRS 286
    1A7 VL R53Y variant

     
    287
    3C8 HVR-H1 N31I variant IYLIE 288
    3C8 HVR-H2 K64L variant VINPGSGDTYYSEKFLG 289
    3C8 VHN31I + K64L variant

     
    290


    [0207] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in U.S. Patent No. 7,550,140. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain comprising the sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYTMNWVRQAPGKGLEWVSAISGSGG STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRYSQVHYALDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 185) and/or a light chain comprising the sequence of DIVMTQSPDSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKAGQSPQLLIYLGSN RASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYNHPTTFGQGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 186). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody 008 as described in U.S. Patent No. 7,550,140. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody 008 as described in U.S. Patent No. 7,550,140.

    [0208] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in U.S. Patent No. 7,550,140. In some aspects, the anti-human OX40 agonist antibody comprises the sequence of DIQMTQSPDSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKAGQSPQLLIYLGSN RASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYNHPTTFGQGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 187). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody SC02008 as described in U.S. Patent No. 7,550,140. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody SC02008 as described in U.S. Patent No. 7,550,140.

    [0209] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in U.S. Patent No. 7,550,140. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain comprising the sequence of EVQLVESGGGLVHPGGSLRLSCAGSGFTFSSYAMHWVRQAPGKGLEWVSAIGTGGG TYYADSVMGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYDNVMGLYWFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:188) and/or a light chain comprising the sequence of EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPAFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:189). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody 023 as described in U.S. Patent No. 7,550,140. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody 023 as described in U.S. Patent No. 7,550,140.

    [0210] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in U.S. Patent No. 7,960,515. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSST IDYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARESGWYLFDYWGQGT LVTVSS (SEQ ID NO:190) and/or a light chain variable region comprising the sequence of DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGGGTKVEIK (SEQ ID NO:191). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody 11D4 as described in U.S. Patent No. 7,960,515. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody 11D4 as described in U.S. Patent No. 7,960,515.

    [0211] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in U.S. Patent No. 7,960,515. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNS GSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDQSTADYYFYYGM DVWGQGTTVTVSS (SEQ ID NO:192) and/or a light chain variable region comprising the sequence of EIVVTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK (SEQ ID NO:193). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody 18D8 as described in U.S. Patent No. 7,960,515. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody 18D8 as described in U.S. Patent No. 7,960,515.

    [0212] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2012/027328. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQGLKWMGWINTE TGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCANPYYDYVSYYAMD YWGQGTTVTVSS (SEQ ID NO:194) and/or a light chain variable region comprising the sequence of DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYLYTG VPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPRTFGQGTKLEIK (SEQ ID NO:195). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody hu106-222 as described in WO 2012/027328. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody hu106-222 as described in WO 2012/027328.

    [0213] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2012/027328. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of EVQLVESGGGLVQPGGSLRLSCAASEYEFPSHDMSWVRQAPGKGLELVAAINSDGG STYYPDTMERRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHYDDYYAWFAYWG QGTMVTVSS (SEQ ID NO:196) and/or a light chain variable region comprising the sequence of EIVLTQSPATLSLSPGERATLSCRASKSVSTSGYSYMHWYQQKPGQAPRLLIYLASNL ESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRELPLTFGGGTKVEIK (SEQ ID NO:197). In some aspects, the antibody comprises at least one, two, three, four, five or six hypervariable region (HVR) sequences of antibody Hu119-122 as described in WO 2012/027328. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody Hu119-122 as described in WO 2012/027328.

    [0214] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2013/028231. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain comprising the sequence of MYLGLNYVFIVFLLNGVQSEVKLEESGGGLVQPGGSMKLSCAASGFTFSDAWMDW VRQSPEKGLEWVAEIRSKANNHATYYAESVNGRFTISRDDSKSSVYLQMNSLRAED TGIYYCTWGEVFYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYITCNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK (SEQ ID NO:198) and/or a light chain comprising the sequence of MRPSIQFLGLLLFWLHGAQCDIQMTQSPSSLSASLGGKVTITCKSSQDINKYIAWYQH KPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLLTFG AGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID NO:199). In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of MYLGLNYVFIVFLLNGVQSEVKLEESGGGLVQPGGSMKLSCAASGFTFSDAWMDW VRQSPEKGLEWVAEIRSKANNHATYYAESVNGRFTISRDDSKSSVYLQMNSLRAED TGIYYCTWGEVFYFDYWGQGTTLTVSS (SEQ ID NO:214) and/or a light chain variable region comprising the sequence of MRPSIQFLGLLLFWLHGAQCDIQMTQSPSSLSASLGGKVTITCKSSQDINKYIAWYQH KPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLLTFG AGTKLELK (SEQ ID NO:215). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody Mab CH 119-43-1 as described in WO 2013/028231. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody Mab CH 119-43-1 as described in WO 2013/028231.

    [0215] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2013/038191. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYN DGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCANYYGSSLSMDYWG QGTSVTVSS (SEQ ID NO:200) and/or a light chain variable region comprising the sequence of DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGV PSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKR (SEQ ID NO:201). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 as described in WO 2013/038191. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody clone 20E5 as described in WO 2013/038191.

    [0216] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2013/038191. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of EVQLQQSGPELVKPGASVKISCKTSGYTFKDYTMHWVKQSHGKSLEWIGGIYPNNG GSTYNQNFKDKATLTVDKSSSTAYMEFRSLTSEDSAVYYCARMGYHGPHLDFDVW GAGTTVTVSP (SEQ ID NO:202) and/or a light chain variable region comprising the sequence of DIVMTQSHKFMSTSLGDRVSITCKASQDVGAAVAWYQQKPGQSPKLLIYWASTRHT GVPDRFTGGGSGTDFTLTISNVQSEDLTDYFCQQYINYPLTFGGGTKLEIKR (SEQ ID NO:203). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 as described in WO 2013/038191. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody clone 12H3 as described in WO 2013/038191.

    [0217] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2014/148895A1. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWMGYINPY NDGTKYNEKFKGRVTITSDTSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWG QGTLVTVSS (SEQ ID NO:204) and/or a light chain variable region comprising the sequence of DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGV PSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:205). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 as described in WO 2014/148895A1. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody clone 20E5 as described in WO 2014/148895A1.

    [0218] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2014/148895A1. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWMGYINPY NDGTKYNEKFKGRVTITSDTSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWG QGTLVTVSS (SEQ ID NO:204) and/or a light chain variable region comprising the sequence of DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSG VPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:206). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 as described in WO 2014/148895A1. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody clone 20E5 as described in WO 2014/148895A1.

    [0219] In some aspects the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2014/148895A1. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINPYN DGTKYNEKFKGRATITSDTSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQ GTLVTVSS (SEQ ID NO:207) and/or a light chain variable region comprising the sequence of DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGV PSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:205). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 as described in WO 2014/148895A1. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody clone 20E5 as described in WO 2014/148895A1.

    [0220] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2014/148895A1. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINPYN DGTKYNEKFKGRATITSDTSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQ GTLVTVSS (SEQ ID NO:207) and/or a light chain variable region comprising the sequence of DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSG VPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:206). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 as described in WO 2014/148895A1. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody clone 20E5 as described in WO 2014/148895A1.

    [0221] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2014/148895A1. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINPYN DGTKYNEKFKGRATLTSDKSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWG QGTLVTVSS (SEQ ID NO:208) and/or a light chain variable region comprising the sequence of DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGV PSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:205). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 as described in WO 2014/148895A1. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody clone 20E5 as described in WO 2014/148895A1.

    [0222] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2014/148895A1. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINPYN DGTKYNEKFKGRATLTSDKSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWG QGTLVTVSS (SEQ ID NO:208) and/or a light chain variable region comprising the sequence of DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSG VPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:206). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 20E5 as described in WO 2014/148895A1. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody clone 20E5 as described in WO 2014/148895A1.

    [0223] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2014/148895A1. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWMGGIYPN NGGSTYNQNFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDV WGQGTTVTVSS (SEQ ID NO:209) and/or a light chain variable region comprising the sequence of DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHT GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:210). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 as described in WO 2014/148895A1. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody clone 12H3 as described in WO 2014/148895A1.

    [0224] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2014/148895A1. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWMGGIYPN NGGSTYNQNFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDV WGQGTTVTVSS (SEQ ID NO:209) and/or a light chain variable region comprising the sequence of DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHT GVPDRFSGGGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:211). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 as described in WO 2014/148895A1. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody clone 12H3 as described in WO 2014/148895A1.

    [0225] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2014/148895A1. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWIGGIYPNN GGSTYNQNFKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDV WGQGTTVTVSS (SEQ ID NO:212) and/or a light chain variable region comprising the sequence of DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHT GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:210 In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 as described in WO 2014/148895A1. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody clone 12H3 as described in WO 2014/148895A1.

    [0226] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2014/148895A1. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWIGGIYPNN GGSTYNQNFKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDV WGQGTTVTVSS (SEQ ID NO:212) and/or a light chain variable region comprising the sequence of DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHT GVPDRFSGGGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:211). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 as described in WO 2014/148895A1. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody clone 12H3 as described in WO 2014/148895A1.

    [0227] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2014/148895A1. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWIGGIYPNN GGSTYNQNFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDV WGQGTTVTVSS (SEQ ID NO:213) and/or a light chain variable region comprising the sequence of DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHT GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:210). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 as described in WO 2014/148895A1. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody clone 12H3 as described in WO 2014/148895A1.

    [0228] In some aspects, the OX40 agonist antibody is an anti-human OX40 agonist antibody described in WO 2014/148895A1. In some aspects, the anti-human OX40 agonist antibody comprises a heavy chain variable region comprising the sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWIGGIYPNN GGSTYNQNFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDV WGQGTTVTVSS (SEQ ID NO:213) and/or a light chain variable region comprising the sequence of DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYWASTRHT GVPDRFSGGGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:211). In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody clone 12H3 as described in WO 2014/148895A1. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody clone 12H3 as described in WO 2014/148895A1.

    [0229] In some aspects, the agonist anti-human OX40 antibody is L106 BD (Pharmingen Product # 340420). In some aspects, the antibody comprises at least one, two, three, four, five or six hypervariable region (HVR) sequences of antibody L106 (BD Pharmingen Product # 340420). In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody L106 (BD Pharmingen Product # 340420).

    [0230] In some aspects, the agonist anti-human OX40 antibody is ACT35 (Santa Cruz Biotechnology, Catalog # 20073). In some aspects, the antibody comprises at least one, two, three, four, five or six hypervariable region (HVR) sequences of antibody ACT35 (Santa Cruz Biotechnology, Catalog # 20073). In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody ACT35 (Santa Cruz Biotechnology, Catalog # 20073).

    [0231] In some aspects, the OX40 agonist antibody is MEDI6469. In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody MEDI6469. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody MEDI6469.

    [0232] In some aspects, the OX40 agonist antibody is MEDI0562. In some aspects, the antibody comprises at least one, two, three, four, five, or six hypervariable region (HVR) sequences of antibody MEDI0562. In some aspects, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody MEDI0562.

    Fc Modifications That Reduce Effector Function



    [0233] In certain aspects, an antibody, subunit, or antigen binding polypeptide described herein comprises one or more amino acid modifications for attenuating effector function (such as CDC and/or ADCC). In exemplary aspects, the modification to attenuate effector function is a modification that alters the glycosylation pattern of the Fc region, e.g., a modification that results in an aglycosylated Fc region. In exemplary aspects, the modification to attenuate effector function is a modification that does not alter the glycosylation pattern of the Fc region. In certain aspects, the modification to attenuate effector function reduces or eliminates binding to human effector cells, binding to one or more Fc receptors, and/or binding to cells expressing an Fc receptor. In an exemplary aspect, the Fc variants described herein comprise an N297G or N297A modification in the Fc region of human IgG1. In an exemplary aspect, the Fc variants described herein comprise the following modifications: L234A, L235A and P329G in the Fc region of human IgG1, that result in attenuated effector function.

    [0234] In various aspects, Fc variants having reduced effector function refer to Fc variants that reduce effector function (e.g., CDC, ADCC, and/or binding to FcR, etc. activities) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more as compared to the effector function achieved by a wild-type Fc region (e.g., an Fc region not having a mutation to reduce effector function, although it may have other mutations). In certain aspects, Fc variants having reduced effector function refer to Fc variants that eliminate all detectable effector function as compared to a wild-type Fc region. Assays for measuring effector function are known in the art and described below.

    [0235] In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcγR binding (hence likely lacking ADCC activity). The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)).

    [0236] Fc variants with reduced effector function include those having amino acid substitutions at one or more of the following amino acid residues: 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc variant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).

    [0237] Certain antibody variants with improved or diminished binding to FcRs are described (see, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

    [0238] In certain aspects, the present disclosure contemplates an antigen binding complex variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the complex in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII and Fc(RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

    [0239] In certain aspects, Fc variants described herein can comprise one or more modifications in the Fc region that result in diminished C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551,

    [0240] WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000). In exemplary aspects, the Fc variants described herein comprise a modification at lysine 322 in the Fc region of human IgGI (EU numbering of residues). In some aspects, the modification(s) result in diminished C1q binding and/or CDC, e.g., as compared to an Fc region without the modification(s). For example, in certain aspects, Fc variants described herein comprise a K322A modification in the Fc region of human IgGI (EU numbering of residues), e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000). Other such exemplary modifications (in the Fc region of human IgG1, and according to EU numbering of residues) include but are not limited to D270K, D270V, P329A, and P331A.

    [0241] In certain aspects, the Fc variants described herein comprise modifications to the Fc region that reduce effector function as described in Strohl, Current Opinion in Biotechnology, 20;685-691 (2009). In exemplary aspects, the Fc variants described herein comprise modifications at one or more amino acid residues selected from the following (EU numbering of residues):
    1. (a) N297A in the Fc region of human IgG1;
    2. (b) 234 and 235 in the Fc region of human IgG1,
    3. (c) 234, 235 and 329 in the Fc region of human IgG1,
    4. (d) 234 and 237 in the Fc region of human IgG2,
    5. (e) 235, 237 and 318 in the Fc region of human IgG4,
    6. (f) 228 and 236 in the Fc region of human IgG4,
    7. (g) 268, 309, 330 and 331 in the Fc region of human IgG2,
    8. (h) 220, 226, 229 and 238 in the Fc region of human IgG1,
    9. (i) 226, 229, 233, 234 and 235 in the Fc region of human IgG1,
    10. (j) 234, 235 and 331 in the Fc region of human IgG1,
    11. (k) 226 and 230 in the Fc region of human IgG1, and
    12. (l) 267 and 328 in the Fc region of human IgG1,
    wherein the modifications reduce effector function of the Fc domain.

    [0242] In other exemplary aspects, the Fc variants described herein comprise modifications that attenuate effector function selected from the following (EU numbering of residues):
    1. (a) N297A in the Fc region of human IgG1;
    2. (b) L234A and L235A in the Fc region of human IgG1,
    3. (c) L234A, L235A and P329G in the Fc region of human IgG1,
    4. (d) V234A and G237A in the Fc region of human IgG2,
    5. (e) L235A, G237A and E318A in the Fc region of human IgG4,
    6. (f) S228P and L236E in the Fc region of human IgG4,
    7. (g) one or more substitutions in the region spanning amino acid residues 118 to 260 in the Fc region of human IgG2 or in the region spanning amino acids 261 to 447 in the Fc region of human IgG4,
    8. (h) H268Q, V309L, A330S and A331S in the Fc region of human IgG2,
    9. (i) C220S, C226S, C229S and P238S in the Fc region of human IgG1,
    10. (j) C226S, C229S, E233P, L234V and L235A in the Fc region of human IgG1,
    11. (k) L234F, L235E and P331S in the Fc region of human IgG1,
    12. (l) C226S and P230S in the Fc region of human IgG1, and
    13. (m) S267E and L328F in the Fc region of human IgG1.


    [0243] In certain aspects, the Fc variants described herein do not comprise an N297A modification to attenuate effector function.

    [0244] In an exemplary aspect, an agonist antigen binding complex provided herein binds to and agonizes OX40 in the absence of FcR binding. In an exemplary aspect, the agonist antigen binding complex provided herein binds to and agonizes OX40 while having reduced FcR binding as compared to the equivalent antigen binding complex that does not contain a mutation in the Fc region to attenuate effector function. In various aspects, the agonist antigen binding complex provided herein binds to and agonizes OX40 while having FcR binding that is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more as compared to the equivalent antigen binding complex that does not contain a mutation in the Fc region to attenuate effector function. In certain aspects, the agonist antigen binding complex provided herein binds to and agonizes OX40 while having FcR binding that is reduced by at least 50%, 75%, 80%, 85%, 90, 95%, 97%, 98% or more as compared to the equivalent antigen binding complex that does not contain a mutation in the Fc region to attenuate effector function.

    III. Production of Antigen Binding Complexes With Agonist Activity



    [0245] Certain aspects of the present disclosure relate to methods of producing an antigen binding complex (e.g., a tetravalent antigen binding complex) having agonist activity for a cell surface receptor.

    [0246] In some aspects, the methods include assembling a first and a second subunit, and coupling the first and the second subunits via a linker. In some aspects, the first and/or the second subunits are bispecific antibodies. A variety of techniques for assembling a bi- or multi-specific antibody are known in the art and described herein (e.g., see infra). For example, in some aspects, the methods include providing a first half-antibody and a second half-antibody, then assembling the first and the second half-antibodies in vitro to form a first subunit or bispecific antibody. In some aspects, the first half-antibody comprises a first antibody heavy chain variable domain (VH1) and a first antibody light chain variable domain (VL1). In some aspects, the second half-antibody comprises a second antibody heavy chain variable domain (VH2) and a second antibody light chain variable domain (VL2). In some aspects, the methods further include providing a third half-antibody and a fourth half-antibody, then assembling the third and the fourth half-antibodies in vitro to form a second subunit or bispecific antibody. In some aspects, the third half-antibody comprises the first antibody heavy chain variable domain (VH1) and the first antibody light chain variable domain (VL1). In some aspects, the fourth half-antibody comprises the second antibody heavy chain variable domain (VH2) and the second antibody light chain variable domain (VL2). In some aspects, one or both of the subunits comprises a first half-antibody (e.g., VH1 and VL1) that specifically binds one epitope (e.g., of a cell surface receptor), and a second half-antibody (e.g., VH2 and VL2) that specifically binds a different epitope (e.g., of the same cell surface receptor). In some aspects, one or both of the subunits comprises a first half-antibody (e.g., VH1 and VL1) that specifically binds one epitope (e.g., of a cell surface receptor), and a second half-antibody (e.g., VH2 and VL2) that specifically binds the same epitope. While this represents an exemplary method for producing an antigen binding complex (e.g., a "c:IgG-IgG" format as illustrated in FIGS. 3A & 3B), the present disclosure contemplates other suitable methods of production using techniques known in the art, including other techniques for generating a bispecific antibody, different linkers, different coupling strategies, and so forth.

    [0247] In some aspects, the first and second subunits are chemically coupled via a linker. A variety of chemical coupling techniques are known in the art and described herein (e.g., see infra). For example, in some aspects, one half-antibody from each subunit includes an engineered free cysteine, and the engineered free cysteines are coupled using techniques known in the art and described herein (e.g., see infra). Thus, this asymmetry allows for a consistent configuration of the complex, as only one pairing is possible. Exemplary free cysteines are described herein and include, without limitation, a cysteine amino acid in the heavy chain selected from T114C, A118C, A140C, L174C, L179C, T187C, T209C, V262C, G371C, Y373C, E382C, S400C, S424C, N434C and Q438C, according to EU numbering; and a cysteine amino acid in the light chain selected from I106C, R108C, R142C, K149C, and V205C, according to Kabat numbering.

    [0248] In some aspects, the first and second subunits are chemically coupled via a bis-maleimido polyethylene glycol (PEG) linker of the present disclosure. For example, in some aspects, coupling the subunits includes reacting the two subunits with a bis-maleimido polyethylene glycol (PEG) linker of the present disclosure and purifying the complex. A variety of purification techniques known in the art may be used. In some aspects, the purification comprises size exclusion chromatography and/or anion exchange chromatography.

    [0249] In some aspects, the methods include providing an antibody and two antibody Fab fragments, then coupling one of the antibody Fab fragments to each of the two half-antibodies via a linker. In some aspects, the antibody comprises two half-antibodies. In some aspects, each half-antibody comprises an antibody heavy chain comprising a first antibody heavy chain variable domain (VH1), and an antibody light chain comprising a first antibody light chain variable domain (VL1). In some aspects, each antibody Fab fragment comprises a second antibody heavy chain variable domain (VH2) and a second antibody light chain variable domain (VL2). In some aspects, the complex comprises a two half-antibodies, each with a VH1 and a VL1 that specifically binds one epitope (e.g., of a cell surface receptor), and two Fab fragments, each with a VH2 and a VL2 that specifically binds a different epitope (e.g., of the same cell surface receptor). In some aspects, the complex comprises a two half-antibodies, each with a VH1 and a VL1 that specifically binds one epitope (e.g., of a cell surface receptor), and two Fab fragments, each with a VH2 and a VL2 that specifically binds the same epitope of the cell surface receptor. While this represents an exemplary method for producing an antigen binding complex (e.g., a "c:Fab-IgG" format as illustrated in FIGS. 3A & 3B), the present disclosure contemplates other suitable methods of production using techniques known in the art, including different linkers (e.g., a polypeptide linker), different coupling strategies (e.g., genetic coupling using single chain antibodies or half-antibodies linked to the Fab fragment(s)), and so forth.

    [0250] In some aspects, the two Fab fragments are each chemically coupled to the antibody via a linker. A variety of chemical coupling techniques are known in the art and described herein (e.g., see infra). For example, in some aspects, each half-antibody and Fab fragment includes an engineered free cysteine, each Fab fragment is coupled to one half-antibody via the engineered free cysteines, and the engineered free cysteines are coupled using techniques known in the art and described herein (e.g., see infra). Exemplary free cysteines are described herein and include, without limitation, a cysteine amino acid in the heavy chain selected from T114C, A118C, A140C, L174C, L179C, T187C, T209C, V262C, G371C, Y373C, E382C, S400C, S424C, N434C and Q438C, according to EU numbering; and a cysteine amino acid in the light chain selected from a C-terminal cysteine, I106C, R108C, R142C, K149C, and V205C, according to Kabat numbering. For example, in certain aspects (e.g., as described in reference to FIGS. 6 & 7), each Fab fragment is chemically coupled to an antibody light chain via a linker, using a C-terminal free engineered cysteine on the Fab fragment and a K149C free engineered cysteine on the antibody light chain.

    [0251] In some aspects, the antibody and the two Fab fragments are chemically coupled via a bis-maleimido polyethylene glycol (PEG) linker of the present disclosure. For example, in some aspects, coupling the subunits includes reacting each of the two antibody Fab fragments with a bis-maleimido polyethylene glycol (PEG) linker to form two bismal-conjugated antibody Fab fragments, removing excess bis-maleimido PEG linker, reacting each of the two bismal-conjugated antibody Fab fragments with the antibody to form the complex, and purifying the complex. A variety of purification techniques known in the art may be used. In some aspects, the purification comprises size exclusion chromatography and/or anion exchange chromatography.

    [0252] In some aspects, the methods include expressing in a host cell two antibody heavy chains. In some aspects, each antibody heavy chain comprises, from N-terminus to C-terminus, a first antibody heavy chain variable domain (VH1), a first antibody heavy chain CH1 domain (CH11), a second heavy chain variable domain (VH2), a second heavy chain CH1 domain (CH12), an antibody heavy chain CH2 domain, and an antibody heavy chain CH3 domain. In some aspects, each VH1 and/or each CH11 comprises a first modification for orthogonal pairing, and each VH2 and/or each CH12 comprises a different, second modification for orthogonal pairing, e.g., as compared to the modification of the VH1 and/or CH11. In some aspects, the methods further include expressing in the host cell two first antibody light chains. In some aspects, each of the two first antibody light chains comprises, from N-terminus to C-terminus, a first antibody light chain variable domain (VL1) and a first antibody light chain constant domain (CL1). In some aspects, the VL1 and/or the CL1 comprises a modification for orthogonal pairing with the first modification of the antibody heavy chains. In some aspects, the methods further include expressing in the host cell two second antibody light chains. In some aspects, each of the two second antibody light chains comprises, from N-terminus to C-terminus, a second antibody light chain variable domain (VL2) and a second antibody light chain constant domain (CL2). In some aspects, the VL2 and/or the CL2 comprises a modification for orthogonal pairing with the second modification of the antibody heavy chains. Upon expression in the host cell, the two antibody heavy chains associate, each of the two heavy chains couples with a first antibody light chain via orthogonal pairing, and each of the two heavy chains couples with a second antibody light chain via orthogonal pairing. In some aspects, the antigen binding complex is biepitopic. While this represents an exemplary method for producing an antigen binding complex (e.g., a "r:Fab-IgG" format as illustrated in FIGS. 3A & 3B), the present disclosure contemplates other suitable methods of production using techniques known in the art, including different linkers (e.g., a polypeptide linker), different coupling strategies (e.g., genetic coupling using single chain antibodies or half-antibodies linked to the Fab fragment(s)), and so forth.

    [0253] Exemplary orthogonal variant pairs are described herein; for further descriptions, see, e.g., PCT/US2016/028850. For example, in some aspects, the first and/or the second modification of the antibody heavy chain are selected from VH-Q39K, VH-Q39E, CH1-S183E, CH1-S183K, CH1-A141I, CH1-F170S, CH1-S181M, CH1-S183A, and CH1-V185A (EU numbering). In some aspects, the modifications of the first and/or the second antibody light chains are selected from VL-Q38E, VL-Q38K, CL-V133K, CL-V133E, CL-F116A, CL-L135V, CL-S174A, CL-S176F, and CL-T178V (EU numbering).

    Bispecific Antibodies



    [0254] In certain aspects, an antigen binding polypeptide, subunit, or antibody provided herein is multispecific, e.g. bispecific. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites or epitopes. In some aspects, a bispecific antibody, subunit, or antigen binding polypeptide has binding specificities for at least two different epitopes of the same target, e.g., a cell surface receptor.

    [0255] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see,e.g. Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

    [0256] In some aspects, e.g., as exemplified infra, a bispecific antibody is created by "knob-in-hole" engineering. For example, two half-antibodies can be assembled into a bispecific antibody in vitro, where a first half-antibody comprises an amino acid modification in its CH3 domain that forms a protuberance, and the second half-antibody comprises an amino acid modification in its CH3 domain that forms a cavity. The protuberance is positionable into the cavity, thereby forming the bispecific antibody upon assembly.

    [0257] In this approach, two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each comprise an interface. An interface of one immunoglobulin polypeptide interacts with a corresponding interface on the other immunoglobulin polypeptide, thereby allowing the two immunoglobulin polypeptides to associate. These interfaces may be engineered such that a "knob" or "protuberance" (these terms may be used interchangeably herein) located in the interface of one immunoglobulin polypeptide corresponds with a "hole" or "cavity" (these terms may be used interchangeably herein) located in the interface of the other immunoglobulin polypeptide. In some aspects, the hole is of identical or similar size to the knob and suitably positioned such that when the two interfaces interact, the knob of one interface is positionable in the corresponding hole of the other interface. Without wishing to be bound to theory, this is thought to stabilize the heteromultimer and favor formation of the heteromultimer over other species, for example homomultimers. In some aspects, this approach may be used to promote the heteromultimerization of two different immunoglobulin polypeptides, creating a bispecific antibody comprising two immunoglobulin polypeptides with binding specificities for different epitopes.

    [0258] In some aspects, a knob may be constructed by replacing a small amino acid side chain with a larger side chain. In some aspects, a hole may be constructed by replacing a large amino acid side chain with a smaller side chain. Knobs or holes may exist in the original interface, or they may be introduced synthetically. For example, knobs or holes may be introduced synthetically by altering the nucleic acid sequence encoding the interface to replace at least one "original" amino acid residue with at least one "import" amino acid residue. Methods for altering nucleic acid sequences may include standard molecular biology techniques well known in the art. The side chain volumes of various amino acid residues are shown in the following table. In some aspects, original residues have a small side chain volume (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine), and import residues for forming a knob are naturally occurring amino acids and may include arginine, phenylalanine, tyrosine, and tryptophan. In some aspects, original residues have a large side chain volume (e.g., arginine, phenylalanine, tyrosine, and tryptophan), and import residues for forming a hole are naturally occurring amino acids and may include alanine, serine, threonine, and valine.
    Table B. Properties of amino acid residues
    Amino acidOne-letter abbreviationMassa (daltons)Volumeb3)Accessible surface areac2)
    Alanine (Ala) A 71.08 88.6 115
    Arginine (Arg) R 156.20 173.4 225
    Asparagine (Asn) N 114.11 117.7 160
    Aspartic Acid (Asp) D 115.09 111.1 150
    Cysteine (Cys) C 103.14 108.5 135
    Glutamine (Gln) Q 128.14 143.9 180
    Glutamic Acid (Glu) E 129.12 138.4 190
    Glycine (Gly) G 57.06 60.1 75
    Histidine (His) H 137.15 153.2 195
    Isoleucine (Ile) I 113.17 166.7 175
    Leucine (Leu) L 113.17 166.7 170
    Lysine (Lys) K 128.18 168.6 200
    Methionine (Met) M 131.21 162.9 185
    Phenylalanine (Phe) F 147.18 189.9 210
    Proline (Pro) P 97.12 122.7 145
    Serine (Ser) S 87.08 89.0 115
    Threonine (Thr) T 101.11 116.1 140
    Tryptophan (Trp) W 186.21 227.8 255
    Tyrosine (Tyr) Y 163.18 193.6 230
    Valine (Val) V 99.14 140.0 155
    aMolecular weight of amino acid minus that of water. Values from Handbook of Chemistry and Physics, 43rd ed. Cleveland, Chemical Rubber Publishing Co., 1961.
    bValues from A.A. Zamyatnin, Prog. Biophys. Mol. Biol. 24:107-123, 1972.
    cValues from C. Chothia, J. Mol. Biol. 105:1-14, 1975. The accessible surface area is defined in Figures 6-20 of this reference.


    [0259] In some aspects, original residues for forming a knob or hole are identified based on the three-dimensional structure of the heteromultimer. Techniques known in the art for obtaining a three-dimensional structure may include X-ray crystallography and NMR. In some aspects, the interface is the CH3 domain of an immunoglobulin constant domain. In these aspects, the CH3/CH3 interface of human IgG1 involves sixteen residues on each domain located on four anti-parallel β-strands. Without wishing to be bound to theory, mutated residues are preferably located on the two central anti-parallel β-strands to minimize the risk that knobs can be accommodated by the surrounding solvent, rather than the compensatory holes in the partner CH3 domain. In some aspects, the mutations forming corresponding knobs and holes in two immunoglobulin polypeptides correspond to one or more pairs provided in the following table.
    Table C. Exemplary sets of corresponding knob-and hole-forming mutations
    CH3 of first immunoglobulinCH3 of second immunoglobulin
    T366Y Y407T
    T366W Y407A
    F405A T394W
    Y407T T366Y
    T366Y:F405A T394W:Y407T
    T366W:F405W T394S:Y407A
    F405W:Y407A T366W:T394S
    F405W T394S
    Mutations are denoted by the original residue, followed by the position using the Kabat numbering system, and then the import residue (all residues are given in single-letter amino acid code). Multiple mutations are separated by a colon.


    [0260] In some aspects, an immunoglobulin polypeptide comprises a CH3 domain comprising one or more amino acid substitutions listed in Table C above. In some aspects, a bispecific antibody comprises a first immunoglobulin polypeptide comprising a CH3 domain comprising one or more amino acid substitutions listed in the left column of Table C, and a second immunoglobulin polypeptide comprising a CH3 domain comprising one or more corresponding amino acid substitutions listed in the right column of Table C.

    [0261] Following mutation of the DNA as discussed above, polynucleotides encoding modified immunoglobulin polypeptides with one or more corresponding knob- or hole-forming mutations may be expressed and purified using standard recombinant techniques and cell systems known in the art. See, e.g., U.S. Pat. Nos. 5,731,168; 5,807,706; 5,821,333; 7,642,228; 7,695,936; 8,216,805; U.S. Pub. No. 2013/0089553; and Spiess et al., Nature Biotechnology 31: 753-758, 2013. Modified immunoglobulin polypeptides may be produced using prokaryotic host cells, such as E. coli, or eukaryotic host cells, such as CHO cells. Corresponding knob- and hole-bearing immunoglobulin polypeptides may be expressed in host cells in co-culture and purified together as a heteromultimer, or they may be expressed in single cultures, separately purified, and assembled in vitro. In some aspects, two strains of bacterial host cells (one expressing an immunoglobulin polypeptide with a knob, and the other expressing an immunoglobulin polypeptide with a hole) are co-cultured using standard bacterial culturing techniques known in the art. In some aspects, the two strains may be mixed in a specific ratio, e.g., so as to achieve equal expression levels in culture. In some aspects, the two strains may be mixed in a 50:50, 60:40, or 70:30 ratio. After polypeptide expression, the cells may be lysed together, and protein may be extracted. Standard techniques known in the art that allow for measuring the abundance of homo-multimeric vs. hetero-multimeric species may include size exclusion chromatography. In some aspects, each modified immunoglobulin polypeptide is expressed separately using standard recombinant techniques, and they may be assembled together in vitro. Assembly may be achieved, for example, by purifying each modified immunoglobulin polypeptide, mixing and incubating them together in equal mass, reducing disulfides (e.g., by treating with dithiothreitol), concentrating, and reoxidizing the polypeptides. Formed bispecific antibodies may be purified using standard techniques including cation-exchange chromatography and measured using standard techniques including size exclusion chromatography. For a more detailed description of these methods, see Speiss et al., Nat Biotechnol 31:753-8, 2013. In some aspects, modified immunoglobulin polypeptides may be expressed separately in CHO cells and assembled in vitro using the methods described above.

    [0262] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is typical to have the first heavy-chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in aspects when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

    [0263] In one aspect of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

    [0264] According to another approach described in WO96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. One interface comprises at least a part of the CH 3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

    [0265] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

    [0266] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

    [0267] Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.

    [0268] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al, J. Immunol, 152:5368 (1994).

    [0269] Another technique for making bispecific antibody fragments is the "bispecific T cell engager" or BiTE® approach (see, e.g., WO2004/106381, WO2005/061547, WO2007/042261, and WO2008/119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain includes two single chain Fv (scFv) fragments, each having a variable heavy chain (VH) and a variable light chain (VL) domain separated by a polypeptide linker of a length sufficient to allow intramolecular association between the two domains. This single polypeptide further includes a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes may be specific for different cell types, such that cells of two different cell types are brought into close proximity or tethered when each scFv is engaged with its cognate epitope. One particular aspect of this approach includes a scFv recognizing a cell-surface antigen expressed by an immune cell, e.g., a CD3 polypeptide on a T cell, linked to another scFv that recognizes a cell-surface antigen expressed by a target cell, such as a malignant or tumor cell.

    [0270] As it is a single polypeptide, the bispecific T cell engager may be expressed using any prokaryotic or eukaryotic cell expression system known in the art, e.g., a CHO cell line. However, specific purification techniques (see, e.g., EP1691833) may be necessary to separate monomeric bispecific T cell engagers from other multimeric species, which may have biological activities other than the intended activity of the monomer. In one exemplary purification scheme, a solution containing secreted polypeptides is first subjected to a metal affinity chromatography, and polypeptides are eluted with a gradient of imidazole concentrations. This eluate is further purified using anion exchange chromatography, and polypeptides are eluted using with a gradient of sodium chloride concentrations. Finally, this eluate is subjected to size exclusion chromatography to separate monomers from multimeric species.

    [0271] Additional descriptions of methods for making multispecific antibodies may be found in Spiess, C. et al. (2015) Mol. Immunol. 67:95-106 (see, e.g., Table 2). These include, without limitation, the following technologies (with exemplary mutations in the first and second heavy chains included): knobs-into-holes (T366W in first heavy chain; T366S, L368A, and Y407V in second heavy chain), DuoBody® (F405L in first heavy chain; K409R in second heavy chain), Azymetric (T350V, L351Y, F405A, and Y407V in first heavy chain; T350V, T366L, K392L, and T394W in second heavy chain), Amgen charge-pair (K409D and K392D in first heavy chain; D399K and E356K in second heavy chain), Rinat-Pfizer charge-pair (D221E, P228E, and L368E in first heavy chain; D221R, P228R, and K409R in second heavy chain), HA-TF (S364H and F405A in first heavy chain; Y349T and T394F in second heavy chain), SEEDBody (IgA/G chimeras in first and second heavy chains), and differential protein A affinity (H435R in first heavy chain).

    Engineered Cysteines



    [0272] In some aspects, two antigen binding polypeptides, antibodies, or subunits are coupled via engineered free cysteines. In some aspects, an antibody and one or more Fab fragments are coupled via engineered free cysteines. For example, in some aspects, two antigen binding polypeptides or subunits, or an antibody and one or more Fab fragments, are coupled via THIOMAB (Genentech) antibody technology. Exemplary descriptions of THIOMAB antibody technology may be found, e.g., in US Patent Nos. 7,521,541; 7,855,275; 8,309,300; and 9,000,130; as well as US PG Pub No. 20160130358. In some aspects, THIOMAB antibody technology is used to couple antigen binding polypeptides, subunits, antibodies, or Fab fragments via a linker.

    [0273] Cysteine thiols are reactive at neutral pH, unlike most amines which are protonated and less nucleophilic near pH 7. Since free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins with cysteine residues often exist in their oxidized form as disulfide-linked oligomers or have internally bridged disulfide groups. Antibody cysteine thiol groups are generally more reactive, i.e. more nucleophilic, towards electrophilic conjugation reagents than antibody amine or hydroxyl groups. Engineering in cysteine thiol groups by the mutation of various amino acid residues of a protein to cysteine amino acids is potentially problematic, particularly in the case of unpaired (free Cys) residues or those which are relatively accessible for reaction or oxidation. In concentrated solutions of the protein, whether in the periplasm of E. coli, culture supernatants, or partially or completely purified protein, unpaired Cys residues on the surface of the protein can pair and oxidize to form intermolecular disulfides, and hence protein dimers or multimers. Disulfide dimer formation renders the new Cys unreactive for conjugation to a drug, ligand, or other label. Furthermore, if the protein oxidatively forms an intramolecular disulfide bond between the newly engineered Cys and an existing Cys residue, both Cys groups are unavailable for active site participation and interactions. Furthermore, the protein may be rendered inactive or nonspecific, by misfolding or loss of tertiary structure (Zhang et al (2002) Anal. Biochem. 311:1-9).

    [0274] Antibodies/subunits/Fab fragments with cysteine substitutions (THIOMAB antibodies) at sites where the engineered cysteines are available for conjugation but do not perturb immunoglobulin folding and assembly or alter antigen binding and effector functions (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729; US 7521541; US 7723485; WO2009/052249).

    [0275] In some aspects, a cysteine engineered antibody/subunit/Fab fragment refers to an antibody in which one or more residues of an antibody are substituted with cysteine residues. The thiol group(s) of the cysteine engineered antibodies can be conjugated via a linker. In particular aspects, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody or Fab fragment to other moieties. For example, a THIOMAB antibody may be an antibody with a single mutation of a non-cysteine native residue to a cysteine in the light chain (e.g., G64C, I106C, R108C, K149C or R142C according to Kabat numbering) or in the heavy chain (e.g., HC-D101C, HC-V184C, or HC-T205C according to Kabat numbering, or HC-T114C, HC-A140C, HC-L174C, HC-L179C, HC-T187C, HC-T209C, HC-V262C, HC-G371C, HC-Y373C, HC-E382C, HC-S424C, HC-N434C, and HC-Q438C according to EU numbering (i.e., HC-A136C according to Kabat numbering is HC-A140C according to EU numbering).

    [0276] In some aspects, a free cysteine amino acid refers to a cysteine amino acid residue which has been engineered into a parent antibody, has a thiol functional group (-SH), and is not paired as an intramolecular or intermolecular disulfide bridge.

    [0277] Exemplary free engineered cysteine amino acids are known in the art and described herein. In some aspects, a heavy chain comprises a cysteine amino acid in the heavy chain selected from T114C, A118C, A140C, L174C, L179C, T187C, T209C, V262C, G371C, Y373C, E382C, S400C, S424C, N434C and Q438C, according to EU numbering. In some aspects, a light chain comprises a cysteine amino acid in the light chain selected from I106C, R108C, R142C, K149C, and V205C, according to Kabat numbering. In some aspects, a Fab fragment comprises a C-terminal cysteine amino acid.

    [0278] It should be noted that a single site mutation yields a single engineered cysteine residue in a ThioFab, while a single site mutation yields two engineered cysteine residues in a THIOMAB antibody, due to the dimeric nature of the IgG antibody. Mutants with engineered cysteine (Cys) residues were evaluated for the reactivity of the newly introduced, engineered cysteine thiol groups. The thiol reactivity value is a relative, numerical term in the range of 0 to 1.0 and can be measured for any cysteine engineered antibody. Thiol reactivity values of cysteine engineered antibodies of the invention are in the range of 0.0 to 1.0. Specifically, the thiol reactivity values of cysteine engineered antibodies of the invention are in the range of 0.1 to 1.0. In certain aspects, the thiol reactivity values of cysteine engineered antibodies of the disclosure are in the ranges of 0.0 to 0.1, 0.1 to 0.5, 0.1 to 0.6, 0.1 to 0.7, 0.1 to 0.8, 0.1 to 0.9, or 0.1 to 1.0. In certain aspects, the thiol reactivity values of cysteine engineered antibodies of the disclosure are in the ranges of 0.2 to 1.0, 0.3 to 1.0, 0.4 to 1.0, 0.5 to 1.0, 0.6 to 1.0, 0.7 to 1.0, or 0.8 to 1.0. In certain aspects, the thiol reactivity values of cysteine engineered antibodies of the disclosure are in the range of 0.6 to 1.0. In certain aspects, the thiol reactivity values of cysteine engineered antibodies of the disclosure are in the ranges of 0.7 to 1.0. In certain aspects, the thiol reactivity values of cysteine engineered antibodies of the invention are in the ranges of 0.8 to 10. In certain aspects, the thiol reactivity values of cysteine engineered antibodies of the disclosure are in the ranges of 0.5 to 0.8. In certain aspects, the thiol reactivity values of cysteine engineered antibodies of the disclosure are in the ranges of 0.5 to 0.9. In certain aspects, the thiol reactivity values of cysteine engineered antibodies of the disclosure are in the ranges of 0.5 to 0.7. In certain aspects, the thiol reactivity values of cysteine engineered antibodies of the present disclosure are in the ranges of 0.5 to 1.0.

    [0279] The design, selection, and preparation methods of the present disclosure enable cysteine engineered antibodies which are reactive with electrophilic functionality. Reactive cysteine residues on an antibody surface allow coupling via a thiol reactive group such as maleimide or haloacetyl. The nucleophilic reactivity of the thiol functionality of a Cys residue to a maleimide group is about 1000 times higher compared to any other amino acid functionality in a protein, such as amino group of lysine residues or the N-terminal amino group. Thiol specific functionality in iodoacetyl and maleimide reagents may react with amine groups, but higher pH (>9.0) and longer reaction times are required (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London).

    [0280] Cysteine engineered antibodies/subunits/Fab fragments of the present disclosure preferably retain the antigen binding capability of their wild type, parent counterparts. Thus, cysteine engineered antibodies are capable of binding, preferably specifically, to antigens, e.g., an epitope of a cell surface receptor.

    [0281] Cysteine engineered antibodies/subunits/Fab fragments of the present disclosure may be site-specifically and efficiently coupled with a thiol-reactive reagent. One example of a thiol-reactive reagent is N-ethyl maleimide (NEM). In an exemplary aspect, reaction of a THIOMAB antibody with a biotin-linker reagent provides a biotinylated THIOMAB antibody by which the presence and reactivity of the engineered cysteine residue may be detected and measured. Reaction of a THIOMAB antibody with a multifunctional linker reagent provides a THIOMAB antibody with a functionalized linker which may be further reacted with an antibody or Fab fragment. In certain aspects, the THIOMAB antibody is a ThioFab.

    [0282] The exemplary methods described here may be applied generally to the identification and production of antibodies, and more generally, to other proteins through application of the design and screening steps described herein.

    [0283] Such an approach may be applied to the conjugation of other thiol-reactive agents in which the reactive group is, for example, a maleimide, an iodoacetamide, a pyridyl disulfide, or other thiol-reactive conjugation partner (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671).

    Vectors, Host Cells and Recombinant Methods



    [0284] For recombinant production of an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) of the present disclosure, or a subunit thereof, provided herein, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antigen binding polypeptide is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, preferred host cells are of either prokaryotic or eukaryotic (generally mammalian, but also including fungi (e.g., yeast), insect, plant, and nucleated cells from other multicellular organisms) origin. In some aspects, the host cell is an isolated host cell, e.g., a host cell derived from a multicellular organism that is grown as an isolated cell, such as a cell line derived from an invertebrate (e.g., insect) or vertebrate (e.g., mouse, human, Chinese hamster ovary (CHO) cell, etc.) organism that is grown in cell culture. In cases where an antigen binding polypeptide is an antibody, it will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species.

    a. Generating antigen binding polypeptides using prokaryotic host cells


    i. Vector construction



    [0285] Polynucleotide sequences encoding polypeptide components of the antigen binding complexes (e.g., a tetravalent antigen binding complex having agonist activity) of the present disclosure or subunit(s) thereof provided herein can be obtained using standard recombinant techniques. Desired polynucleotide sequences may be isolated and sequenced from, for example, antibody producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purpose of the present invention. Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides. The vector components generally include, but are not limited to: an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert and a transcription termination sequence.

    [0286] In some aspects, a polynucleotide sequence of the present disclosure includes a mutation, e.g., one that encodes for an amino acid substitution described herein, such as an engineered free cysteine or a modification for attenuating effector function of an Fc region. DNA encoding an amino acid sequence variant of the starting polypeptide is prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding the polypeptide. Variants of recombinant antibodies may be constructed also by restriction fragment manipulation or by overlap extension PCR with synthetic oligonucleotides. Mutagenic primers encode the cysteine codon replacements). Standard mutagenesis techniques can be employed to generate DNA encoding such mutant cysteine engineered antibodies. General guidance can be found in Sambrook et al Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel et al Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, N.Y., 1993.

    [0287] Site-directed mutagenesis is one method for preparing substitution variants, i.e. mutant proteins. This technique is well known in the art (see for example, Carter (1985) et al Nucleic Acids Res. 13:4431-4443; Ho et al (1989) Gene (Amst.) 77:51-59; and Kunkel et al (1987) Proc. Natl. Acad. Sci. USA 82:488). Briefly, in carrying out site-directed mutagenesis of DNA, the starting DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of such starting DNA. After hybridization, a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of the starting DNA as a template. Thus, the oligonucleotide encoding the desired mutation is incorporated in the resulting double-stranded DNA. Site-directed mutagenesis may be carried out within the gene expressing the protein to be mutagenized in an expression plasmid and the resulting plasmid may be sequenced to confirm the introduction of the desired cysteine replacement mutations (Liu et al (1998) J. Biol. Chem. 273:20252-20260). Site-directed of protocols and formats, including those commercially available, e.g. QuikChange® Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).

    [0288] PCR mutagenesis is also suitable for making amino acid sequence variants of the starting polypeptide. See Higuchi, (1990) in PCR Protocols, pp.177-183, Academic Press; Ito et al (1991) Gene 102:67-70; Bernhard et al (1994) Bioconjugate Chem. 5:126-132; and Vallette et al (1989) Nuc. Acids Res. 17:723-733. Briefly, when small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template.

    [0289] Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al (1985) Gene 34:315-323. The starting material is the plasmid (or other vector) comprising the starting polypeptide DNA to be mutated. The codon(s) in the starting DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the starting polypeptide DNA. The plasmid DNA is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures, wherein the two strands of the oligonucleotide are synthesized separately and then hybridized together using standard techniques. Oligonucleotides are prepared by the phosphoramidite synthesis method (US 4415732; US 4458066; Beaucage, S. and Iyer, R. (1992) "Advances in the synthesis of oligonucleotides by the phosphoramidite approach", Tetrahedron 48:2223-2311). This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 5' and 3' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated DNA sequence. Mutant DNA containing the encoded cysteine replacements can be confirmed by DNA sequencing.

    [0290] Single mutations are also generated by oligonucleotide directed mutagenesis using double stranded plasmid DNA as template by PCR based mutagenesis (Sambrook and Russel, (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; Zoller et al (1983) Methods Enzymol. 100:468-500; Zoller, M.J. and Smith, M. (1982) Nucl. Acids Res. 10:6487-6500).

    [0291] In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides easy means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins. Examples of pBR322 derivatives used for expression of particular antibodies are described in detail in Carter et al., U.S. Patent No. 5,648,237.

    [0292] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as λGEM.TM.-11 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.

    [0293] The expression vector of the invention may comprise two or more promoter- cistron pairs, encoding each of the polypeptide components. A promoter is an untranslated regulatory sequence located upstream (5') to a cistron that modulates its expression. Prokaryotic promoters typically fall into two classes, inducible and constitutive. An inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to changes in the culture condition, e.g., the presence or absence of a nutrient or a change in temperature.

    [0294] A large number of promoters recognized by a variety of potential host cells are well known. The selected promoter can be operably linked to cistron DNA encoding, for example, the light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the invention. Both the native promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of the target genes. In some aspects, heterologous promoters are utilized, as they generally permit greater transcription and higher yields of the expressed target gene as compared to the native target polypeptide promoter.

    [0295] Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the β-galactamase and lactose promoter systems, a tryptophan (trp) promoter system and hybrid promoters such as the tac or the trc promoter. However, other promoters that are functional in bacteria (such as other known bacterial or phage promoters) are suitable as well. Their nucleotide sequences have been published, thereby enabling a skilled worker to operably ligate them to cistrons encoding the genes of the antigen binding polypeptide protein, e.g., the target light and heavy chains (Siebenlist et al., (1980) Cell 20: 269), using linkers or adaptors to supply any required restriction sites.

    [0296] In one aspect, each cistron within the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptides across a membrane. In general, the signal sequence may be a component of the vector, or it may be a part of the target polypeptide DNA that is inserted into the vector. The signal sequence selected should be one that is recognized and processed {i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the signal sequences native to the heterologous polypeptides, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one aspect of the invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

    [0297] In another aspect, the production of the immunoglobulins can occur in the cytoplasm of the host cell, and therefore does not require the presence of secretion signal sequences within each cistron. In that regard, immunoglobulin light and heavy chains are expressed, folded and assembled to form functional immunoglobulins within the cytoplasm. Certain host strains (e.g., the E. coli trxB- strains) provide cytoplasm conditions that are favorable for disulfide bond formation, thereby permitting proper folding and assembly of expressed protein subunits. See Proba and Pluckthun Gene, 159:203 (1995).

    [0298] Prokaryotic host cells suitable for expressing antigen binding polypeptides (e.g., antibodies) of the invention include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one aspect, gram-negative cells are used. In one aspect, E. coli cells are used as hosts for the invention. Examples of E coli strains include strain W31 10 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1 190-1219; ATCC Deposit No. 27,325) and derivatives thereof, including strain 33D3 having genotype W31 10 AfhuA (AtonA) ptr3 lac Iq lacL8 AompTA(nmpc-fepE) degP41 kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof, such as E coli 294 (ATCC 31 ,446), E coli B, E colix 1776 (ATCC 31 ,537) and E coli RV308 (ATCC 31 ,608) are also suitable. In one aspect, E coli Alpp finds particular use. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above-mentioned bacteria having defined genotypes are known in the art and described in, for example, Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select the appropriate bacteria taking into consideration replicability of the replicon in the cells of a bacterium. For example, E coli, Serratia, or Salmonella species can be suitably used as the host when well known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon. Typically the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated in the cell culture.

    ii. Polypeptide Production



    [0299] Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

    [0300] Transformation means introducing DNA into the prokaryotic host so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride is generally used for bacterial cells that contain substantial cell-wall barriers. Another method for transformation employs polyethylene glycol/DMSO. Yet another technique used is electroporation.

    [0301] Prokaryotic cells used to produce the polypeptides of the invention are grown in media known in the art and suitable for culture of the selected host cells. Examples of suitable media include Luria broth (LB) plus necessary nutrient supplements. In some aspects, the media also contains a selection agent, chosen based on the construction of the expression vector, to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to media for growth of cells expressing ampicillin resistant gene.

    [0302] Any necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source. Optionally the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and dithiothreitol.

    [0303] The prokaryotic host cells are cultured at suitable temperatures. For E. coli growth, for example, the preferred temperature ranges from about 20°C to about 39°C, more preferably from about 25°C to about 37°C, even more preferably at about 30°C. The pH of the medium may be any pH ranging from about 5 to about 9, depending mainly on the host organism. For E. coli, the pH is preferably from about 6.8 to about 7.4, and more preferably about 7.0.

    [0304] If an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for the activation of the promoter. In one aspect of the invention, PhoA promoters are used for controlling transcription of the polypeptides. Accordingly, the transformed host cells are cultured in a phosphate-limiting medium for induction. Preferably, the phosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons et al., J. Immunol. Methods (2002), 263:133-147). A variety of other inducers may be used, according to the vector construct employed, as is known in the art.

    [0305] In one aspect of the invention, antigen binding polypeptides (such as, for example, an antibody) production is conducted in large quantity by a fermentation process. Various large-scale fed-batch fermentation procedures are available for production of recombinant proteins. Large-scale fermentations have at least 1000 liters of capacity, preferably about 1 ,000 to 100,000 liters of capacity. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon/energy source). Small scale fermentation refers generally to fermentation in a fermentor that is no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters.

    [0306] In a fermentation process, induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, e.g., an OD550 of about 180-220, at which stage the cells are in the early stationary phase. A variety of inducers may be used, according to the vector construct employed, as is known in the art and described above. Cells may be grown for shorter periods prior to induction. Cells are usually induced for about 12-50 hours, although longer or shorter induction time may be used.

    [0307] To minimize proteolysis of expressed antigen binding polypeptides (especially those that are proteolytically sensitive), certain host strains deficient for proteolytic enzymes can be used for the present invention. For example, host cell strains may be modified to effect genetic mutation(s) in the genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof. Some E. coli protease-deficient strains are available and described in, for example, Joly et al. (1998), Proc. Natl. Acad. Sci. USA 95:2773-2777; Georgiou et al. , U.S. Patent No. 5,264,365; Georgiou et al., U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

    [0308] In one aspect, E. coli strains deficient for proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins are used as host cells in the expression system of the invention. In a second aspect, the E. coli strain is deficient for a lipoprotein of the outer membrane (ΔIρρ).

    iii. Antigen binding polypeptide purification



    [0309] In one aspect, the antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) of the present disclosure or a subunit thereof produced herein is further purified to obtain preparations that are substantially homogeneous for further assays and uses. Standard protein purification methods known in the art can be employed. The following procedures are exemplary of suitable purification procedures: fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

    [0310] In one aspect, Protein A immobilized on a solid phase is used for immunoaffinity purification of, for example, antigen binding polypeptides of the invention. Protein A is a 41 kD cell wall protein from Staphylococcus aureus which binds with a high affinity to the Fc region of antigen binding polypeptides. Lindmark et al. (1983) J. Immunol. Meth. 62:1 -13. The solid phase to which Protein A is immobilized is preferably a column comprising a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column. In some applications, the column has been coated with a reagent, such as glycerol, in an attempt to prevent nonspecific adherence of contaminants.

    [0311] As the first step of purification, the preparation derived from the cell culture as described above is applied onto the Protein A immobilized solid phase to allow specific binding of the antigen binding polypeptide of interest to Protein A. The solid phase is then washed to remove contaminants non-specifically bound to the solid phase. The antigen binding polypeptide (such as, for example, an antibody) is recovered from the solid phase by elution.

    b. Generating antigen binding polypeptides using eukaryotic host cells



    [0312] The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

    i. Signal sequence component



    [0313] A vector for use in a eukaryotic host cell may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available. The DNA for such precursor region is ligated in reading frame to DNA encoding the desired antigen binding polypeptide(s) (e.g., antibodies).

    ii. Origin of replication



    [0314] Generally, an origin of replication component is not needed for mammalian expression vectors. For example, the SV40 origin may typically be used, but only because it contains the early promoter.

    iii. Selection gene component



    [0315] Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, where relevant, or (c) supply critical nutrients not available from complex media.

    [0316] One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

    [0317] Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-l and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

    [0318] For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL- 9096).

    [0319] Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'- phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See, for example, U.S. Patent No. 4,965,199.

    iv. Promoter component



    [0320] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the desired Fc-containing polypeptide(s) (e.g., antibody) nucleic acid. Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.

    [0321] For production of Fc-containing polypeptide(s) (such as, for example, an antibody) transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as, for example, polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, or from heat-shock promoters, provided such promoters are compatible with the host cell systems.

    [0322] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hind 111 E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982) on expression of human β-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

    v. Enhancer element component



    [0323] Transcription of DNA encoding an antigen binding polypeptide(s) (such as, for example, an antibody) by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (e.g., globin, elastase, albumin, a-fetoprotein, and insulin genes). Also, one may use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) for a description of elements for enhancing activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5' or 3' to the antibody polypeptide-encoding sequence, provided that enhancement is achieved, but is generally located at a site 5' from the promoter.

    vi. Transcription termination component



    [0324] Expression vectors used in eukaryotic host cells will typically also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding an antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/1 1026 and the expression vector disclosed therein.

    vii. Selection and transformation of host cells



    [0325] Suitable host cells for cloning or expressing the DNA in the vectors herein include higher eukaryote cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

    [0326] Host cells are transformed with the above-described expression or cloning vectors for desired antigen binding polypeptide(s) (such as, for example, an antibody) production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

    viii. Culturing the host cells



    [0327] The host cells used to produce a desired antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) of the present disclosure or a subunit thereof may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

    ix. Purification of antigen binding polypeptides



    [0328] When using recombinant techniques, the antigen binding complexes (e.g., a tetravalent antigen binding complex having agonist activity) of the present disclosure or subunit(s) thereof can be produced intracellularly, or directly secreted into the medium. If the polypeptide is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Where the polypeptide is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

    [0329] The polypeptide composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1 , y2, or y4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human y3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABXresin (J. T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

    [0330] Following any preliminary purification step(s), the mixture comprising the polypeptide(s) of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt). The production of the antigen binding polypeptides can alternatively or additionally (to any of the foregoing particular methods) comprise dialyzing a solution comprising a mixture of the polypeptides.

    x. Antigen binding polypeptide production using baculovirus



    [0331] Recombinant baculovirus may be generated by co-transfecting a plasmid encoding an antigen binding polypeptide and BaculoGold virus DNA (Pharmingen) into an insect cell such as a Spodoptera frugiperda cell (e.g., Sf9 cells; ATCC CRL 1711) or a Drosophila melanogaster S2 cell using, for example, lipofectin (commercially available from GIBCO-BRL). In a particular example, an antigen binding polypeptide sequence is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-His tags. A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen) or pAcGP67B (Pharmingen). Briefly, the sequence encoding an antigen binding polypeptide may be amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product may then be digested with the selected restriction enzymes and subcloned into the expression vector.

    [0332] After transfection with the expression vector, the host cells (e.g., Sf9 cells) are incubated for 4-5 days at 28°C and the released virus is harvested and used for further amplifications. Viral infection and protein expression may be performed as described, for example, by O'Reilley et al. (Baculovirus expression vectors: A Laboratory Manual. Oxford: Oxford University Press (1994)).

    [0333] Expressed poly-His tagged antigen binding polypeptide can then be purified, for example, by Ni2+- chelate affinity chromatography as follows. Extracts can be prepared from recombinant virus-infected Sf9 cells as described by Rupert et al. (Nature 362:175-179 (1993)). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL HEPES pH 7.9; 12.5 mM MgCl2; 0.1 mM EDTA; 10% glycerol; 0.1 % NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate; 300 mM NaCl; 10% glycerol pH 7.8) and filtered through a 0.45 µm filter. A Ni2+-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water, and equilibrated with 25 mL of loading buffer. The filtered cell extract is loaded onto the column at 0.5 mL per minute. The column is washed to baseline A280 with loading buffer, at which point fraction collection is started. Next, the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM Nal; 10% glycerol pH 6.0), which elutes nonspecifically bound protein. After reaching A280 baseline again, the column is developed with a 0 to 500 mM Imidazole gradient in the secondary wash buffer. One mL fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni2+-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His10-tagged antigen binding polypeptide are pooled and dialyzed against loading buffer.

    [0334] Alternatively, purification of the antigen binding polypeptide can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography. In one aspect, the antigen binding polypeptide of interest may be recovered from the solid phase of the column by elution into a solution containing a chaotropic agent or mild detergent. Exemplary chaotropic agents and mild detergents include, but are not limited to, Guanidine-HCI, urea, lithium perclorate, Arginine, Histidine, SDS (sodium dodecyl sulfate), Tween, Triton, and NP-40, all of which are commercially available.

    [0335] THIOMAB antibodies are full length antibodies that include native cysteine residues that form disulfide bonds within the antibody. Accordingly, these native cysteine residues do not have any reactive thiol groups to conjugate with drug-maleimide (unless treated with a reducing agent). Hence, the newly engineered Cys residue, can remain unpaired, and able to react with, i.e. conjugate to, an electrophilic linker reagent or drug-linker intermediate, such as a drug-maleimide.

    [0336] Thiol reactivity may also be generalized to certain domains of an antibody, such as the light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. Cysteine replacements resulting in thiol reactivity values of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 0.95 and higher may be made in the heavy chain constant domains α, δ, ε, γ, and µ of intact antibodies: IgA, IgD, IgE, IgG, and IgM, respectively, including the IgG subclasses: IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

    Uses of OX40 Agonists



    [0337] In certain aspects, an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) of the present disclosure that binds to OX40 as described herein may be used for enhancing an immune response, treating cancer, preventing cancer, enhancing efficacy of other cancer therapy, enhancing vaccine efficacy, treating a viral or bacterial disease or disorder, or modulating a T cell response in a subject.

    [0338] In one aspect, provided is a method for enhancing immune function (e.g., by upregulating cell-mediated immune responses) in an individual having cancer comprising administering to the individual an effective amount of an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) that binds to OX40 as described herein. In one aspect, provided is a method for enhancing T cell function in an individual having cancer comprising administering to the individual an effective amount of an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) that binds to OX40 as described herein.

    [0339] In some aspects, "enhancing T cell function" includes inducing, causing or stimulating an effector or memory T cell to have a renewed, sustained or amplified biological function. Examples of enhancing T-cell function include: increased secretion of γ-interferon from CD8+ effector T cells, increased secretion of γ-interferon from CD4+ memory and/or effector T-cells, increased proliferation of CD4+ effector and/or memory T cells, increased proliferation of CD8+ effector T-cells, increased antigen responsiveness (e.g., clearance), relative to such levels before the intervention. In one aspect, the level of enhancement is at least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner of measuring this enhancement is known to one of ordinary skill in the art.

    [0340] In one aspect, provided is a method for enhancing immune function (e.g., by reducing immune dysfunction and/or a dysfunctional immune response or immune cell) in an individual having cancer comprising administering to the individual an effective amount of an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) that binds to OX40 as described herein. In some aspects, "dysfunction" in the context of immune dysfunction refers to a state of reduced immune responsiveness to antigenic stimulation. In some aspects, "dysfunctional" also includes refractory or unresponsive to antigen recognition, specifically, impaired capacity to translate antigen recognition into downstream T-cell effector functions, such as proliferation, cytokine production (e.g., gamma interferon) and/or target cell killing.

    [0341] In one aspect, provided is a method for treating tumor immunity and/or enhancing tumor immunogenicity in an individual having cancer comprising administering to the individual an effective amount of an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) that binds to OX40 as described herein. In some aspects, "tumor immunity" refers to the process in which tumors evade immune recognition and clearance. Thus, in some aspects, as a therapeutic concept, tumor immunity is "treated" when such evasion is attenuated, and the tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage and tumor clearance. In some aspects, "immunogenicity" refers to the ability of a particular substance to provoke an immune response. Tumors are immunogenic and enhancing tumor immunogenicity aids in the clearance of the tumor cells by the immune response.

    [0342] In some aspects, an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) that binds to OX40 as described herein enhances CD4+ effector T cell function, for example, by increasing CD4+ effector T cell proliferation and/or increasing gamma interferon production by the CD4+ effector T cell (for example, as compared to proliferation and/or cytokine production prior to treatment with an agonist antigen binding complex that binds to OX40). In some aspects, the cytokine is gamma interferon. In some aspects, an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) that binds to OX40 as described herein increases number of intratumoral (infiltrating) CD4+ effector T cells (e.g., total number of CD4+ effector T cells, or e.g., percentage of CD4+ cells in CD45+ cells), e.g., as compared to number of intratumoral (infiltrating) CD4+ T cells prior to treatment with an agonist antigen binding complex that binds to OX40. In some aspects, an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) that binds to OX40 as described herein increases number of intratumoral (infiltrating) CD4+ effector T cells that express gamma interferon (e.g., total gamma interferon expressing CD4+ cells, or e.g., percentage of gamma interferon expressing CD4+ cells in total CD4+ cells), e.g., as compared to number of intratumoral (infiltrating) CD4+ T cells that express gamma interferon prior to treatment with an agonist antigen binding complex that binds to OX40.

    [0343] In some aspects, an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) that binds to OX40 as described herein increases number of intratumoral (infiltrating) CD8+ effector T cells (e.g., total number of CD8+ effector T cells, or e.g., percentage of CD8+ in CD45+ cells), e.g., as compared to number of intratumoral (infiltrating) CD8+ T effector cells prior to treatment with anti-human OX40 agonist antibody. In some aspects, an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) that binds to OX40 as described herein increases the number of intratumoral (infiltrating) CD8+ effector T cells that express gamma interferon (e.g., percentage of CD8+ cells that express gamma interferon in total CD8+ cells), e.g., compared to number of intratumoral (infiltrating) CD8+ T cells that express gamma interferon prior to treatment with an agonist antigen binding complex that binds to OX40.

    [0344] In some aspects, an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) that binds to OX40 as described herein enhances memory T cell function, for example by increasing memory T cell proliferation and/or increasing cytokine production by the memory cell. In some aspects, the cytokine is gamma interferon.

    [0345] In some aspects, an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) that binds to OX40 as described herein inhibits Treg function, for example, by decreasing Treg suppression of effector T cell function (e.g., effector T cell proliferation and/or effector T cell cytokine secretion). In some aspects, the effector T cell is a CD4+ effector T cell. In some aspects, the anti-human OX40 agonist antigen binding complex reduces the number of intratumoral (infiltrating) Treg (e.g., total number of Treg or e.g., percentage of Fox3p+ cells in CD4+ cells).

    [0346] In one aspect, the present disclosure provides methods for enhancing an immune response in a mammal, comprising administering to the mammal a therapeutically effective amount of an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) that binds to OX40. In certain aspects, the methods involve stimulating, evoking, increasing, improving, or augmenting any response of a mammal's immune system. The immune response may be a cellular response (i.e. cell-mediated, such as cytotoxic T lymphocyte mediated) or a humoral response (i.e. antibody mediated response), and may be a primary or secondary immune response. Examples of enhancement of immune response include increased CD4+ helper T cell activity and generation of cytolytic T cells. The enhancement of immune response can be assessed using a number of in vitro or in vivo measurements known to those skilled in the art, including, but not limited to, cytotoxic T lymphocyte assays, release of cytokines (for example IL-2 production), regression of tumors, survival of tumor bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity. Typically, methods of the disclosure enhance the immune response by a mammal when compared to the immune response by an untreated mammal or an animal not treated using the claimed methods. In one aspect, the method enhances a cellular immune response, particularly a cytotoxic T cell response. In another aspect, the cellular immune response is a T helper cell response. In still another aspect, the immune response is a cytokine production, particularly IL-2 production.

    [0347] In another aspect, the present disclosure provides method of treating cancer in a mammal, comprising administering to the mammal a therapeutically effective amount of an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) that binds to OX40. In certain aspects, the methods involve causing a desirable or beneficial effect in a mammal diagnosed with a cancer. The desirable or beneficial effect may include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of reoccurrence of cancer, reduction of pain associated with the cancer, or improved survival of the animal. Inhibition of reoccurrence of cancer contemplates cancer sites and surrounding tissue which have previously been treated by radiation, chemotherapy, surgery, or other techniques. The effect can be either subjective or objective. For example, if the animal is human, the human may note improved vigor or vitality or decreased pain as subjective symptoms of improvement or response to therapy. Alternatively, the clinician may notice a decrease in tumor size or tumor burden based on physical exam, laboratory parameters, tumor markers or radiographic findings. Some laboratory signs that the clinician may observe for response to treatment include normalization of tests, such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. Additionally, the clinician may observe a decrease in a detectable tumor marker. Alternatively, other tests can be used to evaluate objective improvement, such as sonograms, nuclear magnetic resonance testing and positron emissions testing.

    [0348] In one aspect, the present disclosure provides methods for preventing cancer in a mammal, comprising administering to the mammal a therapeutically effective amount of an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) that binds to OX40. In certain aspects, the method involves delaying, inhibiting, or preventing the onset of a cancer in a mammal in which the onset of oncogenesis or tumorigenesis is not evidenced but a predisposition for cancer is identified whether determined by genetic screening or otherwise. The term also encompasses treating a mammal having premalignant conditions to stop the progression of, or cause regression of, the premalignant conditions towards malignancy. Examples of premalignant conditions include hyperplasia, dysplasia, and metaplasia.

    [0349] In certain aspects, cancers that are amenable to treatment by the antigen binding complexes (e.g., a tetravalent antigen binding complex having agonist activity) that bind to OX40 as described herein include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some aspects, the cancer is selected from: non-small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast carcinoma (e.g. triple-negative breast cancer), gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Yet, in some aspects, the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma and breast carcinoma (e.g. triple-negative breast cancer), including metastatic forms of those cancers. In some aspects, the cancer is Urothelial carcinoma (uBC), melanoma, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), renal, or bladder cancer.

    [0350] In some aspects, examples of cancer further include, but are not limited to, B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), B-cell proliferative disorders, and Meigs' syndrome. More specific examples include, but are not limited to, relapsed or refractory NHL, front line low grade NHL, Stage III/IV NHL, chemotherapy resistant NHL, precursor B lymphoblastic leukemia and/or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and/or prolymphocytic leukemia and/or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and/or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone-MALT lymphoma, nodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasma cell myeloma, low grade/follicular lymphoma, intermediate grade/follicular NHL, mantle cell lymphoma, follicle center lymphoma (follicular), intermediate grade diffuse NHL, diffuse large B-cell lymphoma, aggressive NHL (including aggressive front-line NHL and aggressive relapsed NHL), NHL relapsing after or refractory to autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, Burkitt's lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and/or Sezary syndrome, skin (cutaneous) lymphomas, anaplastic large cell lymphoma, angiocentric lymphoma.

    [0351] In some aspects, examples of cancer further include, but are not limited to, B-cell proliferative disorders, which further include, but are not limited to, lymphomas (e.g., B-Cell Non-Hodgkin's lymphomas (NHL)) and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include e.g. a) follicular lymphomas, b) Small Non-Cleaved Cell Lymphomas/ Burkitt's lymphoma (including endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma and Non-Burkitt's lymphoma), c) marginal zone lymphomas (including extranodal marginal zone B-cell lymphoma (Mucosa-associated lymphatic tissue lymphomas, MALT), nodal marginal zone B-cell lymphoma and splenic marginal zone lymphoma), d) Mantle cell lymphoma (MCL), e) Large Cell Lymphoma (including B-cell diffuse large cell lymphoma (DLCL), Diffuse Mixed Cell Lymphoma, Immunoblastic Lymphoma, Primary Mediastinal B-Cell Lymphoma, Angiocentric Lymphoma-Pulmonary B-Cell Lymphoma), f) hairy cell leukemia, g) lymphocytic lymphoma, Waldenstrom's macroglobulinemia, h) acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL)/ small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, i) plasma cell neoplasms, plasma cell myeloma, multiple myeloma, plasmacytoma, and/or j) Hodgkin's disease.

    [0352] In some aspects of any of the methods, the cancer is a B-cell proliferative disorder. In some aspects, the B-cell proliferative disorder is lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), or mantle cell lymphoma. In some aspects, the B-cell proliferative disorder is NHL, such as indolent NHL and/or aggressive NHL. In some aspects, the B-cell proliferative disorder is indolent follicular lymphoma or diffuse large B-cell lymphoma.

    [0353] In some aspects of any of the methods of the invention, the cancer displays human effector cells (e.g., is infiltrated by human effector cells). Methods for detecting human effector cells are well known in the art, including, e.g., by IHC. In some aspects, the cancer display high levels of human effector cells. In some aspects, human effector cells are one or more of NK cells, macrophages, monocytes. In some aspects, the cancer is any cancer described herein. In some aspects, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast carcinoma (e.g. triple-negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.

    [0354] Antigen binding complexes (e.g., a tetravalent antigen binding complex having agonist activity) described herein can be used either alone or in combination with other agents in a therapy. For instance, an antigen binding complex (e.g., a tetravalent antigen binding complex having agonist activity) described herein may be co-administered with at least one additional therapeutic agent.

    [0355] Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antigen binding polypeptide (e.g., antibody) or complex described herein can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent or agents. In one aspect, administration of the antigen binding polypeptide (e.g., antibody) or complex described herein and administration of an additional therapeutic agent occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other. Antigen binding polypeptides (e.g., antibodies) or complexes described herein can also be used in combination with radiation therapy.

    [0356] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a chemotherapy or chemotherapeutic agent. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a radiation therapy or radiotherapeutic agent. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a targeted therapy or targeted therapeutic agent. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an immunotherapy or immunotherapeutic agent, for example a monoclonal antibody.

    [0357] "Chemotherapeutic agent" includes chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech/OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), disulfiram, epigallocatechin gallate , salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca), sunitib (SUTENT®, Pfizer/Sugen), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), finasunate (VATALANIB®, Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin, Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), Lonafamib (SCH 66336), sorafenib (NEXAVAR®, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), AG1478, alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including topotecan and irinotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); adrenocorticosteroids (including prednisone and prednisolone); cyproterone acetate; 5α-reductases including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat dolastatin; aldesleukin, talc duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ω1I (Angew Chem. Intl. Ed. Engl. 1994 33:183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® (docetaxel, doxetaxel; Sanofi-Aventis); chloranmbucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

    [0358] Chemotherapeutic agent also includes (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, iodoxyfene , 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all transretionic acid, fenretinide, as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN®, rIL-2; a topoisomerase 1 inhibitor such as LURTOTECAN®; ABARELIX® rmRH; and (ix) pharmaceutically acceptable salts, acids and derivatives of any of the above.

    [0359] Chemotherapeutic agent also includes antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth). Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and the anti-interleukin-12 (ABT-874/J695, Wyeth Research and Abbott Laboratories) which is a recombinant exclusively human-sequence, full-length IgG1 λ antibody genetically modified to recognize interleukin-12 p40 protein.

    [0360] Chemotherapeutic agent also includes "EGFR inhibitors," which refers to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity, and is alternatively referred to as an "EGFR antagonist." Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, US Patent No. 4,943, 533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBUTIX®) and reshaped human 225 (H225) (see, WO 96/40210, Imclone Systems Inc.); IMC-11F8, a fully human, EGFR-targeted antibody (Imclone); antibodies that bind type II mutant EGFR (US Patent No. 5,212,290); humanized and chimeric antibodies that bind EGFR as described in US Patent No. 5,891,996; and human antibodies that bind EGFR, such as ABX-EGF or Panitumumab (see WO98/50433, Abgenix/Amgen); EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF-alpha for EGFR binding (EMD/Merck); human EGFR antibody, HuMax-EGFR (GenMab); fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6. 3 and E7.6. 3 and described in US 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanized mAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). The anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g., EP659,439A2, Merck Patent GmbH). EGFR antagonists include small molecules such as compounds described in US Patent Nos: 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, as well as the following PCT publications: WO98/14451, WO98/50038, WO99/09016, and WO99/24037. Particular small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech/OSIPharmaceuticals); PD 183805 (CI 1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®) 4-(3'-Chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol); (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine); CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU 5271; Pfizer); dual EGFR/HER2 tyrosine kinase inhibitors such as lapatinib (TYKERB®, GSK572016 or N-[3-chloro-4-[(3 fluorophenyl)methoxy]phenyl]-6[5[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).

    [0361] Chemotherapeutic agents also include "tyrosine kinase inhibitors" including the EGFR-targeted drugs noted in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724,714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisense agent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1 signaling; non-HER targeted TK inhibitors such as imatinib mesylate (GLEEVEC®, available from Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib (SUTENT®, available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787/ZK222584, available from Novartis/Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035,4-(3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrol opyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d] pyrimidines; curcumin (diferuloyl methane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lamber); antisense molecules (e.g. those that bind to HER-encoding nucleic acid); quinoxalines (US Patent No. 5,804,396); tryphostins (US Patent No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); imatinib mesylate (GLEEVEC®); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone), rapamycin (sirolimus, RAPAMUNE®); or as described in any of the following patent publications: US Patent No. 5,804,396; WO 1999/09016 (American Cyanamid); WO 1998/43960 (American Cyanamid); WO 1997/38983 (Warner Lambert); WO 1999/06378 (Warner Lambert); WO 1999/06396 (Warner Lambert); WO 1996/30347 (Pfizer, Inc); WO 1996/33978 (Zeneca); WO 1996/3397 (Zeneca) and WO 1996/33980 (Zeneca).

    [0362] Chemotherapeutic agents also include dexamethasone, interferons, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, and pharmaceutically acceptable salts thereof.

    [0363] Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate and fluprednidene acetate; immune selective anti-inflammatory peptides (ImSAIDs) such as phenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG) (IMULAN BioTherapeutics, LLC); anti-rheumatic drugs such as azathioprine, ciclosporin (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomideminocycline, sulfasalazine, tumor necrosis factor alpha (TNFα) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), golimumab (Simponi), Interleukin 1 (IL-1) blockers such as anakinra (Kineret), T cell costimulation blockers such as abatacept (Orencia), Interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA®); Interleukin 13 (IL-13) blockers such as lebrikizumab; Interferon alpha (IFN) blockers such as Rontalizumab; Beta 7 integrin blockers such as rhuMAb Beta7; IgE pathway blockers such as Anti-M1 prime; Secreted homotrimeric LTa3 and membrane bound heterotrimer LTa1/β2 blockers such as Anti-lymphotoxin alpha (LTa); radioactive isotopes (e.g., At211, 1131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu); miscellaneous investigational agents such as thioplatin, PS-341, phenylbutyrate, ET-18- OCH3, or farnesyl transferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechine gallate, theaflavins, flavanols, procyanidins, betulinic acid and derivatives thereof; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin); podophyllotoxin; tegafur (UFTORAL®); bexarotene (TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine; perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome inhibitor (e.g. PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone; farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASARTM); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATINTM) combined with 5-FU and leucovorin.

    [0364] Chemotherapeutic agents also include non-steroidal anti-inflammatory drugs with analgesic, antipyretic and anti-inflammatory effects. NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase. Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, rofecoxib, and valdecoxib. NSAIDs can be indicated for the symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthropathies, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhoea, metastatic bone pain, headache and migraine, postoperative pain, mild-to-moderate pain due to inflammation and tissue injury, pyrexia, ileus, and renal colic.

    [0365] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a PARP inhibitor (e.g., Olaparanib, Rucaparib, Niraparib, Cediranib, BMN673, Veliparib), Trabectedin, nab-paclitaxel (albumen-bound paclitaxel, ABRAXANE), Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine (e.g., FOLFOX, FOLFIRI), IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, Torisel (temsirolimus), Inlyta (axitinib, Pfizer), Afinitor (everolimus, Novartis), Nexavar (sorafenib, Onyx / Bayer), Votrient, Pazopanib, axitinib, IMA-901, AGS-003, cabozantinib, Vinflunine, Hsp90 inhibitor (e.g., apatorsin), Ad-GM-CSF (CT-0070), Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid (VELCADE), amrubicine, carfilzomib, pralatrexate, and/or enzastaurin.

    [0366] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a PD-1 axis binding antagonist. A PD-1 axis binding antagonist includes but is not limited to a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist. Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PD-L2" include B7-DC, Btdc, and CD273. In some aspects, PD-1, PD-L1, and PD-L2 are human PD-1 , PD-L1 and PD-L2. In some aspects, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect the PD-1 ligand binding partners are PD-L1 and/or PD-L2. In another aspect, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific aspect, PD-L1 binding partners are PD-1 and/or B7- 1. In another aspect, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partners. In a specific aspect, a PD-L2 binding partner is PD-1. The antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. In some aspects, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some aspects, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (nivolumab, OPDIVO), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA), CT- 011 (Pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, BGB-108, and BGB-A317. In some aspects, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some aspects, the PD-1 binding antagonist is AMP-224. In some aspects, the PD-L1 binding antagonist is anti-PD-Ll antibody. In some aspects, the anti-PD-L1 binding antagonist is selected from the group consisting of YW243.55.S70, MPDL3280A (atezolizumab), MEDI4736 (durvalumab), MDX-1105, and MSB0010718C (avelumab). MDX-1105, also known as BMS-936559, is an anti-PD-Ll antibody described in WO2007/005874. Antibody YW243.55.S70 (heavy and light chain variable region sequences shown in SEQ ID Nos. 20 and 21, respectively) is an anti-PD-Ll described in WO 2010/077634 A1. MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558 or nivolumab, is an anti-PD-1 antibody described in WO2006/121168. Merck 3475, also known as MK-3475, SCH-900475 or pembrolizumab, is an anti-PD-1 antibody described in WO2009/114335. CT-011, also known as hBAT, hBAT-1 or pidilizumab, is an anti- PD-1 antibody described in WO2009/101611. AMP-224, also known as B7-DCIg, is a PD-L2- Fc fusion soluble receptor described in WO2010/027827 and WO201 1/066342. In some aspects, the anti-PD-1 antibody is MDX- 1106. Alternative names for "MDX- 1106" include MDX-1 106-04, ONO-4538, BMS-936558 or nivolumab. In some aspects, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4).

    [0367] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an agonist directed against an activating co-stimulatory molecule. In some aspects, an activating co-stimulatory molecule may include CD40, CD226, CD28, GITR, CD137, CD27, HVEM, or CD127. In some aspects, the agonist directed against an activating co-stimulatory molecule is an agonist antibody that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antagonist directed against an inhibitory co-stimulatory molecule. In some aspects, an inhibitory co-stimulatory molecule may include CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase. In some aspects, the antagonist directed against an inhibitory co-stimulatory molecule is an antagonist antibody that binds to CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3 (e.g., LAG-3-IgG fusion protein (IMP321)), B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase.

    [0368] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antagonist directed against CTLA-4 (also known as CD152), e.g., a blocking antibody. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with ipilimumab (also known as MDX-010, MDX-101, or Yervoy®). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with tremelimumab (also known as ticilimumab or CP-675,206). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antagonist directed against B7-H3 (also known as CD276), e.g., a blocking antibody. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with MGA271. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antagonist directed against a TGF beta, e.g., metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299.

    [0369] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a treatment comprising adoptive transfer of a T cell (e.g., a cytotoxic T cell or CTL) expressing a chimeric antigen receptor (CAR). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with UCART19. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with WT128z. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with KTE-C19 (Kite). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with CTL019 (Novartis). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a treatment comprising adoptive transfer of a T cell comprising a dominant-negative TGF beta receptor, e.g, a dominant-negative TGF beta type II receptor. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a treatment comprising a HERCREEM protocol (see, e.g., ClinicalTrials.gov Identifier NCT00889954).

    [0370] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antagonist directed against CD19. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with MOR00208. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antagonist directed against CD38. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with daratumumab.

    [0371] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an agonist directed against CD137 (also known as TNFRSF9, 4-1BB, or ILA), e.g., an activating antibody. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with urelumab (also known as BMS-663513). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an agonist directed against CD40, e.g., an activating antibody. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with CP-870893. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an agonist directed against OX40 (also known as CD134), e.g., an activating antibody. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a different anti-OX40 antibody (e.g., AgonOX). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an agonist directed against CD27, e.g., an activating antibody. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with CDX-1127. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antagonist directed against indoleamine-2,3-dioxygenase (IDO). In some aspects, with the IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT).

    [0372] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an agonist directed against CD137 (also known as TNFRSF9, 4-1BB, or ILA), e.g., an activating antibody. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with urelumab (also known as BMS-663513). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an agonist directed against CD40, e.g., an activating antibody. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with CP-870893 or RO7009789. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an agonist directed against OX40 (also known as CD134), e.g., an activating antibody.). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an agonist directed against CD27, e.g., an activating antibody. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with CDX-1127 (also known as varlilumab). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antagonist directed against indoleamine-2,3-dioxygenase (IDO). In some aspects, with the IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT). In some aspects, the IDO antagonist is an IDO antagonist shown in WO2010/005958. In some aspects the IDO antagonist is 4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide (e.g., as described in Example 23 of WO2010/005958). In some aspects the IDO antagonist is



    [0373] In some aspects, the IDO antagonist is INCB24360. In some aspects, the IDO antagonist is Indoximod (the D isomer of 1-methyl-tryptophan). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antibody-drug conjugate. In some aspects, the antibody-drug conjugate comprises mertansine or monomethyl auristatin E (MMAE). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A, RG7599 or lifastuzumab vedotin). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine, or KADCYLA®, Genentech). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an anti-MUC16 antibody-MMAE conjugate, DMUC5754A. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an anti-MUC16 antibody-MMAE conjugate, DMUC4064A. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antibody-drug conjugate targeting the endothelin B receptor (EDNBR), e.g., an antibody directed against EDNBR conjugated with MMAE. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antibody-drug conjugate targeting the lymphocyte antigen 6 complex, locus E (Ly6E), e.g., an antibody directed against Ly6E conjugated with MMAE, (also known as DLYE5953A). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with polatuzumab vedotin. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antibody-drug conjugate targeting CD30. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with ADCETRIS (also known as brentuximab vedotin). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with polatuzumab vedotin.

    [0374] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an angiogenesis inhibitor. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antibody directed against a VEGF, e.g., VEGF-A. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with bevacizumab (also known as AVASTIN®, Genentech). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antibody directed against angiopoietin 2 (also known as Ang2). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with MEDI3617. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antibody directed against VEGFR2. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with ramucirumab. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a VEGF Receptor fusion protein. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with aflibercept. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with ziv-aflibercept (also known as VEGF Trap or Zaltrap®). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a bispecific antibody directed against VEGF and Ang2. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with RG7221 (also known as vanucizumab). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an angiogenesis inhibitor and in conjunction with a PD-1 axis binding antagonist (e.g., a PD-1 binding antagonist such as an anti-PD-1 antibody, a PD-L1 binding antagonist such as an anti-PD-Ll antibody, and a PD-L2 binding antagonist such as an anti-PD-L2 antibody). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with bevacizumab and a PD-1 axis binding antagonist (e.g., a PD-1 binding antagonist such as an anti-PD-1 antibody, a PD-L1 binding antagonist such as an anti-PD-Ll antibody, and a PD-L2 binding antagonist such as an anti-PD-L2 antibody). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with bevacizumab and MDX-1106 (nivolumab, OPDIVO). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with bevacizumab and Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with bevacizumab and CT-011 (Pidilizumab). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with bevacizumab and MEDI-0680 (AMP-514). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with bevacizumab and PDR001. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with bevacizumab and REGN2810. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with bevacizumab and BGB-108. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with bevacizumab and BGB-A317. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with bevacizumab and YW243.55.S70. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with bevacizumab and MPDL3280A. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with bevacizumab and MEDI4736. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with bevacizumab and MDX-1105. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with bevacizumab and MSB0010718C (avelumab).

    [0375] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antineoplastic agent. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an agent targeting CSF-1R (also known as M-CSFR or CD115). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with anti-CSF-1R antibody (also known as IMC-CS4 or LY3022855) In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with anti-CSF-1R antibody, RG7155 (also known as RO5509554 or emactuzumab). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an interferon, for example interferon alpha or interferon gamma. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with Roferon-A (also known as recombinant Interferon alpha-2a). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with GM-CSF (also known as recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, sargramostim, or Leukine®). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with IL-2 (also known as aldesleukin or Proleukin®). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with IL-12. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with IL27. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with IL-15. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with ALT-803. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antibody targeting CD20. In some aspects, the antibody targeting CD20 is obinutuzumab (also known as GA101 or Gazyva®) or rituximab. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antibody targeting GITR. In some aspects, the antibody targeting GITR is TRX518. In some aspects, the antibody targeting GITR is MK04166 (Merck).

    [0376] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of Bruton's tyrosine kinase (BTK). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with ibrutinib. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of Isocitrate dehydrogenase 1 (IDH1) and/or Isocitrate dehydrogenase 2 (IDH2). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with AG-120 (Agios).

    [0377] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with obinutuzumab and a PD-1 axis binding antagonist (e.g., a PD-1 binding antagonist such as an anti-PD-1 antibody, a PD-L1 binding antagonist such as an anti-PD-Ll antibody, and a PD-L2 binding antagonist such as an anti-PD-L2 antibody).

    [0378] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a cancer vaccine. In some aspects, the cancer vaccine is a peptide cancer vaccine, which in some aspects is a personalized peptide vaccine. In some aspects the peptide cancer vaccine is a multivalent long peptide, a multi-peptide, a peptide cocktail, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci, 104:14-21, 2013). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an adjuvant. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a treatment comprising a TLR agonist, e.g., Poly-ICLC (also known as Hiltonol®), LPS, MPL, or CpG ODN. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with tumor necrosis factor (TNF) alpha. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with IL-1. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with HMGB1. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an IL-10 antagonist. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an IL-4 antagonist. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an IL-13 antagonist. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an IL-17 antagonist. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an HVEM antagonist. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an ICOS agonist, e.g., by administration of ICOS-L, or an agonistic antibody directed against ICOS. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a treatment targeting CX3CL1. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a treatment targeting CXCL9. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a treatment targeting CXCL10. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a treatment targeting CCL5. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an LFA-1 or ICAM1 agonist. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a Selectin agonist.

    [0379] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of B-Raf. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with vemurafenib (also known as Zelboraf®). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with dabrafenib (also known as Tafinlar®). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with encorafenib (LGX818).

    [0380] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an EGFR inhibitor. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with erlotinib (also known as Tarceva®). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of EGFR-T790M. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with gefitinib. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with afatinib. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with cetuximab (also known as Erbitux®). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with panitumumab (also known as Vectibix®). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with rociletinib. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with AZD9291. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of a MEK, such as MEK1 (also known as MAP2K1) and/or MEK2 (also known as MAP2K2). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with cobimetinib (also known as GDC-0973 or XL-518). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with trametinib (also known as Mekinist®). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with binimetinib.

    [0381] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction an inhibitor of B-Raf (e.g., vemurafenib or dabrafenib) and an inhibitor of MEK (e.g., MEK1 and/or MEK2 (e.g., cobimetinib or trametinib). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of ERK (e.g., ERK1/2). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with GDC-0994). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of B-Raf, an inhibitor of MEK, and an inhibitor of ERK1/2. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of EGFR, an inhibitor of MEK, and an inhibitor of ERK1/2. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with one or more MAP kinase pathway inhibitor. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with CK127. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of K-Ras.

    [0382] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of c-Met. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with onartuzumab (also known as MetMAb). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of anaplatic lymphoma kinase (ALK). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with AF802 (also known as CH5424802 or alectinib). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with crizotinib. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with ceritinib. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of a phosphatidylinositol 3-kinase (PI3K). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjuction with buparlisib (BKM-120). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with pictilisib (also known as GDC-0941). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with buparlisib (also known as BKM-120). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with perifosine (also known as KRX-0401). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a delta-selective inhibitor of a phosphatidylinositol 3-kinase (PI3K). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with idelalisib (also known as GS-1101 or CAL-101). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with taselisib (also known as GDC-0032). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with BYL-719. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of an Akt. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with MK2206. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with GSK690693. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with ipatasertib (also known as GDC-0068). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of mTOR. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with sirolimus (also known as rapamycin). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with temsirolimus (also known as CCI-779 or Torisel®). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with everolimus (also known as RAD001). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with ridaforolimus (also known as AP-23573, MK-8669, or deforolimus). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with OSI-027. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with AZD8055. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with INK128. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with a dual PI3K/mTOR inhibitor. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with XL765. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with GDC-0980. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with BEZ235 (also known as NVP-BEZ235). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with BGT226. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with GSK2126458. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with PF-04691502. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with PF-05212384 (also known as PKI-587).

    [0383] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an agent that selectively degrades the estrogen receptor. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with GDC-0927. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of HER3. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with duligotuzumab. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of LSD1. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of MDM2. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of BCL2. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with venetoclax. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of CHK1. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with GDC-0575. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an inhibitor of activated hedgehog signaling pathway. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with ERIVEDGE.

    [0384] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with radiation therapy. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with gemcitabine. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with nab-paclitaxel (ABRAXANE). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with trastuzumab. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with TVEC. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with IL27. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with cyclophosphamide. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an agent that recruits T cells to the tumor. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with lirilumab (IPH2102/BMS-986015). In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with Idelalisib. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antibody that targets CD3 and CD20. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with REGN1979. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an antibody that targets CD3 and CD19. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with blinatumomab.

    [0385] In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with an oncolytic virus. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with carboplatin and nab-paclitaxel. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with carboplatin and paclitaxel. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with cisplatin and pemetrexed. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with cisplatin and gemcitabine. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with FOLFOX. In some aspects, an antigen binding polypeptide (e.g., antibody) or complex described herein may be administered in conjunction with FOLFIRI.

    [0386] Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antigen binding polypeptide (e.g., antibody) or complex described herein can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant. Antibodies or complexes of the invention can also be used in combination with radiation therapy.

    [0387] An antibody or complex of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

    [0388] Antibodies or complexes of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody or complex need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

    [0389] For the prevention or treatment of disease, the appropriate dosage of an antibody or complex of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody or complex is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 µg/kg to 40 mg/kg of antibody or complex can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 µg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

    Screening Assays



    [0390] Also provided herein are methods for identifying polypeptides that have agonist activity. In particular, an antigen binding polypeptide may not have agonist activity when expressed as an individual polypeptide (e.g., an individual antibody, antibody fragment, ligand, etc.), however, when the same polypeptide is presented in the context of a tetravalent complex as described herein, the complex may exhibit agonist activity. Therefore, by screening for agonist activity of individual polypeptides, there may be a number of candidates that are discarded as false negatives, e.g., polypeptides that have the ability to act as an agonist when contained in a complex but do not exhibit such activity when presented in isolated form. Therefore, the antigen binding complexes as described herein may be used in an initial screen of candidate polypeptides to identify those having agonist activity.

    [0391] Accordingly, in certain aspects, the present disclosure provides novel methods for identifying an antigen binding complex (e.g., a tetravalent antigen binding complex) having agonist activity. The methods include providing a plurality of antigen binding complexes (e.g., a tetravalent antigen binding complex having agonist activity) as described herein, screening the antigen binding complexes against a cell surface receptor, and selecting antigen binding complexes having agonist activity for the cell surface receptor. In certain aspects, the antigen binding complexes may be provided as libraries of antigen binding complexes whose amino acid sequences differ from each other. Such libraries provide a tremendously useful resource for identifying antigen binding complex which bind to the cell surface receptor and has agonist activity for the cell surface receptor.

    [0392] In certain aspects, the antigen binding complexes useful in such a screening assay may be a library wherein each antigen binding complex is tetravalent. In some aspects, the complex is monospecific or monoepitopic, wherein each antigen binding polypeptide in a given complex binds the same epitope, and each tetravalent complex contains a different antibody that binds to the same target, e.g., essentially a library of complexes of monospecific antibodies raised to a given cell surface target. In some aspects, the complex is biepitopic, wherein each complex comprises two or more antigen binding polypeptides that bind different epitopes (e.g., of the same target), and each tetravalent complex targets a different combination of epitopes, e.g., essentially a library of complexes of monospecific antibodies raised to a given cell surface target. Such libraries would be useful, for example, for identifying an antibody (or antigen binding complex) that binds to cell surface receptor and agonizes the receptor.

    [0393] The antigen binding complexes or the antigen binding polypeptides described herein can be characterized for their physical/chemical properties and biological functions by various assays known in the art. For example, as exemplified herein, complex formation may be assayed, e.g., using Size Exclusion Chromatography (SEC) to monitor the formation and/or purity of complexes.

    [0394] In certain aspects, antigen binding complexes or antigen binding polypeptides can be characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non- denaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain digestion.

    [0395] In certain aspects, antigen binding complexes or antigen binding polypeptides may be analyzed for biological activity, such as, for example, antigen binding activity. Antigen binding assays are known in the art and can be used herein including, for example, any direct or competitive binding assays using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immnosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, fluorescent immunoassays, and protein A immunoassays.

    [0396] In another aspect, competition assays may be used to identify an antibody that competes with a reference antibody for binding to a target. In certain aspects, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by the reference antibody. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

    [0397] In an exemplary competition assay, an immobilized target polypeptide is incubated in a solution comprising a first labeled antibody that binds to a target polypeptide (e.g., OX40) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to the target polypeptide. The second antibody may be present in a hybridoma supernatant. As a control, immobilized target polypeptide is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to the target polypeptide, excess unbound antibody is removed, and the amount of label associated with immobilized target polypeptide is measured. If the amount of label associated with immobilized target polypeptide is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to target polypeptide. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

    [0398] In other aspects, the antigen binding complexes or antigen binding polypeptides described herein may be analyzed for agonist activity. In some aspects, the agonist activity is tested in vitro (e.g., in a cell-free or cell-based assay, as opposed to an in vivo assay in an intact mammal). In certain aspects, agonist activity of the antigen binding complexes or antigen binding polypeptides described herein can be determined by analyzing whether or not a cell that is depending on a ligand for growth will grow in the same way when an antigen binding complex or polypeptide is added during cell culture as compared to when a ligand is added. If the cell grows in the same or in a similar manner, then the antigen binding complex or polypeptide is determined to have agonistic activity. In certain aspects, agonist activity of the antigen binding complexes or antigen binding polypeptides described herein can be determined by analyzing whether or not a cell line having intrinsic ligand-dependent activities (not limited to growth) shows the same reaction when an antigen binding complex or polypeptide is added during cell culture as compared to when the ligand is added. If the cell line shows the same or a similar reaction as for the ligand, then the antigen binding complex or polypeptide is determined to have agonistic activity.

    [0399] In certain aspects, cells capable of transducing the above-mentioned cell growth signals express the receptors responsive to the ligand on the cell surface. These cells transduce cell growth signals when the ligand (or agonist antigen binding complex or polypeptide) binds to the receptor. In certain aspects, cells useful for screening for agonist activity proliferate or transduce a signal upon binding of a ligand to a cell surface receptor on the cell. In other aspects, when the cell surface receptor is one that does not transduce a signal into the cell, then chimeric receptors made by fusing the extracellular domain (e.g., ligand binding domain) of a non-transducing receptor to the intracellular domain of a receptor that does transduce a signal into the cell. Receptors suitable for constructing chimeric receptors by fusion with ligand-binding receptors include any receptor that transduces a signal, including, for example, the G-CSF receptor, mpl, neu, GM-CSF receptor, EPO receptor, c-Kit, and FLT-3 receptors. Cells used to express such receptors include, for example, BaF3, NFS60, FDCP-1, FDCP-2, CTLL-2, DA-1, and KT-3.

    [0400] In certain aspects, agonistic activity refers to any activity caused by ligand (or antigen binding complex or polypeptide) binding that induces a specific reaction in a cell, such as, for example, inducing a change in a certain physiological activity by transmitting a signal into a cell. Such physiological activities include, for example, growth activities, growth-inducing activities, survival activities, differentiation activities, differentiation-inducing activities, transcriptional activities, membrane transport activities, binding activities, proteolytic activities, phosphorylation/dephosphorylation activities, oxidation-reduction activities, transfer activities, nucleolytic activities, dehydration activities, cell death-inducing activities, and apoptosis-inducing activities.

    [0401] The agonistic activities described herein can be determined by methods known to those skilled in the art. For example, agonistic activity can be evaluated by methods which use cell growth as an indicator. More specifically, an antigen binding complex or polypeptide whose agonistic activity is to be determined is added to cells that show agonist-dependent growth, and the cells are cultured. Next, a reagent that shows a color reaction at a particular wavelength depending on viable cell count, such as WST-8, is added, and the absorbance is measured. The agonistic activity can be determined using the measured absorbance as an indicator.

    [0402] In certain aspects, agonist activity is determined using an indicator that can monitor quantitative and/or qualitative changes in the cell upon exposure to a ligand (or antigen binding complex or polypeptide). For example, it is possible to use cell-free assay indicators, cell-based assay indicators, tissue-based assay indicators, and in vivo assay indicators. Indicators that can be used in cell-free assays include enzymatic reactions, quantitative and/or qualitative changes in proteins, DNAs, or RNAs. Such enzymatic reactions include, for example, amino acid transfers, sugar transfers, dehydrations, dehydrogenations, and substrate cleavages. Alternatively, protein phosphorylations, dephosphorylations, dimerizations, multimerizations, hydrolyses, and dissociations; DNA or RNA amplifications, cleavages, and extensions can be used as the indicator in cell-free assays. For example, protein phosphorylations downstream of a signal transduction pathway may be used as a detection indicator. Alterations in cell phenotype, for example, quantitative and/or qualitative alterations in products, alterations in growth activity, alterations in cell number, morphological alterations, or alterations in cellular properties, can be used as the indicator in cell-based assays. The products include, for example, secretory proteins, surface antigens, intracellular proteins, and mRNAs. The morphological alterations include, for example, alterations in dendrite formation and/or dendrite number, alteration in cell flatness, alteration in cell elongation/axial ratio, alterations in cell size, alterations in intracellular structure, heterogeneity/homogeneity of cell populations, and alterations in cell density. Such morphological alterations can be observed under a microscope. Cellular properties to be used as the indicator include anchor dependency, cytokine-dependent response, hormone dependency, drug resistance, cell motility, cell migration activity, pulsatory activity, and alteration in intracellular substances. Cell motility includes cell infiltration activity and cell migration activity. The alterations in intracellular substances include, for example, alterations in enzyme activity, mRNA levels, levels of intracellular signaling molecules such as Ca2+ and cAMP, and intracellular protein levels. When a cell membrane receptor is used, alterations in the cell proliferating activity induced by receptor stimulation can be used as the indicator. The indicators to be used in tissue-based assays include functional alterations adequate for the subject tissue. In in vivo assays, alterations in tissue weight, alterations in the blood system (for example, alterations in blood cell counts, protein contents, or enzyme activities), alterations in electrolyte levels, and alterations in the circulating system (for example, alterations in blood pressure or heart rate).

    [0403] Any suitable method for measuring such detection indicators may be used in connection with the methods described herein. For example, absorbance, luminescence, color development, fluorescence, radioactivity, fluorescence polarization, surface plasmon resonance signal, time-resolved fluorescence, mass, absorption spectrum, light scattering, and fluorescence resonance energy transfer may be used. These measurement methods are known to those skilled in the art and may be selected appropriately depending on the purpose. For example, absorption spectra can be obtained by using a conventional photometer, plate reader, or such; luminescence can be measured with a luminometer or such; and fluorescence can be measured with a fluorometer or such. Mass can be determined with a mass spectrometer. Radioactivity can be determined with a device such as a gamma counter depending on the type of radiation. Fluorescence polarization can be measured with BEACON (TaKaRa). Surface plasmon resonance signals can be obtained with BIACORE. Time-resolved fluorescence, fluorescence resonance energy transfer, or such can be measured with ARVO or such. Furthermore, a flow cytometer can also be used for measurements. It is possible to use one of the above methods to measure two or more different types of detection indicators. A greater number of detection indicators may also be examined by using two or more measurement methods simultaneously and/or consecutively. For example, fluorescence and fluorescence resonance energy transfer can be measured at the same time with a fluorometer.

    OX40 Assays



    [0404] As described above, certain aspects of the present disclosure relate to agonist activity for a cell surface receptor. As will be recognized by one of skill in the art, the particular assay(s) used to determine agonist activity for a cell surface receptor may depend upon the particular cell surface receptor. Exemplary assays related to determining OX40 activity are provided below. Based on this guidance and common knowledge in the art, one of skill in the art may suitably identify assays for other cell surface receptors described herein.

    [0405] In one aspect, assays are provided for identifying an agonist antigen binding complex that binds to OX40 having biological activity. Biological activity may include, e.g., binding OX40 (e.g., binding human and/or cynomolgus OX40), increasing OX40-mediated signal transduction (e.g., increasing NFkB-mediated transcription), depleting cells that express human OX40 (e.g., T cells), depleting cells that express human OX40 by ADCC and/or phagocytosis, enhancing T effector cell function (e.g., CD4+ effector T cell), e.g., by increasing effector T cell proliferation and/or increasing cytokine production (e.g., gamma interferon) by effector T cells, enhancing memory T cell function (e.g., CD4+ memory T cell), e.g., by increasing memory T cell proliferation and/or increasing cytokine production by memory T cells (e.g., gamma interferon), or inhibiting regulatory T cell function (e.g., by decreasing Treg suppression of effector T cell function (e.g., CD4+ effector T cell function).

    [0406] In certain aspects, the agonist antigen binding complex that binds to OX40 has one or more of the listed biological activities in the absence of binding to human effector cells. In certain aspects, the agonist antigen binding complex that binds to OX40 has one or more of the listed biological activities and does bind to human effector cells. Antibodies having such biological activity in vivo and/or in vitro are also provided.

    [0407] In certain aspects, an antibody of the invention is tested for such biological activity.

    [0408] T cell costimulation may be assayed using methods known in the art and exemplary methods are disclosed herein. For example, T cells (e.g., memory or effector T cells) may be obtained from peripheral white blood cells (e.g., isolated from human whole blood using Ficoll gradient centrifugation). Memory T cells (e.g., CD4+ memory T cells) or effector T cells (e.g. CD4+ Teff cells) may be isolated from PBMC using methods known in the art. For example, the Miltenyi CD4+ memory T cell isolation kit or Miltenyi naive CD4+ T cell isolation kit may be used. Isolated T cells are cultured in the presence of antigen presenting cells (e.g., irradiated L cells that express CD32 and CD80), and activated by addition of anti-CD3 antibody in the presence or absence of an agonist antigen binding complex that binds to OX40. The effect of an agonist antigen binding complex that binds to OX40 antibody on T cell proliferation may be measured using methods well known in the art. For example, the CellTiter-Glo® kit (Promega) may be used, and results read on a Multilabel Reader (Perkin Elmer). The effect of an agonist antigen binding complex that binds to OX40 on T cell function may also be determined by analysis of cytokines produced by the T cell. In one aspect, production of interferon gamma by CD4+ T cells is determined, e.g., by measurement of interferon gamma in cell culture supernatant. Methods for measuring interferon gamma are well-known in the art.

    [0409] Treg cell function may be assayed using methods known in the art and exemplary methods are disclosed herein. In one example, the ability of Treg to suppress effector T cell proliferation is assayed. T cells are isolated from human whole blood using methods known in the art (e.g., isolating memory T cells or naive T cells). Purified CD4+ naive T cells are labeled (e.g., with CFSE) and purified Treg cells are labeled with a different reagent. Irradiated antigen presenting cells (e.g., L cells expressing CD32 and CD80) are co-cultured with the labeled purified naive CD4+ T cells and purified Tregs. The co-cultures are activated using anti-CD3 antibody and tested in the presence or absence of an agonist antigen binding complex that binds to OX40. Following a suitable time (e.g., 6 days of coculture), the level of CD4+ naive T cell proliferation is tracked by dye dilution in reduced label staining (e.g., reduced CFSE label staining) using FACS analysis.

    [0410] OX40 signaling may be assayed using methods well known in the art and exemplary methods are disclosed herein. In one aspect, transgenic cells are generated that express human OX40 and a reporter gene comprising the NFkB promoter fused to a reporter gene (e.g., beta luciferase). Addition of an agonist antigen binding complex that binds to OX40 to the cells results in increased NFkB transcription, which is detected using an assay for the reporter gene.

    [0411] Phagocytosis may be assayed, e.g., by using monocyte-derived macrophages, or U937 cells (a human histiocytic lymphoma cells line with the morphology and characteristics of mature macrophages). OX40 expressing cells are added to the monocyte-derived macrophages or U937 cells in the presence or absence of an agonist antigen binding complex that binds to OX40. Following culturing of the cells for a suitable period of time, the percentage of phagocytosis is determined by examining percentage of cells that double stain for markers of 1) the macrophage or U937 cell and 2) the OX40 expressing cell, and dividing this by the total number of cells that show markers of the OX40 expressing cell (e.g., GFP). Analysis may be done by flow cytometry. In another aspect, analysis may be done by fluorescent microscopy analysis.

    [0412] Cells for use in any of the above in vitro assays include cells or cell lines that naturally express OX40 or that have been engineered to express OX40. Such cells include activated T cells, Treg cells and activated memory T cells that naturally express OX40. Such cells also include cell lines that express OX40 and cell lines that do not normally express OX40 but have been transfected with nucleic acid encoding OX40. Exemplary cell lines provided herein for use in any of the above in vitro assays include transgenic BT474 cells (a human breast cancer cell line) that express human OX40.

    IV. Pharmaceutical Compositions



    [0413] The antigen binding complexes (e.g., a tetravalent antigen binding complex having agonist activity) of the present disclosure or subunit(s) thereof as described herein may be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual subject, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The "therapeutically effective amount" of the complexes or proteins to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat a particular disorder (for example, a cancer, allergic or inflammatory disorder, or autoimmune disorder). In certain aspects, the complexes and proteins described herein may optionally be formulated with one or more agents currently used to prevent or treat the disorder. The effective amount of such other agents depends on the amount of complexes or proteins present in the formulation, the type of disorder or treatment, and other factors discussed above.

    [0414] Therapeutic formulations are prepared using standard methods known in the art by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (20th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA). Acceptable carriers, include saline, or buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagines, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN, PLURONICS, or PEG.

    [0415] In certain aspects, the formulation contains a pharmaceutically acceptable salt, preferably sodium chloride, and preferably at about physiological concentrations. Optionally, the formulations of the invention can contain a pharmaceutically acceptable preservative. In some aspects the preservative concentration ranges from 0.1 to 2.0%, typically v/v. Suitable preservatives include those known in the pharmaceutical arts. Benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben are preferred preservatives. Optionally, the formulations of the invention can include a pharmaceutically acceptable surfactant at a concentration of 0.005 to 0.02%.

    [0416] The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

    [0417] The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

    [0418] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antigen binding complex or the antigen binding polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antigen binding complex(es) or antigen binding polypeptide(s) remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio- disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

    [0419] The complexes described herein may be administered to a human subject, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Local administration may be particularly desired if extensive side effects or toxicity is associated with antagonism to the target molecule recognized by the proteins. An ex vivo strategy can also be used for therapeutic applications. Ex vivo strategies involve transfecting or transducing cells obtained from the subject with a polynucleotide encoding a protein or complex of this invention. The transfected or transduced cells are then returned to the subject. The cells can be any of a wide range of types including, without limitation, hematopoietic cells (e.g., bone marrow cells, macrophages, monocytes, dendritic cells, T cells, or B cells), fibroblasts, epithelial cells, endothelial cells, keratinocytes, or muscle cells.

    V. Articles of Manufacture



    [0420] Another aspect of the invention is an article of manufacture containing one or more antigen binding complexes (e.g., a tetravalent antigen binding complex having agonist activity) as described herein, and materials useful for the treatment or diagnosis of a disorder (for example, an autoimmune disease or cancer). The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition that is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antigen binding complex or polypeptide as described herein. The label or package insert indicates that the composition is used for treating the particular condition. The label or package insert will further comprise instructions for administering the antigen binding complex or polypeptide composition to the subject. Articles of manufacture and kits comprising combinatorial therapies described herein are also contemplated.

    [0421] Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

    [0422] Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials considered from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

    EXAMPLES


    Example 1. Exploration of Biepitopic Antibodies to Enhance Agonist Activity



    [0423] There is growing interest in discovering antibodies that mediate agonist activity against target receptors. Agonist antibodies engage a receptor in a manner that is productive for signaling, in effect acting as a surrogate ligand. For some targets antibody-mediated agonism may be possible via bivalent engagement of the receptor, taking advantage of the homodimeric nature of IgG. However some receptor systems require multivalent cross-linking to elicit activity, whereby receptors are pulled together into a cluster to elicit optimal signaling. For these receptors, antibody cross-linking in vivo is typically enabled by engagement of antibody Fc with Fc receptors (Wilson et al., 2011, Cancer Cell 19:101-13; Kim & Ashkenazi, 2013, J Exp Med 210:1647-51; Stewart et al., 2014 Journal for ImmunoTherapy of Cancer 2:1-10).

    [0424] The potential of biepitopic engagement of OX40 to enhance antibody-mediated receptor agonism was explored. OX40 is a TNFRSF member co-stimulatory molecule expressed on antigen experienced effector T (Teff) and regulatory T (Treg) cells, including infiltrating cells in mouse and human tumors. Activation of OX40 by agonist antibodies has been shown to promote anti-tumor immunity by enhancing Teff activation and inhibiting Treg mediated suppression (Voo et al., 2013, J. Immunol, 191:3641-50). Agonism by anti-OX40 antibodies has been shown to provide costimulatory activity similar to the natural ligand OX40L, but in a manner that requires cross-linking in vitro or via Fc/FcγR-mediated cross-linking in vivo (Voo et al., 2013, J. Immunol, 191:3641-50).

    Materials and methods



    [0425] A biepitopic anti-OX40 antibody was generated by constructing a bispecific antibody comprised of two separate variable regions. One variable region comprises an anti-OX40 humanized antibody with VH and VL sequences as set forth in SEQ ID NOs: 56 and 57, respectively. While the variable region comprising these domains is more completely described as 1A7.gr. 1, herein this variable region will be abbreviated as 1A7. In other words, 1A7 herein refers to the humanized 1A7.gr.1 variable region as defined by SEQ ID NOs: 56 and 57. One variable region comprises an anti-OX40 humanized antibody with VH and VL sequences as set forth in SEQ ID NOs: 126 and 129, respectively. While the variable region comprising these domains is more completely described as 3C8.gr.5.SG, herein this variable region will be abbreviated as 3C8. In other words, 3C8 herein refers to the humanized 3C8.gr.5.SG variable region as defined by SEQ ID NOs: 126 and 129.

    [0426] Bispecific antibody was generated using knobs-into-hole variants and in vitro assembly of separate half-antibodies (Spiess et al., 2013, Nat Biotechnol 31(8):753-8). DNA encoding antibody heavy and light chains were constructed in the pRK mammalian expression vector (Eaton et al., 1986, Biochemistry 25:8343-8347) using standard molecular biology techniques. Plasmids encoding heavy chain and light chain of antibodies with mutations for expi293 or CHO cell expression were constructed through gene synthesis (Genewiz) or through mutagenesis using QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Cat. 210514) and Q5 Site-Directed Mutagenesis Kit (New England BioLab Cat. E0554S). DNA encoding the 1A7 VH region was subcloned into a variant human IgG1 (hIgG1) comprising "knob" mutation T366W (EU numbering), a C-terminal 6His tag, and with or without N297G (EU numbering) mutation. DNA encoding the 3C8 VH region was subcloned into a variant human IgG1 (hIgG1) comprising "hole" mutations T366S/L368A/Y407V (EU numbering), a C-terminal Flag tag, and with or without N297G mutation.

    [0427] A variety of mutational strategies have been described for reducing the effector function properties of monoclonal antibodies (Strohl, 2009, Curr Opin in Biotech 20:685-691). Two approaches were used to engineer an effector-attenuated version of the antibodies of the present disclosure. First, antibodies were constructed in the context of an aglycosylated Fc region by combining them with the substitution N297G, which removes the conserved N-linked glycosylation site at position 297 of the Fc region. The second approach utilized substitutions L234A, L235A, and P329G (EU numbering) (the L234A/L235A/P329G triple variant is referred to as LALAPG), which has previously been shown to reduce binding to Fc receptors and complement (see e.g., US PG Publication No. 2012/0251531). DNA encoding VL regions for both antibodies were constructed with human Ckappa (hCk) constant chains.

    [0428] pRK vector DNA encoding 1A7 and 3C8 light and heavy (native and half antibodies) were cotransfected into HEK293 cells for expression, and resulting protein was purified. After expression, the Expi293 cells were settled under gravity. The supernatants were transferred to a 50 mL Falcon tube (Corning, Corning, New York) containing 0.2mL (50% slurry) of MabSelect Sure resin (GE Healthcare Life Sciences, Pittsburgh, PA) using a Freedom EVO 200 liquid handling workstation (Tecan, Morrisville, NC). The supernatant/resin mixtures were incubated overnight on an Innova 2000 platform shaker (New Brunswick Scientific, Enfield, CT) at 270 RPM. After settling, the resin was transferred to a 96-well 2 ml filter plate with a 25-µm membrane (Thompson Instruments, Oceanside, CA) and washed twice with 1 mL PBS buffer pH 7.4 to remove any unbound protein and medium components by centrifugation at 2,000 rpm for 5 minutes using a Sorvall HT6 Centrifuge (Thermo Scientific, Waltham, MA). The plate containing resin was stacked on top of 0.2 µm 96-well filter plate (Orochem, Naperville, IL, USA) and 96-well elution capture plate (Orochem, Naperville, IL). The bound half-antibodies were eluted from the resin with elution buffer (50 mM Phosphoric Acid, pH 2.9) in 2 sequential elution steps (total volume of elution - 0.668 mL) by centrifugation at 2,000 rpm for 5 minutes and pH of the eluate was raised by addition of 1/12th volume of neutralization buffer (1 M Arginine, 0.685 M Succinate, pH 5.0). Concentration of the half-antibody preps was calculated after measuring the absorbance at 280 nm using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE).

    [0429] Half-antibodies were assembled into biepitopic antibodies. Half-antibodies purified as described above were normalized to the same concentration and mixed in a 1:1 ratio using a Hamilton Star liquid handling workstation (Hamilton Robotics, Reno, NV). The pH of the mixture of half-antibodies was raised to 8.0 by adding 1M Arginine pH 9.5 solution. Reduced L-Glutathione in 0.5 M Stock in 1 M Arginine, pH 9.5 (Sigma-Aldrich, St. Louis, MO) was added so that final L-Glutathione concentration was 200 times excess over the amount of protein in the solution. This mixture was incubated at 32°C for 24 hours to allow the half-antibodies to assemble into biepitopic antibodies.

    [0430] The desired biepitopic antibodies (heterodimer composed of knob and hole half-antibodies) generated during assembly were purified away from other species (unassembled half-antibodies and knob-knob or hole-hole homodimers) by a 2-step process involving His-tag purification in a first step followed by Flag-tag purification in a second step. The first step involved use of 1.2 mL liquid handling tips (Dynamic Devices LLC, Wilmington, DE) that were custom-packed (Glygen Corp, Columbia, MD) with 0.1 mL of Ni-NTA Agarose resin (Qiagen, Valencia, CA). Using a Lynx LM1200 liquid handling workstation, all His-tagged species (including the unassembled Knob half-antibody, Knob-Knob homodimer and Knob-Hole heterodimer) were captured onto the resin tips by pipetting for 15 cycles. The resin tips were washed with 1 mL PBS pH 7.4 and the His-Tagged species were eluted from the tips in 2 sequential elution steps (total volume of elution = 0.6 mL) with elution buffer (50 mM Sodium Phosphate, 500 mM Sodium Chloride, 300 mM Imidazole pH 7.5). The eluted samples were diluted 1:1 with reagent-grade water to prepare them for the next step.

    [0431] To the solution obtained after first post assembly step purification, 0.4 mL of a 50% anti-FLAG antibody resin slurry was added using a Freedom EVO 200 liquid handling workstation and the resulting mixture was incubated on a Forma Orbital shaker (Thermo Electron Corporation, Madison, WI) at 250 RPM for 3 hours at 4°C. The resin was separated from the supernatant by centrifuging at 2,000 RPM for 5 minutes and transferred to a filter plate to be washed with 1 mL PBS pH 7.4 to remove any unbound proteins. The plate was stacked on top of 0.2 µm 96-well filter plate and 96-well elution capture plate. The bound biepitopic antibody (heterodimer) was eluted off the resin in 3 sequential elutions using a total of 1.57 mL elution buffer (50 mM Phosphoric Acid, pH 2.9) by centrifuging at 2,000 rpm for 5 minutes and neutralized with 0.11 mL of 20x PBS, pH 11.0 to adjust the pH of the purified biepitopic antibody solution to 6.0. Concentration of the biepitopic antibody was calculated after measuring the absorbance at 280 nm using a NanoDrop 2000 spectrophotometer.

    [0432] Quality of the final purified biepitopic antibodies was assessed using analytical SEC HPLC and mass spectrometry. SEC was carried out by injecting 0.03 mL onto a TSKGel SuperSW3000 column (Tosoh Bioscience, King of Prussia, PA) attached to a Dionex UltiMate 3000 HPLC System (Thermo Scientific, Waltham, MA). The analysis was done with an isocratic gradient (200mM Potassium Phosphate, 250mM Potassium Chloride, pH 7.2). UV detection was set at 280 nM. Mass spectrometric data was acquired using an Agilent 6224 TOF LC-MS system (Agilent Technologies, Santa Clara, CA, USA). Biepitopic antibody samples were reduced with 100 mM dithiothreitol (G-Biosciences, St. Louis, MO) at 37°C for 20 minutes. The polypeptide chains were separated with a PLRP-S reversed phase column (Agilent Technologies, Santa Clara, CA, USA). Intact masses of the reduced light and heavy chains were obtained by Maximun Entropy Deconvolution using MassHunter software (Qualitative Analysis B.03.01).

    Results



    [0433] The ability of biepitopic 1A7/3C8 antibody to form higher-order complexes in the presence of OX40 was investigated using aSEC. 1A7 or 1A7/3C8 biepitopic antibody was mixed with human OX40 (G&P Biosciences FCL2479) at 2:1 ratio, incubated at RT for 2 hours, the mixtures were run on TSKGel SuperSW3000 column. The data are shown in FIG. 1. At the 2:1 antibody:OX40 ratio, monoepitopic 1A7 IgG1 antibody forms three species: free antibody, antibody with one arm bound, and antibody with 2 arms bound. In contrast, 1A7/3C8 forms species larger than 1:2 antibody:OX40 stoichometry. The results indicate that biepitopic anti-OX40 antibody is able to promote higher order complexes in the presence of target antigen relative to monoepitopic antibody.

    Example 2. Design of Multivalent Monoepitopic and Biepitopic Antibody Formats



    [0434] A combination of multivalency and multiepitopic targeting was explored in attempt to engineer antibody formats with enhanced agonist activities. FIG. 2 depicts the structure of an IgG antibody that illustrates the elements used in engineering novel multivalent and multiepitopic antibody formats. IgG comprises a heavy chain and a light chain. Heavy chain comprises variable heavy domain (VH), constant heavy domain 1 (CH1), hinge, constant heavy domain 2 (CH2), and constant heavy domain 3 (CH3). Light chain comprises variable light domain (VL) and constant light domain (CL). CL can be constant kappa (Ck) or constant lambda (CI). Fv region with specificity for target comprises variable heavy (VH) and variable light (VL) domains. Fab region with specificity for target comprises variable heavy (VH) and variable light (VL) domains, and constant heavy domain 1 (CH1) and constant light (CL). The IgG region excluded by the Fab region is heavy chain Hinge-CH2-CH3. FIGS. 3A & 3B show a series of multivalent and multiepitopic antibody formats that were engineered and characterized. Beyond a bivalent native IgG, four tetravalent (valency = 4) formats were engineered: a coupled IgG-IgG (c:IgG-IgG) wherein two full length IgGs are chemically coupled, a coupled Fab-IgG (c:Fab-IgG) wherein two Fabs are chemically coupled to a full length IgG, a recombinant Fab-IgG (r:Fab-IgG) wherein two additional Fab arms are coupled genetically to an IgG, and a recombinant Fv-IgG (r:Fv-IgG) wherein two additional Fv regions are coupled genetically to an IgG. For each of these formats constructs were explored wherein all four variable regions bind the same epitope on the target antigen (referred to herein as monoepitopic), or wherein two variable regions bind one epitope and the other variable regions bind a distinct epitope on the target antigen (referred to herein as biepitopic).

    [0435] All four variable regions may bind the same epitope on the target antigen, referred to herein as monoepitopic. For example all four variable regions may be the anti-OX40 1A7 variable region, or all four variable regions may be the anti-OX40 3C8 variable region. Alternatively, two variable regions may bind one epitope and the other two variable regions may bind a distinct epitope on the target antigen, referred to herein as biepitopic. For example two of the variable regions may be the 1A7 variable region, and the other two variable regions may be the 3C8 variable region.

    [0436] A coupled IgG-IgG (c:IgG-IgG) format is described herein wherein two full length IgGs are chemically coupled. FIG. 4 illustrates the components of one version of a biepitopic c:IgG-IgG. In this version c:IgG-IgG comprises an IgGEp1 with specificity for epitope 1 chemically coupled to an IgGEp2 with specificity for epitope 2. IgGEp2 is linked to IgGEp1 through a chemical linker L. A variety of linkers may be used to connect IgGEp1 and IgeGEp2. The site of attachment of IgGEp1 and IgGEp2 may vary. The c:IgG-IgG format described comprises four separate protein chains: IgGEp1 heavy chain (HC) composed of VH1-Hinge-CH2-CH3, IgGEp1 light chain (LC) composed of VL1-CL, IgGEp2 HC composed of VH2-CH1-Hinge-CH2-CH3, and IgGEp2 LC composed of VL2-CL. FIG. 5 illustrates the components of another version of a biepitopic c:IgG-IgG. In this version c:IgG-IgG comprises a biepitopic IgGEp1&2 with specificity for both epitopes 1 and 2 chemically coupled to itself. IgGEp1&2 is coupled through a chemical linker L. A variety of linkers may be used to couple IgGEp1&2. The site of attachment of IgGEp1&2 may vary. The c:IgG-IgG format described comprises four separate protein chains: IgGEp1 heavy chain (HC) composed of VH1-Hinge-CH2-CH3, IgGEp1 light chain (LC) composed of VL1-CL, IgGEp2 HC composed of VH2-CH1-Hinge-CH2-CH3, and IgGEp2 LC composed of VL2-CL. In the examples described herein, c:IgG-IgG's are constructed and tested that comprise the variable regions of the 1A7 and 3C8 antibodies. In a preferred aspect, IgGEp1&2 comprises the 1A7 variable region and the 3C8 variable region, referred to as c:IgG-IgG 1A7/3C8-1A7/3C8.

    [0437] A coupled Fab-IgG (c:Fab-IgG) format is described herein wherein two Fabs are chemically coupled to a full length IgG. FIG. 6 illustrates the components of the c:Fab-IgG format. c:Fab-IgG comprises an IgGEp1 with specificity for epitope 1 chemically coupled to FabEp2 with specificity for epitope 2. FabEp2 is linked to IgGEp1 through a chemical linker L. A variety of linkers may be used to connect IgGEp1 and FabEp2. The site of attachment of IgGEp1 and FabEp2 may vary. The c:Fab-IgG format comprises four separate protein chains: IgGEp1 heavy chain (HC) composed of VH1-Hinge-CH2-CH3, IgGEp1 light chain (LC) composed of VL1-CL, FabEp2 HC composed of VH2-CH1, and FabEp2 LC composed of VL2-CL.

    [0438] In the examples described herein, c:Fab-IgG's were constructed and tested that comprise the variable regions of the 1A7 and 3C8 antibodies. In some aspects, IgGEp1 comprises the 1A7 variable region and FabEp2 comprises the 3C8 variable region, referred to as c:Fab-IgG 3C8-1A7. In some aspects, IgGEp1 comprises the 3C8 variable region and FabEp2 comprises the 1A7 variable region, referred to as c:Fab-IgG 1A7-3C8.

    [0439] A recombinant Fv-IgG (r:Fv-IgG) is described herein wherein two additional variable regions are genetically linked to an IgG. FIG. 7 illustrates the components of the r:Fv-IgG format. r:Fv-IgG comprises a single heavy chain and a single light chain. r:Fv-IgG comprises an IgGEp2 with specificity for epitope 2 linked N-terminally to FvEp1 with specificity for epitope 1. FvEp1 is linked to IgGEp2 through heavy chain linker LH and light chain linker LL. A variety of linkers may be used to connect FvEp2 to FvEp1. The heavy chain comprises regions VH1-LH-VH2-CH1-Hinge-CH2-CH3. The light chain comprises regions VL1-LL-VL2-CL. In this format, FvEp2 with specificity for epitope 2 comprises VH2 and VL2 domains, and FvEp1 with specificity for epitope 1 comprises VH1 and VL1 domains.

    [0440] In the examples described herein, r:Fv-IgG's were constructed and tested that comprise the variable regions of the 1A7 and 3C8 antibodies. In some aspects, FvEp1 comprises the 3C8 variable region and FvEp2 comprises the 1A7 variable region, referred to as r:Fv-IgG 3C8-1A7. In some aspects, FvEp1 comprises the 1A7 variable region and FvEp2 comprises the 3C8 variable region, referred to as r:Fv-IgG 1A7-3C8. Sequences of the heavy and light chains of exemplary r:Fv-IgG's are listed in SEQ IDs 240-243. Exemplary linkers LH and LL are provided in SEQ IDs 270-278 and described below in Example 16. Alternate exemplary r:Fv-IgG's with different linkers LH and LL, described below in Example 16, are listed in SEQ IDs 250-257. Alternate exemplary r:Fv-IgG's comprising Fv's with differing OX40 affinities, as described below in Example 17, are listed in SEQ IDs 258-267.

    [0441] A recombinant Fab-IgG (r:Fab-IgG) is described herein wherein two additional Fab arms are linked genetically to an IgG. FIG. 8 illustrates the components of one version of the r:Fab-IgG format comprising one homodimeric HC and two LC's. This r:Fab-IgG version (referred to herein as r:Fab-IgG VI) comprises IgGEp2 with specificity for epitope 2 linked N-terminally to FabEp1 with specificity for epitope 1. FabEp1 is linked to IgGEp2 through heavy chain linker LH. A variety of linkers may be used to connect FabEp1 to IgGEp2. The HC in r:Fab-IgG VI comprises VH1-CH1-LH-VH2-CH1-Hinge-CH2-CH3. One LC comprises VL1-CL, and the other LC comprises VH2-CL. Correct pairing between heavy and light chains may be controlled using engineered variants in VH, CH1, VL, and/or CL domains, as described in the examples herein. In this format FvEp2 with specificity for epitope 2 comprises VH2 and VL2 domains, and FvEp1 with specificity for epitope 1 comprises VH1 and VL1 domains.

    [0442] In the examples described herein, r:Fab-IgG VI antibodies were constructed and tested that comprise the variable regions of the 1A7 and 3C8 antibodies. In some aspects, FvEp1 comprises the 3C8 variable region and FvEp2 comprises the 1A7 variable region, referred to as r:Fab-IgG 3C8-1A7. In some aspects, FvEp1 comprises the 1A7 variable region and FvEp2 comprises the 3C8 variable region, referred to as r:Fab-IgG 1A7-3C8. Sequences of the heavy and light chains of exemplary anti-OX40 r:Fab-IgG's are listed in SEQ IDs 244-249.

    [0443] FIG. 9 illustrates the components of an alternate version of the r:Fab-IgG format comprising a heterodimeric HC and two LC's. This r:Fab-IgG version (referred to herein as r:Fab-IgG V2) comprises FabEp2 linked to one arm of IgGEp1&2 through heavy chain linker LH, and FabEp1 is linked to the other arm of IgGEp1&2 through heavy chain linker LH. A variety of linkers may be used to connect FabEp1 to IgGEp1&2 and FabEp2 to IgGEP1&2. One HC of this format comprises VH2-CH1-LH-VH2-CH1-Hinge-CH2-CH3, and the other HC of this format comprises VH1-CH1-LH-VH1-CH1-Hinge-CH2-CH3. One LC of this format comprises VL2-CL, and the other LC comprises VH1-CL. FvEp2 with specificity for epitope 2 comprises VH2 and VL2 domains. FvEp1 with specificity for epitope 1 comprises VH1 and VL1 domains. Engineered variants may be used to promote heterodimer formation of the heavy chains.

    [0444] A variety of linkers may be used to connect FabEp1 to IgGEp2. Linkers may comprise sequences that substantially comprise serine, glycine, or serine and glycine. Such Gly-Ser linkers may comprise greater than 50% glycine and/or serine, greater than 70% glycine and/or serine, greater than 90% glycine and/or serine, or 100% glycine and/or serine. Serine and glycine are commonly used as linkers due to their flexibility and favorable solution properties. Linkers may otherwise or also comprise sequences that substantially comprise native antibody sequence. In these linkers, native sequences that compose antibody sequences are used as linkers. Particularly useful native antibody sequences for the r:Fab-IgG are heavy chain sequences that reside in the hinge region of a native antibody between heavy chain constant regions CH1 and CH2. The utility of native antibody sequence linkers is that they can be used to reduce some non-native sequence within a tetravalent antibody format. The length of a given linker can vary, from 1 residue to 20 or more residues. Greater length typically provides greater flexibility between linked immunoglobulin domains. Exemplary recombinant linkers for use in the r:Fab-IgGs of the present disclosure are described in SEQ ID NO:268 and 269.

    SEQ ID NO:268. LH Linker for r:Fab-IgG: DKTHT

    SEQ ID NO:269. LH Linker for r:Fab-IgG: DKTHTGGGGSGG



    [0445] While r:Fab-IgG formats are described herein wherein a Fab is linked to an IgG through a single heavy chain linker (LH), it is contemplated that r:Fab-IgGs may also comprise linkers between light chains. Indeed linked light chain constructs were explored for the r:Fab-IgG format, but resulted in larger a higher population of aggregated species upon expression and purification (data not shown) and so were not characterized further.

    [0446] Exemplary sequences of heavy and light chains of the anti-OX40 IgG and tetravalent formats described herein are provided below. Linkers are underlined where appropriate.

    r:Fv-IgG Format

    SEQ ID NO:240. r:Fv-IgG 1A7-3C8 Heavy Chain.

    SEQ ID NO:241. r:Fv-IgG 1A7-3C8 Light Chain.

    SEQ ID NO:242. r:Fv-IgG 3C8-1A7 Heavy Chain.

    SEQ ID NO:243. r:Fv-IgG 3C8-1A7 Light Chain

    SEQ ID NO:250. r: Fv-IgG 3C8-1A7 GS Short Linker Heavy Chain.

    SEQ ID NO:251. r: Fv-IgG 3C8-1A7 GS Short Linker Light Chain.

    SEQ ID NO:252. r: Fv-IgG 3C8-1A7 GS Long Linker Heavy Chain.

    SEQ ID NO:253. r: Fv-IgG 3C8-1A7 GS Long Linker Light Chain.

    SEQ ID NO:254. r: Fv-IgG 3C8-1A7 ES Short Elbow Linker Heavy Chain.

    SEQ ID NO:255. r: Fv-IgG 3C8-1A7 ES Short Elbow Linker Light Chain.

    SEQ ID NO:256. r: Fv-IgG 3C8-1A7 EL Long Elbow Linker Heavy Chain.

    SEQ ID NO:257. r: Fv-IgG 3C8-1A7 EL Long Elbow Linker Light Chain.

    SEQ ID NO:258. r: Fv-IgG 3C8-1A7(High) Heavy Chain.

    SEQ ID NO:259. r: Fv-IgG 3C8-1A7(High) Light Chain.

    SEQ ID NO:260. r: Fv-IgG 3C8(High)-1A7 Heavy Chain.

    SEQ ID NO:261. r: Fv-IgG 3C8(High)-1A7 Light Chain

    SEQ ID NO:262. r: Fv-IgG 3C8(High)-1A7(High) Heavy Chain

    SEQ ID NO:263. r: Fv-IgG 3C8(High)-1A7(High) Light Chain

    SEQ ID NO:264. r: Fv-IgG 3C8(High)-1A7(Low) Heavy Chain

    SEQ ID NO:265. r: Fv-IgG 3C8(High)-1A7(Low) Light Chain

    SEQ ID NO:266. r: Fv-IgG 3C8-1A7(Low) Heavy Chain

    SEQ ID NO:267. r: Fv-IgG 3C8-1A7(Low) Light Chain

    r:Fab-IgG Format

    SEQ ID NO:244. r:Fab-IgG 1A7-3C8 Heavy Chain.

    SEQ ID NO:245. r:Fab-IgG 1A7-3C8 Light Chain 1.

    SEQ ID NO:246. r:Fab-IgG 1A7-3C8 Light Chain 2.

    SEQ ID NO:247. r: Fab-IgG 3C8-1A7 Heavy chain.

    SEQ ID NO:248. r: Fab-IgG 3C8-1A7 Light chain 1.

    SEQ ID NO:249. r: Fab-IgG 3C8-1A7 Light chain 2.

    c:Fab-IgG Format

    SEQ ID NO:232. 3C8_IgG1 (Full Length Heavy Chain).

    SEQ ID NO:237. 3C8_Ckappa(K149C) (Light Chain).

    SEQ ID NO:239. 1A7_CH1-SPPC (Fab Heavy Chain).

    SEQ ID NO:231. 1A7_Ckappa (Light Chain).

    SEQ ID NO:230. 1A7_IgG1 (Full Length Heavy Chain).

    SEQ ID NO:236. 1A7_Ckappa(K149C) (Light Chain).

    SEQ ID NO:238. 3C8_CH1-SPPC (Fab Heavy Chain).

    SEQ ID NO:233. 3C8_Ckappa (Light Chain).


    Sequences



    [0447] 

    SEQ ID NO:230. 1A7_IgG1 (Full Length Heavy Chain).



    SEQ ID NO:231. 1A7_Ckappa (Light Chain).

    SEQ ID NO:232. 3C8_IgG1 (Full Length Heavy Chain).

    SEQ ID NO:233. 3C8_Ckappa (Light Chain).

    SEQ ID NO:234. 1A7_CH1 (Fab Heavy Chain).

    SEQ ID NO:235. 3C8_CH1 (Fab Heavy Chain).

    SEQ ID NO:236. 1A7_Ckappa(K149C) (Light Chain).

    SEQ ID NO:237. 3C8_Ckappa(K149C) (Light Chain).

    SEQ ID NO:238. 3C8_CH1-SPPC (Fab Heavy Chain).

    SEQ ID NO:239. 1A7_CH1-SPPC (Fab Heavy Chain).

    SEQ ID NO:240. r:Fv-IgG 1A7-3C8 Heavy Chain.

    SEQ ID NO:241. r:Fv-IgG 1A7-3C8 Light Chain.

    SEQ ID NO:242. r:Fv-IgG 3C8-1A7 Heavy Chain.

    SEQ ID NO:243. r:Fv-IgG 3C8-1A7 Light Chain.

    SEQ ID NO:244. r:Fab-IgG 1A7-3C8 Heavy Chain.



    SEQ ID NO:245. r:Fab-IgG 1A7-3C8 Light Chain 1.

    SEQ ID NO:246. r:Fab-IgG 1A7-3C8 Light Chain 2.

    SEQ ID NO:247. r: Fab-IgG 3C8-1A7 Heavy chain.

    SEQ ID NO:248. r: Fab-IgG 3C8-1A7 Light chain 1.

    SEQ ID NO:249. r: Fab-IgG 3C8-1A7 Light chain 2.

    SEQ ID NO:250. r: Fv-IgG 3C8-1A7 GS Short Linker Heavy Chain, as described in Example 16.

    SEQ ID NO:251. r: Fv-IgG 3C8-1A7 GS Short Linker Light Chain, as described in Example 16.

    SEQ ID NO:252. r: Fv-IgG 3C8-1A7 GS Long Linker Heavy Chain, as described in Example 16.



    SEQ ID NO:253. r: Fv-IgG 3C8-1A7 GS Long Linker Light Chain, as described in Example 16.

    SEQ ID NO:254. r: Fv-IgG 3C8-1A7 ES Short Elbow Linker Heavy Chain, as described in Example 16.

    SEQ ID NO:255. r: Fv-IgG 3C8-1A7 ES Short Elbow Linker Light Chain, as described in Example 16.

    SEQ ID NO:256. r: Fv-IgG 3C8-1A7 EL Long Elbow Linker Heavy Chain, as described in Example 16.

    SEQ ID NO:257. r: Fv-IgG 3C8-1A7 EL Long Elbow Linker Light Chain, as described in Example 16.

    SEQ IDs 258 and 259 describe heavy and light chain sequences for r:Fv-IgG 3C8-1A7(High), which comprises 1A7 VH mutation M34I and VL mutation R53Y, described in Example 17.

    SEQ ID NO:258. r: Fv-IgG 3C8-1A7(High) Heavy Chain.



    SEQ ID NO:259. r: Fv-IgG 3C8-1A7(High) Light Chain.

    SEQ IDs 260 and 261 describe heavy and light chain sequences for r:Fv-IgG 3C8(High), which comprises 3C8 VH mutations M31I and K64L, as described in Example 17.

    SEQ ID NO:260. r: Fv-IgG 3C8(High)-1A7 Heavy Chain.

    SEQ ID NO:261. r: Fv-IgG 3C8(High)-1A7 Light Chain

    SEQ IDs 262 and 263 describe heavy and light chain sequences for r:Fv-IgG 3C8(High)-1A7(High), which comprises 3C8 VH mutations M31I and K64L, 1A7 VH mutation M34I, and 1A7 VL mutation R53Y, described in Example 17.

    SEQ ID NO:262. r: Fv-IgG 3C8(High)-1A7(High) Heavy Chain

    SEQ ID NO:263. r: Fv-IgG 3C8(High)-1A7(High) Light Chain

    SEQ IDs 264 and 265 describe heavy and light chain sequences for r:Fv-IgG 3C8(High)-1A7(Low), which comprises 3C8 VH mutations M31I and K64L and 1A7 VH mutation P96A, described in Example 17.

    SEQ ID NO:264. r: Fv-IgG 3C8(High)-1A7(Low) Heavy Chain

    SEQ ID NO:265. r: Fv-IgG 3C8(High)-1A7(Low) Light Chain

    SEQ IDs 266 and 267 describe heavy and light chain sequences for r:Fv-IgG 3C8-1A7(Low), which comprises 1A7 VH mutation P96A, described in Example 17.

    SEQ ID NO:266. r: Fv-IgG 3C8-1A7(Low) Heavy Chain

    SEQ ID NO:267. r: Fv-IgG 3C8-1A7(Low) Light Chain



    [0448] Exemplary heavy chain constant regions (CH1-Hinge-CH2-CH3) and light chain constant regions (CL) that may find use in the present disclosure are provided in SEQ ID NO: 279-288. As is well known in the art, polymorphic, allotypic, and haplotypic variants of human constant chains exist, and these variants may find use in the present disclosure.

    SEQ ID NO:279. Human IgG1 Heavy Constant Region (CH1-Hinge-CH2-CH3)

    SEQ ID NO:295. Human IgG2 Heavy Constant Region (CH1-Hinge-CH2-CH3)

    SEQ ID NO:296. Human IgG3 Heavy Constant Region (CH1-Hinge-CH2-CH3)

    SEQ ID NO:297. Human IgG4 Heavy Constant Region (CH1-Hinge-CH2-CH3)

    SEQ ID NO:298. Human Ckappa Light Constant Region (CL-kappa).

    SEQ ID NO:299. Human Clambda 1 Light Constant Region (CL-lambda1).

    SEQ ID NO:300. Human Clambda 2 Light Constant Region (CL-lambda2).

    SEQ ID NO:301. Human Clambda 3 Light Constant Region (CL-lambda3).

    SEQ ID NO:302. Human Clambda 6 Light Constant Region (CL-lambda6).

    SEQ ID NO:303. Human Clambda 7 Light Constant Region (CL-lambda7).


    Example 3. Engineering and Characterization of Coupled IgG-IgG Antibodies


    Materials and methods



    [0449] Monoepitopic and biepitopic coupled IgG-IgG (c:IgG-IgG) anti-OX40 antibodies were generated using the 1A7 and 3C8 variable regions. c:IgG-IgG antibodies were constructed by engineering an asymmetric thiomab, that is a bispecific antibody in which only one of the half-antibodies has a free cysteine. In the present experiments the substitution K149C on the human Ckappa constant region was used, but the present disclosure contemplates the use of an engineered cysteine at any antibody residue. Bispecific antibodies comprising a single free cysteine at K149C were generated as described above using knobs-into-hole variants (reference) and in vitro assembly of half-antibodies (Spiess et al., 2013, Nat Biotechnol 31(8):753-8). Antibodies were constructed in the pRK vector, and all antibody heavy chains contained the N297G mutation to remove glycosylation and attenuate binding to Fc receptors. 1A7 and 3C8 half-antibodies contained either Knob/His or Hole/Flag heavy chain, and native or K149C light chain depending on the desired construct. pRK vector DNA encoding heavy and light chains for each antibody were cotransfected into HEK293 cells for expression, resulting protein was purified from the supernatant, and bispecific antibodies were assembled and purified as described above.

    [0450] c:IgG-IgG antibodies were generated by chemically coupling asymetric thiomab bispecific antibodies using a bis-maleimido polyethylene glycol linker (Bis-maleimido(PEG). Asymetric thiomab bispecific antibodies were generated using knob-into-hole technology mentioned above with a K149C mutation on the light chain of knob or hole. The IgG-IgG antibodies were made through the K149C site on knob or hole by linkers contained either 2 or 3 PEG units for the present experiments, although it is contemplated that a variety of PEG lengths could be utilized.

    Results



    [0451] c:IgG-IgG antibodies were tested for their ability to agonize OX40 receptor. CD4+ memory T cells (CD4+ CD45RO+) were sorted from buffy coat, and irradiated L cells expressing either CD80 (B7-1) and CD32a (FcyRIIa) or CD32a (FcγRIIa) only were used as surrogate antigen presenting cells (APCs). CD4+ memory T cells were incubated with L cells, stimulated with soluble anti-CD3 antibody (mouse anti-human CD3 clone SP34) and increasing concentrations of anti-OX40 antibodies or anti-Her2 antibodies as control. Cells were cultured for 7 days, harvested, and assayed for T cell proliferation by CellTiter-Glo® (Promega) and cytokine release by ELISA.

    [0452] T cell proliferation data are shown in FIG. 10. The results demonstrate that while tetravalent monoepitopic 1A7(N297G)-1A7(N297G) and bivalent biepitopic 1A7/3C8(N297G) provide a modest levels of agonist activity in the absence of FcR-mediated crosslinking, tetravalent biepitopic 1A7/3C8(N297G)-1A7/3C8(N297G) promotes a strong level of agonist activity.

    [0453] The assay was repeated wherein tetravalent monoepitopic 3C8(N297G)-3C8(N297G) was included in the test set. These data are shown in FIG. 11. The results again demonstrated the potent agonism of the 1A7/3C8 tetravalent format in the absence of crosslinking (N297G), and illustrated the synergistic combination of targeting 1A7 and 3C8 epitopes in the context of tetravalent engagement. Importantly, the data also demonstrate that the 1A7/3C8 c:IgG-IgG format is inactive in the absence of CD3 stimulation, indicating that despite its potent activity TCR engagement is still required.

    Example 4. Engineering and Characterization of Coupled FAb-IgG Antibodies


    Materials and methods



    [0454] Monoepitopic and biepitopic coupled Fab-IgG (c:Fab-IgG) anti-OX40 antibodies were generated based on the 1A7 and 3C8 variable regions. c:Fab-IgG antibodies were constructed by coupling a 1A7 full length human IgG1 comprising a free cysteine at K149C to a 3C8 Fab engineered with a C-terminal free cysteine. In the present experiments the substitution K149C on the human Ckappa constant region was used, but the present disclosure contemplates the use of an engineered cysteine at any antibody residue.

    [0455] c:IgG-IgG antibodies were generated by chemically coupling Fab and IgG antibodies using a bis-maleimido polyethylene glycol linker (Bis-maleimido(PEG). Linkers contained either 2 or 3 PEG units for the present experiments. It is contemplated that a variety of PEG lengths could be utilized. 3C8 Fab was reacted with bismal BMPEG3, and then excess BMPEG3 was removed. Conjugated 3C8 Fab was then reacted with 1A7 IgG1 K149C, and coupled Fab-IgG was purified using an S200 SEC column followed by a Mono-Q column using conventional chromatography methods. Quality of the final purified c:Fab-IgG antibodies was assessed using analytical Size Exclusion Chromatography coupled with Multiple Angle Light Scattering (SEC-MALS) as well as mass spectrometry. SEC-MALS was run using an Xbridge BEH column in PBS at 08. ml/min.

    Results



    [0456] The results are shown in FIG. 12. Fits of the data resulted in final radii and molecular weights as follows: c:Fab-IgG 1A7/3C8 had a Rh(Q)z (nm) of 7.3 nm +/- 4.6% and a MW of 248.6 +/- 15% KDa; c:Fab-IgG 1A7(N297G)/3C8 had an Rh(Q)z of 7.3 nm +/-4.3% and a MW of 238.2 KDa +/- 16.7%. Mass spectrometry confirmed (data not shown) confirmed that the purified c:Fab-IgG's had the predicted MW

    [0457] c:IgG-IgG antibodies were tested for their ability to agonize OX40 receptor using the primary T cell assay as described above, utilizing L cells expressing CD32a as surrogate antigen presenting cells (APCs). T cell proliferation data for the human IgG1 and human IgG1(N297G) versions are shown in FIG. 13 and FIG. 14 respectively. The results demonstrate that the 1A7/3C8 c:Fab-IgG format promotes a strong level of agonist activity, in contrast to the weak activity mediated by bivalent 1A7 IgG, bivalent 3C8 IgG, biepitopic 1A7/3C8 IgG. Together with the data on the c:IgG-IgG formats (included in the same assay), the results demonstrate the benefit of targeting two epitopes (in this example 1A7 and 3C8 OX40 epitopes) in the context of tetravalency.

    Example 5. Engineering and Characterization of Recombinant Fv-IgG Antibodies


    Materials and methods



    [0458] Monoepitopic and biepitopic recombinant Fv-IgG (r:Fv-IgG) anti-OX40 antibodies were generated based on the 1A7 and 3C8 variable regions. r:Fv-IgG antibodies were constructed by genetically engineering an additional VL region N-terminal to a native VL region, and an additional VH region N-terminal to a native VH region. The r:Fv-IgG constructed resulted in either tetravalent monoepitopic (1A7-1A7 and 3C8-3C8), or tetravalent biepitopic (1A7-3C8 and 3C8-1A7) formats. Heavy and light chain amino acid sequences of r:Fv-IgG 1A7-3C8 used in these examples are provided in SEQ ID NOs:240-241 respectively, and heavy and light chain amino acid sequences of r:Fv-IgG 3C8-1A7 used in these examples are provided in SEQ ID NOs:242-243 respectively. Antibodies were constructed in the pRK vector using conventional molecular biology methods. All antibodies comprised a native IgG1 constant region. pRK vector DNA encoding heavy and light chains for each r:Fv-IgG were cotransfected into HEK293 cells for expression, and resulting protein was purified from the supernatant using MabSelect Sure resin.

    [0459] r:Fv-IgG antibodies were tested for their ability to agonize OX40 receptor using OX40 expressing Jurkat cells with a luciferase reporter (clone 2A4 Jurkat-OX40-luc). Cells were seeded in Corning 96 well plate (cat# 3603) at 2 x 105 cells/well in 50 uL AIM-V media (Thermo Fischer Scientific, Cat. # 12055-091) in 96 well tissue culture plate (Corning Inc., Cat# 3603). Anti-OX40 antibodies were serially diluted in AIM-V media at 2X concentration and 50 uL of the diluted antibodies were added to the each well and incubated in 37° C for 16-18 hours at 5% CO2. 100 uL Bright Glo (Promega cat# E2610) was added and mixed at room temperature for 10 minutes. Luminescence was detected on an Infinite M1000 Pro plate reader (Tecan). In contrast to the primary T cell assay described above, this Jurkat reporter assay does not include co-incubation with FcR+ L cells. Agonist activity in this assay thus reflects only cross-linking mediated by the antibody in the absence of any artificial cross-linking.

    Results



    [0460] The data are shown in FIG. 15. In the absence of any crosslinking, the 1A7 IgG1 antibody mediated no OX40 receptor agonism. The tetravalent monoepitopic constructs (r:Fv-IgG 1A7-1A7 and r:Fv-IgG 3C8-3C8) mediated a moderate level of crosslink-independent activity. The strongest level of agonist activity was mediated by the tetravalent biepitopic constructs (r:Fv-IgG 3C8-1A7 and r:Fv-IgG 1A7-3C8).

    [0461] r:Fv-IgG antibodies were tested for their ability to agonize OX40 receptor using the primary T cell assay as described above, utilizing L cells expressing CD32a. T cell proliferation data for the human IgG1 versions are shown in FIG. 16. Consistent with the Jurkat luciferase reporter assay, the biepitopic versions (1A7-3C8 and 3C8-1A7) promote receptor activation that is substantially greater than the activity of the monoepitopic versions (1A7-1A7 and 3C8-3C8). Together with the data on the c:IgG-IgG formats and c:Fab-IgG formats, the r:FAb-IgG results further support the benefit of targeting two epitopes (in this example 1A7 and 3C8 OX40 epitopes) in the context of tetravalency.

    Example 6. Engineering and Characterization of Recombinant Fab-IgG Antibodies


    Materials and methods



    [0462] Monoepitopic and biepitopic recombinant Fab-IgG (r:Fab-IgG's) anti-OX40 antibodies were generated comprising the 1A7 and 3C8 variable regions. r:Fab-IgG antibodies were constructed by genetically engineering an additional heavy chain Fab region (VH-CH1) N-terminal to the native heavy chain Fab (VH-CH1) region. Tetravalent monoepitopic versions of the r:Fab-IgG were engineered by constructing either the 1A7 or the 3C8 Fabs in tandem (i.e. 1A7-1A7 or 3C8-3C8) and co-expressing 1A7 or 3C8 light chains (VL-Ckappa) respectively. Tetravalent monoepitopic r:Fab-IgG's are a "two-chain system" in the same way as native IgG's, i.e. there is a single heavy chain and a single light chain. Tetravalent biepitopic versions of the r:Fab-IgG required additional engineering to control the heavy chain / light chain pairing. Biepitopic versions are made by co-expressing the tandem heavy chain (e.g. 1A7-3C8 or 3C8-1A7) with the light chains for both 1A7 and 3C8, resulting in a "three-chain system" (SEQ ID NOs: 244-246 for the 1A7-3C8 orientation or 247-249 for the 3C8-1A7 orientation). The variable regions do not on their own ensure proper pairing, and therefore orthogonal variants were incorporated into the VH/VL and CH1/CL regions as described in International Pub. No. WO2016172485. VH/VL regions utilized variant pairs VH-Q39K / VL-Q38E or VH Q39E / VL Q38K (Kabat numbering), and CH1/CL regions utilized variant pairs -CH1-S183E / CL-V133K or CH1-S183K / CL-V133E (EU numbering). Other contemplated variants for dictating proper heavy chain / light chain pairing include CH1 A141I, F170S, S181M, S183A, V185A, and CL F116A, L135V, S174A, S176F, and T178V (EU numbering). Antibodies were constructed in the pRK vector using conventional molecular biology methods. pRK vector DNA encoding heavy and light chains (one light chain [1A7 or 3C8] for monoepitopic and two light chains [1A7 and 3C8] for biepitopic) for each r:Fab-IgG were cotransfected into HEK293 cells for expression. Resulting protein was purified from the supernatant using MabSelect Sure resin.

    [0463] Quality of the final purified r:Fab-IgG antibodies was assessed using analytical Size Exclusion Chromatography coupled with Multiple Angle Light Scattering (SEC-MALS) as well as mass spectrometry. SEC-MALS was run as described above using an Xbridge BEH column in PBS.

    Results



    [0464] The results are shown in FIG. 17 and FIG. 18 for the biepitopic r:Fab-IgG versions 3C8-1A7 and 1A7-3C8, respectively. Fits of the data resulted in final radii and molecular weights as follows: r:Fab-IgG 3C8-1A7 had a Rh(Q)z (nm) of 7.6 nm and a MW of approximately 236 KDa, and the r:Fab-IgG 1A7-3C8 had a Rh(Q)z of 7.2 nm and a MW of approximately 233.3 KDa.

    [0465] r:Fab-IgG antibodies were tested for their ability to agonize OX40 receptor using OX40 expressing Jurkat cells with a luciferase reporter (2A4 Jurkat-OX40-luc) as described above. The data are shown in FIG. 19. The tetravalent monoepitopic constructs (r:Fab-IgG 1A7-1A7 and r:Fab-IgG 3C8-3C8) mediated no crosslink-independent activity. In constrast, strong agonist activity was mediated by the tetravalent biepitopic constructs (r:Fab-IgG 3C8-1A7 and r:Fab-IgG 1A7-3C8).

    [0466] r:Fab-IgG antibodies were tested for their ability to agonize OX40 receptor using the primary T cell assay as described above, utilizing L cells expressing CD32a. T cell proliferation data for the human IgG1 versions are shown in FIG. 20. Tetravalent monoepitopic versions showed no (1A7-1A7) or modest activity (3C8-3C8). In contrast, and consistent with the Jurkat luciferase reporter assay, the biepitopic versions (1A7-3C8 and 3C8-1A7) promoted strong receptor activation. Together with the data on the c:IgG-IgG, c:Fab-IgG, and r:Fv-IgG formats, the r:FAb-IgG results further support the benefit of targeting two epitopes (in this example 1A7 and 3C8 OX40 epitopes) in the context of tetravalency.

    Example 7. Large scale production of tetravalent formats



    [0467] Tetravalent formats were expressed and purified at large scale for deeper in vitro and in vivo characterization. DNA of all new variants described below were constructed in the pRK mammalian expression vector through gene synthesis or mutagenesis as described above. Heavy and light chain DNAs were cotransfected into 293 or CHO cells for expression as described above. DNAs encoding Fabs were expressed in E. coli.

    Purification of r:Fab-IgG and r:Fv-IgG anti-OX40 antibodies



    [0468] Harvested CHO media was loaded onto a 5 mL MabSelect SuRe column (GE Healthcare, #17-5438-01). After the sample was loaded, the column was washed with 10 column volumes (CV) of Tris buffer (25 mM Tris pH 7.0, 150 mM NaCl, 5 mM EDTA, 2 mM sodium azide (NaN3)), 5 CVs of Triton X-114 buffer (25 mM Tris pH 7.0, 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-114, 2 mM NaN3), 10 CVs of Tris buffer, 2 CVs of KP wash buffer (0.4 M potassium phosphate pH 7.0, 5 mM EDTA, 0.02% Polysorbate Tween 20), and finally 10 CVs of Tris buffer. Protein was eluted with 5 CVs of elution buffer (50 mM sodium citrate pH 3.0, 150 mM NaCl) and immediately neutralized with 1M Tris pH 8.0 buffer. The neutralized elution was concentrated and purified over a HiLoad 16/600 Superdex 200 size exclusion chromatography (SEC) column (GE Healthcare, #28-983-36) with Arginine buffer (200 mM Arginine, 137 mM Succinic acid, 1 mM NaN3) as the mobile phase. Eluted fractions were collected in 1 mL increments and analyzed by SDS-PAGE. The final eluant pool was concentrated and formulated into 20 mM Histidine Acetate (HisOAc) pH 5.5, 240 mM Sucrose, 0.02% Polysorbate Tween 20.

    Production of c:Fab-IgG



    [0469] Hinge-cysteine-Fabs (Fab-SPPC) were expressed in E. coli and were purified as previously described (Scheer, J. M. et al. Reorienting the Fab domains of trastuzumab results in potent HER2 activators. PLoS One 2012, 7, e51817). THIOMAB antibodies were recombinantely expressed in CHO cells and purified as previously described (Sadowsky, J. D. et al. Development of efficient chemistry to generate site-specific disulfide-linked protein- and peptide-payload conjugates: Application to THIOMAB™ antibody-drug conjugates. Bioconjug. Chem. 2017).

    [0470] Hinge-cysteine-Fabs were functionalized with bis-maleimide crosslinker. Fresh bis-maleimide crosslinker (BMPEG) (ThermoFisher Scientific, 22337) was reconstituted to final concentration of 50mM in N,N-Dimethylacetamide (Sigma Aldrich, D5511). The purified Fab-SPPC was then reacted with 10 mole equivalents of BMPEG at pH 5.0 for 2 hours at room temperature and reaction progress was monitored by mass spectrometry. After incubation for 2 hours, the reaction was complete and excess crosslinker was removed by simple buffer exchange with 10,000 MWCO Amicon Ultra-15 centrifugal filter units (UFC901096, Millipore) into conjugation buffer (25 mM sodium acetate (NaOAc) pH 5.0, 2 mM NaN3). Post buffer exchange, the final functionalized Fab (Fab-BMPEG) was analyzed with mass spectrometry.

    [0471] c:Fab-IgG was conjugated and purified. Purified THIOMAB antibody (5 mg/mL) was reacted with five molar excess of Fab-BMPEG (5 mg/mL). The reaction mixture was conditioned with conjugation buffer to a final pH of 5.0 and incubated at room temperature for 21 hours. Reaction progress was monitored with mass spectrometry. After incubation for 21 hours, the reaction was complete and the reaction mixture was purified with hydrophobic interaction chromatography (HIC). Briefly, the sample was conditioned with 1.5M ammonium sulfate and loaded onto a ProPac HIC-10 column (5 µm, 7.8 mm x 75 mm) (Thermo Scientific, 063665). After sample load, the column was washed with 5 CVs of HIC-Buffer A (50 mM potassium phosphate pH 7.0, 1 M ammonium sulfate) and eluted with a linear gradient from 0% to 80% HIC-Buffer B (50 mM potassium phosphate pH 7.0, 20% isopropanol) over 60 CVs. Eluted fractions were collected and analyzed by LC/MS prior to formulation. The final conjugate was formulated into 20 mM HisOAc pH 5.5, 150 mM NaCl buffer and quality of final conjugate was assessed with LC/MS, SDS-PAGE, and SEC.

    Production of c:IgG-IgG



    [0472] ThioMab_KiH (knob-into-hole) antibody was expressed and purified. Half antibodies were expressed, purified, and annealed as previously described (Atwell, S., Ridgway, J. B., Wells, J. a & Carter, P. Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. J. Mol. Biol. 1997, 270, 26-35) to form the knob-into-hole THIOMAB antibody (ThioMab _KiH). Due to the annealing process, the engineered cysteine on the ThioMab _KiH antibody was blocked and thus unreactive towards maleimide crosslinker. To remove the adduct, the ThioMab _KiH antibody was reduced and reoxidized as previously described (Sadowsky, J. D. et al. Development of efficient chemistry to generate site-specific disulfide-linked protein- and peptide-payload conjugates: Application to THIOMAB™ antibody-drug conjugates. Bioconjug. Chem. 2017). The final deprotected ThioMab _KiH antibody was formulated into conjugation buffer and stored at 4 °C.

    [0473] ThioMab_KiH antibody was coupled. Deprotected ThioMab _KiH antibody was reacted with 10 mole equivalents of freshly prepared BMPEG (see above) at pH 5.0 for 2 hours and reaction progress was monitored by mass spectrometry. After the reaction was completed, excess crosslinker was removed by ion exchange chromatography (IEX). Briefly, the reaction mixture was diluted 10 fold with IEX-Buffer A (25 mM NaOAc pH 5.0, 2 mM NaN3) and loaded onto a pre-equilibrated Hi-Trap SPHP (GE Healthcare, 17115201) column. After sample load, the column was washed with 5 CVs of IEX-Buffer A and eluted with a step gradient from 0 % -100% over 1 CV with IEX-Buffer B (25 mM NaOAc pH 5.0, 1000 mM NaCl, 2 mM NaN3). Fractions were collected and analyzed by mass spectrometry. The final sample (ThioMab_KiH_BMPEG) was concentrated to 5 mg/mL and formulated into conjugation buffer.

    [0474] The purified ThioMab _KiH _BMPEG was then added to 1.5 molar excess of ThioMab_KiH in the same buffer to form the coupled IgG. The reaction mixture was incubated at room temperature for 48 hours and reaction progress was monitored with LC/MS analysis. After 48 hours the reaction was complete and the mixture was purified with HIC as described earlier. The final purified CIgG was formulated into 20 mM HisOAc pH 5.5, 150 mM NaCl.

    Example 8. Intrinsic agonist activity of tetravalent biepitopic antibody formats



    [0475] r:Fv-IgG antibodies were tested for their ability to activate T cells in the absence of crosslinking. r:Fv-IgG biepitopic versions 3C8-1A7 and 1A7-3C8, and monoepitopic versions 1A7-1A7 and 3C8-3C8 were engineered and produced with the LALAPG effector attentuation variants as described above. r:Fv-IgG's were tested in the primary human T cell assay using FcγRIIa+ L cells (L cells did not express B7-1) as described above.

    [0476] T cell proliferation data are shown in FIG. 21. The biepitopic r:Fv-IgG's 3C8-1A7 and 1A7-3C8 demonstrated the highest level of activity and showed no dependence on Fc-mediated crosslinking - the curves looked virtually the same regardless of whether the r:Fv-IgG's were IgG1 or LALAPG. The monoepitopic r:Fv-IgG versions 1A7-1A7 and 3C8-3C8 showed enhanced activity relative to IgG1 1A7, but weaker activity relative to the biepitopic r:Fv-IgG formats. Moreover, in the absence of Fc-mediated crosslinking (LALAPG versions on the right panel), the monoepitopic r:Fv-IgG's showed no activity. Similar results were obtained from human IgG1 and human IgG1 LALAPG versions of r:Fab-IgG anti-OX40 antibodies (data not shown).

    Example 9. Activity comparison of monoepitopic and biepitopic tetravalent formats



    [0477] All four tetravalent formats were tested in the Jurkat reporter assay as described above. As described, agonist activity in this assay reflects the intrinsic activity of the antibody in the absence of any extrinsic cross-linking. Data in FIG. 22 and FIG. 23 demonstrate the clear superiority of tetravalent biepitopic over monoepitopic formats. Moreover, the bivalent biepitopic 1A7/3C8 antibody tested in this assay illustrates that a bivalent IgG targeting two epitopes alone is insufficient for agonist activity. It is the targeting of two epitopes in the context of tetravalency that provides optimal activity.

    [0478] Tetravalent biepitopic formats were tested head-to-head in the Jurkat reporter assay. Data in FIG. 24 demonstrate that the recombinant biepitopic formats r:Fv-IgG and r:Fab-IgG provide greater activity relative to the bioconjugated biepitopic c:Fab-IgG and c:IgG-IgG formats. In this assay the r:Fv-IgG 3C8-1A7 antibody showed slight superiority to the other r:Fv-IgG and r:Fab-IgG formats.

    Example 10. Measurement of antibody: OX40 complex formation



    [0479] The ability of tetravalent monepitopic and biepitopic antibody formats to form high-order (immune) complexes upon binding to target OX40 was tested using Size Exclusion Chromatography linked in-line with Multiple Angle Light Scattering (SEC-MALS). Purified antibodies and human OX40 (G&P Biosciences FCL2479) were mixed in varying molar ratios in PBS. The mixtures were run over a Waters xBrdige BEH200A SEC 3.5um (7.8 x 300 mm) column and analyzed by an Agilent 1200 HPLC connected to Wyatt Technology detectors DAWN HELEOS-II multiangle laser light scattering photometer and Optilab T-rEX differential refractive index detector.

    [0480] FIG. 25 shows SEC chromatograms for r:Fv-IgGs. A much greater early shift in elution time, which is directly proportional to size, was observed for complexes formed between the biepitopic r:Fv-IgG 1A7-3C8 relative to the monoepitopic r:Fv-IgG 1A7-1A7. Similar results were obtained for r:Fab-IgG and c:Fab-IgG formats (data not shown), suggesting that tetravalent biepitopic formats are able to form larger immune complexes with target protein than tetravalent monoepitopic formats.

    [0481] FIG. 26 shows the calculated size of the antibody/OX40 complex from the MALS data from these experiments. The data further support the larger immune complexes formed by target binding to tetravalent biepitopic formats relative to both tetravalent monoepitopic and bivalent biepitopic formats. These data are consistent with the superior agonist activity observed from targeting two epitopes in the context of valency greater than 2.

    Example 11. Pharmacokinetics (PK) of tetravalent anti-OX40 antibodies in mice



    [0482] PK of tetravalent antibody formats was tested in C57BL-6 or C.B-17 SCID (severe combined immunodeficiency) mice. Mouse PK studies described in this section were approved by the Institutional Animal Care and Use Committee (IACUC). All antibodies tested comprised native human IgG1 heavy constant and Ckappa light constant chains. PK of IgG1 1A7, r:Fv-IgG 1A7-1A7, r:Fv-IgG 1A7-3C8, r:Fab-IgG 1A7-1A7, r:Fab-IgG 1A7-3C8, c:Fab-IgG 1A7-1A7, c:Fab-IgG 3C8-1A7 and cIgG-cIgG 3C8-3C8 antibodies were evaluated in immune-deficient C.B-17 SCID mice. In addition, PK of 1A7 IgG1, r:Fv-IgG 1A7-3C8, r:Fab-IgG 1A7-3C8, cIgG-IgG 1A7-3C8 and c:Fab-IgG 1A7-3C8 antibodies were evaluated in immune-competent C57BL-6 mice. Anti-gD IgG1 antibody was included as isotype control. The mouse PK studies were performed with single intravenous (i.v) dose of 10 mg/kg of the anti-OX40 antibodies and serum samples (n=3/time point/group) were collected at various time points for PK analysis out to Day 21 post-dose.

    [0483] A sandwich ELISA with a colorimetric detection system was used to quantitate anti-OX40 huIgG1 antibodies (IgG1, r:Fv-IgG, r:Fab-IgG, c:Fab-IgG, or c:IgG-IgG) and isotype control anti-gD huIgG1 antibody in C57BL-6 or C.B-17 SCID mouse serum. Microtiter plates were coated with sheep anti-human IgG to capture anti-OX40. Diluted samples, standards, and controls were added to the plate and incubated. Subsequently, goat anti-human IgG-HRP was added for detection and incubated. A peroxidase substrate (tetramethyl benzidine) was added to develop color, and the reaction was stopped by adding 1 M phosphoric acid. The plates were read at 450 nm for detection absorbance and 620 nm for reference absorbance. Sample concentration was determined by entering data into a four-parameter logistic curve-fitting program. The reporting range for the assay was 0.156-20 ng/mL anti-OX40. The minimum sample dilution was 1/100, resulting in a minimum quantifiable concentration of 15.6 ng/mL for C57BL-6 mouse serum or C.B-17 SCID mouse serum.

    [0484] As shown in FIG. 27 and FIG. 28, anti-OX40 antibody formats including IgG1, r:Fv-IgG, r:Fab-IgG, c:Fab-IgG1 and cIgG1 showed similar PK properties, which is comparable to control anti-gD IgG1 antibody in C.B-17 SCID mice. All antibody formats showed a bi-exponential decline after i.v. dosing with a short distribution phase and a long terminal elimination phase. PK for IgG1, r:Fv-IgG, r:Fab-IgG and c:Fab-IgG were in general comparable to anti-gD control until day 10 in C57BL-6 mice (FIG. 29). Higher variability in PK for r:Fv-IgG, r:Fab-IgG and c:Fab-IgG1 at day 14 and 21 can be possibly because of anti-drug antibody development. c:IgG-IgG exposure is slightly lower compared to IgG1 until day 3, but rapid decline in exposure was observed from day 7 possibly because of anti-drug antibody development.

    Example 12. Pharmacodynamic (PD) studies in human OX40-knockin (hOX40ki) mice



    [0485] The in vivo PD of the anti-OX40 antibody formats was tested for their ability to stimulate T cell activation in response to immunization with keyhole limpet hemocyanin (KLH) antigen. On Day 0, human OX40-knockin mice (Genentech, Inc.) were subcutaneously (tail base) injected with PBS or 50 µg Imject mcKLH Subunits (ThermoFisher Scientific; Cat. No. 77649) emulsified in CFA H37 Ra (BD Difco; Cat. No. 231131) at 1:1 ratio for a total volume of 100 µL per mouse. On Day +1, animals were grouped and intravenously administered 10 mg/kg of anti-human OX40 antibodies (all human IgG1 heavy chain constant region) or anti-gD human IgG1 as a control. To quantify the anti-KLH IgG antibody responses, mice from different groups were bled on Day +14 and serum anti-KLH IgG levels were measured by ELISA kit according to the manufacturer's instruction (Life Diagnostics, Inc; Cat. No. KLHG-1).

    [0486] To access memory responses, mice were intraperitoneally rechallenged on Day +16 with 50 µg soluble KLH and draining lymph nodes were profiled 4 days post-rechallenge (Day +20). In some experiments, CD4+ T cells were purified from draining lymph nodes on Day +20 and stimulated in vitro with 10 µg/mL soluble KLH in the presence of purified CD11c+ splenic DCs from WT mice. After 2 days, supernatants were collected and IFN-γ production was assayed using Luminex. Statistical significance was analyzed by Student's t test. Unless otherwise indicated, data represent the mean ± SEM, with p < 0.05 considered statistically significant.

    [0487] FIG. 30, FIG. 31, and FIG. 32 show results from the KLH immunization PD experiment comparing r:Fv-IgG 1A7-3C8 to IgG1 versions of 1A7 and 3C8. The tetravalent biepitopic antibody format superior CD4 T cell expansion and anti-KLH IgG response (FIG. 30), and CD8 T cell expansion and total cell expansion in the draining lymph node (dLN) (FIG. 31). The tetravalent biepitopic format also stimulated greater interferon gamma (IFN-γ) after ex vivo restimulation of purified CD4+ T cells with KLH (FIG. 32).

    [0488] A repeat study directly compared r:Fv-IgG, c:Fab-IgG, and r:Fab-IgG tetravalent biepitopic formats, along with IgG1 versions of 1A7 and 3C8 antibodies. FIG. 33 shows the data from this experiment. r:Fv-IgG and c:Fab-IgG demonstrated clear superiority in expansion of CD4 and CD8 T cells, with r:Fab-IgG showing more modest levels of T cell activation. Altogether these results highlight that the superior in vitro agonist activity of the tetravalent biepitopic antibodies translates into greater T cell activation in vivo.

    Example 13. Anti-tumor activity of tetravalent antibodies



    [0489] Tetravalent antibodies were tested for anti-tumor activity in a syngeneic E.G7-OVA (EL4 expressing chicken ovalbumin (OVA)) lymphoma tumor model in female human OX40 (TNFRSF4) knockin C57BL/6N mice (huOX40.tnfrsf4.ki.B6N) generated at Genentech. Mice were inoculated subcutaneously into the right unilateral flank with 3 million E.G7-OVA tumor cells in 100 microliters of HBSS+matrigel. Mice were allowed to grow tumors until they achieved a mean tumor volume of ∼250 mm3 (5 days after inoculation). At this point (Day 0), mice were recruited into 3 groups, n=10 for all groups. Anti-gD binds to glycoprotein D of Herpes Simplex Virus and serves as a negative control. Antibodies were diluted in sterile PBS and dose volume was 100 ul. All groups were given a single 10 mg/kg dose intravenously on day 1. All antibodies tested comprised human IgG1 heavy chain constant region. Blood was collected 24hrs post dose from 5 mice/group/timepoint. Blood was collected 6 days post dose from the other 5 mice/group. Blood was collected by orbital bleed (collection volume did not exceed 100 ul), under isofluorane-induced anesthesia (inhalation to effect). Serum was harvested from the blood for PK analysis. Measurements and weights were collected 2X/week. Animals exhibiting weight loss of >15% were weighed daily and euthanized if they lost >20% body weight. Animals showing adverse clinical issues were observed more frequently, up to daily depending on severity, and euthanized if moribund. However, no mice in this study showed adverse clinical signs. Mice were euthanized if tumor volumes exceeded 2,000 mm3. The remaining tumors were measured and weighed 2x/week.

    [0490] FIG. 34 shows tumor volume data from the experiment. Tetravalent biepitopic r:Fv-IgG 3C8-1A7 provided superior anti-tumor activity relative to the more modest activity of native IgG1 1A7. These results are consistent with the superior in vitro and in vivo PD activity of this antibody format, and demonstrate the utility of the r:Fv-IgG and other tetravalent biepitopic formats as therapeutic agents.

    Example 14. Structure determination and epitope mapping


    Expression and purification of anti-OX40 ligand Fabs and OX40 ligand ECD



    [0491] Two Fab fragments of anti-OX40 ligand were express and purified in the same way as following. The Fabs were expressed in E. coli. The proteins were purified in two steps.

    [0492] First, the cell lysate supernatant was loaded onto a G sepharose column and eluted with 0.6% acetic acid. Second, Fab-containing fractions were pooled and purified using a SP sepharose column (GE Healthcare). The loading buffer contained 2-(N-morpholino)ethanesulfonic acid (MES) at pH 5.5 and the protein was eluted on a NaCl gradient.

    [0493] Human OX40 ligand extracellular domain (ECD, residues L29-D170) was co-expressed in SF9 insect cells with endoglycosidase H. OX40 ligand containing medium was separated from cell debris by centrifugation. Protein was purified by first using a 10 mL Ni-NTA Superflow (Qiagen) column. OX40 ligand was eluted with buffer containing 20 mM tris(hydroxymethyl)aminomethane (Tris) at pH 8.0, 300 mM NaCl and 300 mM imidazole. Fractions containing the OX40 ligand were pooled and purified by size exclusion chromatography (SEC) on a S-75 column (Pharmacia) equilibrated in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Hepes) pH 7.2 and 200 mM NaCl. The N-terminal his-tag was cleaved by overnight incubation with Thrombin (Sigma) at 4°C. The cleaved OX40 ligand protein was collected in the flow through fraction of a 1.0 mL Ni-NTA Sepharose (Qiagen) column and was further purified by SEC on a S-75 column (Pharmacia) equilibrated in the same S-75 buffer mentioned above.

    Generation and Crystallization of Anti-OX40 Ligand Fabs and antigen complex



    [0494] Equimolar amounts of OX40 ECD (PUR#124725), 1A7 anti-OX40 ligand Fab (PUR#115810) and 3C8 anti-OX40 ligand Fab (PUR#109639) were mixed together and incubated overnight at 4°C. The ternary complex was then purified by SEC on a S200 16/60 column (GE Healthcare) equilibrated in 25 mM Tris pH 7.5 and 0.15 M NaCl. The purified complex was analyzed by SDS PAGE and liquid chromatography mass spectrometry (LC-MS), which revealed all three components in the high molecular weight (HMW) peak fractions of the final SEC step. The HMW peak fractions were concentrated to 10 mg/mL using a Vivaspin concentrator with 10,000 molecular weight cutoff.

    [0495] Crystallization trials were performed using sitting-drop vapor diffusion method with commercially available sparse-matrix screens in 96-well format. Initial crystal hits were observed mostly in high salt conditions. An additive screen (Hampton Research) was performed to improve crystal diffraction quality. The final crystallization condition contained 0.1 M Tris pH 8.5, 1.5 M D-L malic acid pH 7.0 and 0.15 mM dimethyl ethylammonium propane sulfonate (also known as NDSB-195). Crystals were preserved for data collection by brief soaking in a cryo-protectant buffer (25% glycerol added to the reservoir solution), followed by sudden immersion into liquid nitrogen.

    X-ray diffraction data collection and structure determination



    [0496] A diffraction data set of the ternary complex crystal was collected using monochromatic X-rays at Advanced Light Source (ALS) beam line 5.0.2 using a Pilatus-6M detector. Rotation method was applied to a single crystal for the complete data set. Data reduction was performed using program XDS (Kabsch W., Acta Crystallogr D Biol Crystallogr, 2010, 66: 125-132) and the final statistics at 2.6Å are shown in Table 1. The structure was solved by molecular replacement (MR) method using program Phaser (McCoy A. J., Grosse-Kunstleve R. W., Adams P. D., MWinn. D., Storoni L. C., Read R. J., Journal of applied crystallography 2007, 40, 658-674). The two Fab fragments elbow angles were determined by rotation searches against a series of Fab structure models (generated based PDB entry 1FVD) with varying elbow angles ranging from +90° to -10° at 5° interval. Models of elbow angles at +70° and 0° gave the highest signal to noise ratio (3x). A full MR search was then performed using the two best IFVD-based models (+70°, 0° elbow angles) and a previously determined OX40 ECD- 3C8 anti-OX40 fab complex structure (CRY#21829) that had the constant domain (CH1 and CL) of the Fab removed (Compaan D.M., Hymowitz S.G., The Crystal Structure of the costimulatory OX40-OX40L complex, Structure, 2006, 14(8): 1321-30). This MR search resulted in solution of the ternary complex. Inspection of the electron density suggested the Fab model with 0° elbow angle give the best solution and corresponded to the Fab of 1A7. Further adjustment of the elbow angle to the 3C8 fab was required to fit the CH1 and CL domains. The correct 1A7 Fab sequences we built into the initial IFVD-based MR solution using graphics modeling program COOT (Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66, 486-501 (2010)). The structure was further refined using program Phenix (Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 213-221 (2010)) and BUSTER (Dsafa Bricogne G., Blanc E., Brandl M., Flensburg C., Keller P., Paciorek W., Roversi P, Sharff A., Smart O.S., Vonrhein C., Womack T.O. (2016); BUSTER version 2.11.5. Cambridge, United Kingdom: Global Phasing Ltd) in combination with manual model building in COOT (Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66, 486-501 (2010)) in an iterative manner. The computational refinement protocols included minimization of maximum likelihood target functions, anisotropic individual B-factor refinement and TLS refinement methods. The final refinement statistics are shown in Table 1.
    Table 1 Data collection and refinement statistics
     Anti-OX40 1A7, 3C8-OX40 ECD Ternary complexAnti-OX40 3C8 -OX40 ECD Binary complex
    Data collection ALS 5.0.2 ALS 5.0.2
    Space group 1222 P4122
    Cell dimensions    
      a, b, C (Å) 109.8,125.2,197.5 117.0,117.0,118.9
      α, β, γ (°) 90.0, 90.0, 90.0 90.0, 90.0, 90.0
    Resolution (Å) 105.8 -2.61(2.70 - 2.61) * 42.0 - 2.1(2.17 -2.1)*
    Rsym or Rmerge 0.097 (1.13) 0.068 (0.69)
    I/σI 11.08 (1.55) 20.6 (3.65)
    Completeness (%) 100 (100) 99.9 (99.4)
    Redundancy 6.4 (6.2) 12.7 (12.4)
         
    Refinement    
    Resolution (Å) 105.8 -2.61 42.0-2.1
    No. reflections 41721 48695
    Rwork/ Rfree 20.4/25.7 21.8/24.2
    No. atoms 7690 4341
      Protein 7470 4053
      Ligand/ion 14 0
      Water 206 268
    B-factors 62.3 55.9
      Protein 62.5 55.5
      Ligand/ion 93.1  
      Water 52.9 62.0
    R.m.s deviations    
      Bond lengths (Å) 0.019 0.013
      Bond angles (º) 1.84 1.82
    *Highest resolution shell is shown in parenthesis.


    [0497] FIG. 35 shows the structure of the ternary complex of the two Fabs 1A7 and 3C8 bound to OX40. 1A7 and 3C8 bind on diametrically opposed regions of the OX40 receptor. The calculated distance between the C-termini of the 1A7 and 3C8 Fabs in the structure is ∼120 Å. In contrast, the antibody hinge constrains the C-termini of two IgG Fab arms to a maximal distance of ∼35 Å. This analysis suggests that the OX40 binding epitopes and as a consequence orientation of the two Fabs in the structure does not permit intra-IgG OX40 binding. This further suggests a model in which a single OX40 receptor is engaged by two separate r:Fv-IgG molecules, each of has three other Fv's that are binding other OX40 receptors. The result would in theory be immune complexation. This model is consistent with the SEC-MALS data described in Example 10, and the intrinsic agonist activity of the tetravalent biepitopic antibodies.

    [0498] The OX40 epitopes bound by the 1A7 and 3C8 antibodies were determined by visual inspection of the ternary complex structure. FIG. 36 shows the sequence of human OX40 highlighting the binding epitopes of 1A7 (grey boxes) and 3C8 (black outlined boxes). Position numbering is according to that listed in UniProt (The UniProt Consortium, UniProt: the universal protein knowledgebase, Nucleic Acids Res. 45: D158-D169 (2017)). The 1A7 antibody binds to OX40 residues 114-119, 124, 126-127, 129-130, 132, 140, and 142. The 3C8 antibody binds to OX40 residues 68-71, 83-90, 95, and 98. The current disclosure provides data and structural/sequence analysis that suggests that antibodies that bind to these or overlapping epitopes may be good pairs for producing tetravalent biepitopic formats for agonizing the OX40 receptor.

    Example 15. Expansion of anti-OX40 antibody set and OX40 epitope space



    [0499] An antibody discovery campaign was executed to increase the set of antibodies for tetravalent biepitopic targeting of OX40, and to expand coverage to other OX40 epitopes.

    Materials and Methods



    [0500] ClonaCell-HY MediumA (Cat#03801), Medium B (Cat# 03802), Medium C (Cat# 03803), and Medium E (Cat# 03805) are from StemCell Technologies. Cytofusion Medium C (Cat# LCM-C) used for electrofusion is from Cyto Pulse Sciences. SP2ab fusion partner that expresses surface IgG is from Enzo Life Science ((ENZ-70008-0001), Goat anti-rat IgG Fc-HRP conjugated antibody is from Bethyl (A110-236P). TMB one component HRP microwell substrate (Cat# TMBW-1000-01) and TMB stop reagent (Cat# BSTP-1000-01) are from BioFx Laboratories.

    [0501] Sprague Dawley rats were primed once with 50ug human and cyno OX40 antigens following with 25ug injection using CFA/IFA adjuvant for each rat, 6 total boosts had been performed at a week intervals. Three days after the final pre-fusion boost, lymphocytes from immunized rats were harvested.

    [0502] Isolated rat lymphocytes were fused with SP2ab myeloma cells by using the Cyto Pulse CEEF-50 apparatus (Cyto Pulse Sciences). Briefly, after B cell enrichment and IgM+ rat B cells depletion, the isolated IgM- rat B cells and SP2ab cells were mixed at a 1:1 ratio and then resuspended at 10 million cells/ml in Cytofusion Medium C, electrofusion was performed according to manufacturer's guidance. Fused cells were cultured in ClonaCell-HY Medium C with HAT selection at 37°C in a 7% CO2 incubator. After 6 days culture, the fused cells were stained with Goat anti-rat IgG- Alexa Fluor® 647 (Jackson 112-606-071) for 30 mins, then washed twice with FACS buffer, after that, single rat IgG+ hybridoma cell was sorted by FACSAria into 96-well cell culture plates (#353075, Becton Dickinson) with 200 µL/well ClonaCell-HY Medium E. After 7 days culture, hybridoma supernatants were screened by ELISA and all ELISA positive hybridoma clones against both human and cyno OX40 were picked to new 96-well cell culture plates.

    [0503] ELISA assay was performed as follows. 96-well microtiter ELISA plates (Greiner, Germany) were coated with 100 µL/well of either human/cyno OX40 at 1 □g/ml in 0.05 M carbonate buffer (pH 9.6) at 4°C overnight. After washing three times with wash buffer (0.05% Tween 20 in PBS, Sigma), plates were blocked with 200 µL ELISA assay diluents with BSA. 100 µL of cultured supernatants or diluted purified mAbs were added and incubated for 1h at room temperature. The plates were washed three times and incubated with HRP conjugated Goat anti-rat IgG Fc for 1 hour. After washing three times, bound enzyme was detected by addition of 100 µL/well the TMB substrate (BioFX Laboratories, MD, USA) for 5 min. The reactions were stopped by adding 100 µL/well of stop reagent (BioFX, Laboratories, MD, USA) and detection of color at A630 nm.

    [0504] Jurkat cells overexpressing huOX40 were washed twice with phosphate-buffered saline (PBS) containing 1% fetal bovine serum (FBS) and then resuspended in FACS buffer (PBS containing 1% FBS) to final concentration of 5x106 cells/ml. 100 µl cells were added to each well of U-bottom 96 well tissue culture plate (#353077, Becton Dickinson), purified mAbs were added, after 30mins incubation on ice, cells were washed twice with FACS buffer, and subsequently treated with Goat anti-rat IgG- Alexa Fluor® 647 (Jackson Lab, 112-606-071) at 1:400 dilution for 30min. After twice washing with FACS buffer, stained cells were analyzed using FACS Calibur (BD Biosciences), flow cytometry data were analyzed using the FlowJo software (Tree Star, Inc.)

    [0505] The hybridoma supernatants were purified by Protein A affinity chromatography, then sterile filtered (0.2 µm pore size, Nalge Nunc International, NY, USA) and stored at 4°C in PBS. The purified mAbs were confirmed by ELISA before further testing in functional assays.

    [0506] Epitope bins were determined by 96 X 96 array-based SPR imaging system (Carterra USA) as follows. Purified rat anti-OX40 antibodies were diluted at 10 ug/ml in 10 mM sodium acetate buffer pH 4.5(Wasatch Microfluidics, CBNaOAc-pH4.5-0.1L). Using amine coupling, antibodies were directly immobilized onto a SPR sensorprism CMD 200M chip (XanTec Bioanalytics, Germany) using a Continuous Flow Microspotter (Carterra, USA) to create an array of 96 antibodies.

    [0507] For antibody binning, printed chip was loaded on IBIS MX96 SPRi (Carterra USA) and human OX40 protein, diluted to 50 nM in HBS-P buffer, was injected over the chip for 4 minutes at 25°C followed by a second injection of purified antibody, diluted at 10 ug/ml in HBS-P buffer (GE, BR-1006-71) for 4 minutes. The epitope binning data was processed using Wasatch binning software tool.

    [0508] For antibody kinetics, antibodies were immobilized as described above, serial diluted human or cyno OX40 protein, from 300nM, 100nM, 33.3nM, 11.1 nM to 3.7 nM in HBS-P buffer, were injected over the chip and allowed to associate for 3 minutes and disassociate for 10 minutes at 25°C. The kinetic data was processed using Scrubber software

    (BioLogic Software)


    Results



    [0509] 318 hybridoma-produced rat anti-OX40 antibodies were characterized for epitope bin, affinity for OX40 in solution, binding to cell surface OX40, and functional activity alone and when crosslinked with anti-Fc antibody. While numerous antibodies promoted agonism of OX40 when extrinsically crosslinked, none of the 318 antibodies mediated intrinsic agonism on their own (data not shown). Based on the binning, affinity and binding, and activity data, antibodies were cloned from hybridomas into chimeric rat VH and VL domains with human IgG1 heavy and human Ckappa light chains. Antibodies were expressed in HEK293 cells and purified as described above.

    [0510] Table 2 summarizes the epitope bin and equilibrium dissociation constants (KDs) for 34 of the chimeric IgG1 anti-OX40 antibodies, measured using the array-based SPR imaging system (Carterra USA, Wasatch Microfluidics) as described above. Table 2 also shows affinities for the 1A7 and 3C8 IgG1 antibodies measured using a Biacore T200 instrument (GE Healthcare) by capturing antibody and testing binding to analyte OX40.
    Table 2. Properties of expanded anti-OX40 antibody set as IgGs and r:Fv-IgGs
    IgG Formatr:Fv-IgG Format
    AntibodyBinKD (nM)PairKD (nM)Maximal Activity (RLU)
    1A7 1A7 0.15      
    3C8 3C8 1.2 3C8-1A7 0.23 1850
    2A9 8 1.96 2A9-1A7 0.23 1421
    2B4 9 129.3 2B4-1A7 0.91 420
    2B5 11 16.5 2B5-1A7 0.31 1600
    2D8 11 0.912 2D8-1A7 0.35 1555
    2G7 10 1.79 2G7-1A7 0.27 1473
    3F5 11 14.67 3F5-1A7 0.35 1392
    3G5 10 22.58 3G5-1A7 0.59 1735
    3G8 11 18.15   0.66  
    2C7 6 50      
    2A2 2 5.506      
    2F10 10 5.828      
    2E12 4 0.01      
    2D5 10 ND      
    2B12 2 7.266      
    3C2 2 2.399      
    3B1 2 5.95      
    2C10 5 0.763      
    2B7 ND ND      
    2F6 2 30.31      
    2E4 2 0.091      
    3E10 2 2.146      
    2H5 5 0.493      
    2A3 5 5.52      
    3A12 ND ND      
    2C9 10 5.15      
    2D10 ND ND      
    3H12 ND ND      
    2D3 4 0.241      
    3H4 ND ND      
    3D12 ND 5.855      
    3D1 2 ND      
    2E1 2 6.625      
    2H1 4 53      
    2A6 7 288.5      


    [0511] 27 lead antibodies were selected from non-1A7 epitope bins (i.e. antibodies that did not block binding of 1A7 to OX40) and constructed as r:Fv-IgGs paired with the 1A7 variable region. r:Fv-IgG DNAs were constructed and proteins were expressed and purified as described above. Binding affinity of the first 8 r:Fv-IgGs was measured by surface plasmon resonance (SPR) technology using a Biacore T200 instrument (GE Healthcare) as described above. Table 2 shows OX40 binding affinities (KDs) of the r:Fv-IgGs. IgG and r:Fv-IgG versions of the first 8 of these antibodies were tested for the ability to agonize OX40 receptor in the OX40+ Jurkat cell luciferase assay (FIG. 37). As expected, none of the new antibodies mediate agonist activity as IgGs in the absence of extrinsic crosslinking (left graph). In contrast, all but one of the antibody variable regions promotes OX40 agonism when paired with the 1A7 variable region in the r:Fv-IgG format (right graph). The exception is the 2B4 antibody, which did not mediate activity in the context of an r:Fv-IgG when paired with 1A7. The maximal activity of the r:Fv-IgGs are provided in Table 2.

    Example 16. Engineered linker variants of r:Fv-IgG



    [0512] A critical element of the antibody formats of the present disclosure is the use of linkers to connect immunoglobulin domains. A series of r:Fv-IgGs with variant linkers were designed. A first set of linkers comprised sequences that substantially comprise serine, glycine, or serine and glycine. Serine and glycine are commonly used as linkers due to their flexibility and favorable solution properties. A second set of linkers comprised sequences that substantially comprise native antibody sequence. In these linkers, native sequences that compose antibody sequences are used as linkers. Two particularly useful native antibody sequences are heavy chain sequences that reside at the elbow between VH1 and CH1 domains, and light chain sequences that reside at the elbow between the VL and CL domains. The utility of native antibody sequence linkers is that they can be used to reduce some non-native sequence within a tetravalent antibody format. The length of a given linker can vary, from 1 residue to 20 or more residues. Greater length typically provides greater flexibility between linked immunoglobulin domains. Table 3 describes the linkers that were engineered into the r:Fv-IgG 3C8-1A7 tetravalent biepitopic format.
    Table 3. Description of linkers
    NameHeavy Chain Linker (LH)LH SEQ IDLight Chain Linker (LL)LL SEQ ID
    Glycine/Serine Linkers      
    GS(Short) GGGGSG 270 GGSGG 271
    GS(Medium) GGGGSGGGGS 272 GGGGSGGGGS 272
    GS(Long) GGGGSGGGGS GGGG 273 GGSGGGGSGGGGS 274
    Native Antibody Sequence Linkers  
    Elbow(Short) ASTKGP 275 RTVAAP 276
    Elbow(Long) ASTKGPSVFPLAP 277 RTVAAPSVFIFPP 278


    [0513] DNAs encoding variant linkers of r:Fv-IgG 3C8/1A7 were constructed as described above, and proteins were expressed in HEK293 cells and purified as described above. r:Fv-IgGs were tested for OX40 agonist activity in both the primary human T cell and Jurkat reporter assay as described above. Data in FIG. 38 demonstrate that while glycine-serine linkers provided marginally enhanced agonism of OX40 relative to native antibody elbow linkers in the Jurkat reporter assay, the data highlight that all engineered linkers provide effective links between Fv and IgG portions of the r:Fv-IgG to enable agonism of the OX40 receptor.

    Example 17. Affinity engineering of tetravalent formats



    [0514] Variants of both the 1A7 and 3C8 variable regions were engineered in order to explore the dependence of agonist activity of tetravalent biepitopic formats on the affinity of individual OX40 binding components. The affinity engineering approach leveraged NNK-walk library generation coupled with phage display and Next Generation Sequencing (NGS) (described more fully in Koenig et al., J Biol Chem 2015, 290(36):21773-21786).

    [0515] Variable domain NNK-Walk libraries of 1A7 and 3C8 were designed and constructed. 1A7 and 3C8 Fabs were subcloned into a phagemid construct. Diversified 1A7 VH residues were 28-35, 49-65, 93-102 and VL residues were 28-34, 50-56, 91-96. Diversified 3C8 VH residues were 28-35, 50-65, 95-97 and VL residues 28-34, 50-56, 89-96. Diversified residues in the phagemid construct were randomized by NNK walk, each with an NNK codon at one position targeted for randomization. NNK encodes for all 20 amino acids (n = G, A, T, and C; K= G and T, in equal proportions). The template for each mutagenesis reaction was generated by introducing stop codons (TAA) at all of the positions targeted for randomization in each reaction. VH and VL libraries were generated separately by electroporating the pool of DNA products from mutagenesis reactions into Escherichia coli XL1 cells, yielding ∼109 transformants.

    [0516] Phage encoding 1A7 and 3C8 libraries were panned against recombinant OX40 protein. OX40-binding clones were selected using 96 well ELISA plate. Phage display libraries were incubated with 5ug/ml plate bound OX40 as 1st round, followed by a few rounds of solution bound OX40 selection at 50, 10, 2, 0.5, 0.1 nM, and 0.1nM biotinlayted + 100nM non-biotinylated on Streptavidin coated plate. Bound phage from each run was washed and eluted in 100 mM HCl for 20 min, then neutralized with 1/10 volume of 1 M Tris pH 11.0 and used to infect E. coli for amplification and next round selection. Phage titer was monitored at each run. Panning was stopped at the run at which enrichment was not observed. DNA was extracted from the bacteria pellet from the previous run.

    [0517] Enriched NNK walk libraries post-panning were deep sequenced. Phagemid DNA was isolated from E. coli XLI cells from both the unselected and the selected 1A7 and 3C8 NNK library and used for PCR amplification of VL and VH regions using Phusion polymerase (New England BioLabs). PCR products were purified to generate libraries using the TruSeq Nano DNA library preparation kit (Illumina). Multiplexed adapter-ligated libraries with unique barcodes were sequenced on an Illumina MiSeq sequencing system for 300-bp paired-end sequencing. The sequences for enrichment analysis were from the panning at 0.1nM and 0.5nM biotinlated human OX40 for 1A7 and 3C8 respectively.

    [0518] For analysis of NGS results, sequencing reads were splitted into R1/R2 reads if the amplicon size was greater than 300bps. In that case, a merging step was conducted using the FLASH program[Magoč, T. & Salzberg, S. L. Bioinformatics 2011] to reconstruct the full amplicon. For each reconstructed full amplicon, nucleotide sequences (9bps long each) flanking the mutational region were used to locate and extract the mutated section sequence. The extracted sequence was checked against a known template length to screen for unwanted indel sequences. Remaining sequences were translated to amino acids and could be further filtered depending on the researchers' needs. (i.e. keeping only extracted sequence with only 1 mutation, or discarding extracted sequence with mutation on known unmutated position). After screening and keeping those amino acids sequences that satisfied the criteria (i.e. the ones listed above), these sequences were counted based on their unique amino acids sequence. Then, only amino acids sequence above certain cutoffs (i.e. standard >2 counts or Vivian's >20 counts) could be used to further discard unwanted amino acids sequence. These "cleaned" amino acids sequence would then be used to calculate the Log2 ratio of the amino acids' frequencies in the mutated section. In particular, the frequency of each of the 20 residues in each position was determined from the "cleaned" amino acids sequences for the pre-panned and the post-panned libraries. The pre-panned library was sometime the first round panned libraries given the major under-sampling issue with the very initial library. Thereafter, log2 was taken to the frequency ratio of the post- over pre-panned library. Log2 ratio of at least 4-5 (i.e. >=16x-32x enrichments) was considered real enrichment.

    [0519] Based on the NNK walk phage panning NGS results, substitutions were select to both increase and reduce the affinity of the 1A7 and and 3C8 variable regions. DNAs encoding heavy (human IgG1 constant region) and light chain (Ckappa constant region) variants were constructed as described above, and antibodies were expressed in HEK293 cells and purified as described above. Binding of variants to OX40 was tested using Biacore as described above. Table 4 and Table 5 present the variant name, substitution(s), and OX40 affinities (KDs) of all variants generated and tested. Positions of all substitutions are numbered according to the Kabat convention.
    Table 4. 1A7 Affinity Variants
    VariantAlso CalledSubstitution(s)KD (M)
    1A7 1A7 WT 1.40E-10
    1A7.HC.T28E   T28E 1.36E-10
    1A7.LC.N31Q   N31Q 1.00E-10
    1A7VH.001   S32H;N54Q;V102Y 3.46E-10
    1A7VH.003   T30F;N54L;L94H 5.84E-10
    1A7VH.004   M34L;N60Q;S101A 2.26E-10
    1A7VH.005   T30I;E65S 2.39E-10
    1A7VH.006   S32A;V93S 2.32E-10
    1A7VH.007   T30G;D56E 2.76E-10
    1A7VH.008   D31R;D53Y;L94F 4.92E-10
    1A7VH.009   K62V;V102R 1.33E-10
    1A7VH.010   F29L;S58L;S101V 2.21E-10
    1A7VH.01   T28S 1.89E-10
    1A7VH.02   T30E 2.17E-10
    1A7VH.03   T30Q 1.65E-09
    1A7VH.04   D31E 2.42E-10
    1A7VH.05   S32Q 1.07E-09
    1A7VH.06   S32T 3.68E-10
    1A7VH.07   Y33L 5.08E-09
    1A7VH.08   Y33P 1.98E-10
    1A7VH.09   M34I 5.83E-11
    1A7VH.10   M34S 5.84E-10
    1A7VH.12   N54Q 2.52E-10
    1A7VH.13   G55P 3.38E-10
    1A7VH.14   G55Q 2.05E-10
    1A7VH.15   D56E 1.27E-10
    1A7VH.16   D56N 4.95E-10
    1A7VH.17   S57A 1.37E-10
    1A7VH.18   S57N 3.93E-10
    1A7VH.19   S58Q 2.21E-10
    1A7VH.20   N60T 1.46E-10
    1A7VH.21   F63S 1.84E-10
    1A7VH.22   R64N 1.96E-10
    1A7VH.23   E65Q 1.02E-10
    1A7VH.24   V93I 1.84E-10
    1A7VH.25   L94F 1.57E-10
    1A7VH.26   L94I 1.62E-10
    1A7VH.27   L94Y 4.49E-10
    1A7VH.28   A95T 3.38E-10
    1A7VH.29   R97S 2.83E-08
    1A7VH.30   W98F 8.78E-11
    1A7VH.31   W98L 2.91E-09
    1A7VH.32   W98N 4.04E-08
    1A7VH.33   W98Y 5.19E-10
    1A7VH.34   Y199N 3.63E-10
    1A7VH.35   F100A 2.87E-09
    1A7VH.36   S101N 1.52E-10
    1A7VH.37   V102A 1.68E-10
    1A7VH.38   V102Q 1.95E-10
      1A7 (Low) P96A 4.89E-09
    1A7VH09/VL19 1A7 (High) M34I/R53Y 9.00E-11
    1A7-VL001   S30L;L54I;L94E 6.12E-11
    1A7-VL002   N31M;S56A;P96V 3.03E-10
    1A7-VL003   Y32F;L54A;L94S 1.60E-10
    1A7-VL004   S30G;S52I;H92Q 1.74E-10
    1A7-VL005   L33I;S56V 1.48E-10
    1A7-VL006   S56M;P96A 3.76E-10
    1A7-VL007   N31S;L54S;L94S 1.22E-10
    1A7-VL008   D28S;L54N;T93G 3.73E-10
    1A7-VL009   S30G;R53A;T93G 1.38E-10
    1A7-VL010   N31L;L54S;T93S 1.06E-10
    1A7-VL01   D28E 1.07E-10
    1A7-VL02   D28Q 1.81E-10
    1A7-VL03   I29N 3.31E-10
    1A7-VL04   I29Q 6.52E-10
    1A7-VL05   S30D 1.39E-10
    1A7-VL06   S30G 1.46E-10
    1A7-VL07   S30N 9.58E-11
    1A7-VL08   N31E 1.19E-10
    1A7-VL09   Y32A 3.33E-08
    1A7-VL10   L33A 1.66E-10
    1A7-VL11   L33I 1.30E-10
    1A7-VL12   N34S 1.90E-09
    1A7-VL13   Y50A 3.20E-10
    1A7-VL14   Y50Q 2.53E-09
    1A7-VL15   T51G 1.40E-10
    1A7-VL16   S52D 1.20E-10
    1A7-VL17   R53F 7.75E-11
    1A7-VL18   R53N 9.34E-11
    1A7-VL19   R53Y 4.80E-11
    1A7-VL20   L54I 1.57E-10
    1A7-VL21   G91E 1.86E-09
    1A7-VL23   H92N 2.47E-10
    1A7-VL24   L94I 1.60E-10
    1A7-VL25   L94N 1.38E-10
    1A7-VL26   L94Q 1.39E-10
    1A7-VL27   P95A 1.30E-10
    1A7-VL28   P96S 4.06E-10
    Table 5. 3C8 Affinity Variants
    VariantAlso CalledSubstitution(s)KD (M)
    3C8_hIgG1 3C8 WT 1.23E-09
    3C8VH-01   A28K 1.39E-09
    3C8VH-02   F29E 8.98E-09
    3C8VH-03   T30H 7.99E-10
    3C8VH-04   T30K 2.13E-09
    3C8VH-05   Y32L 9.81E-08
    3C8VH-06   Y32V 4.18E-08
    3C8VH-07   L33S 0.0000018
    3C8VH-08   E35I 3.59667E-08
    3C8VH-09   V50F 7.735E-09
    3C8VH-10   V50S 2.01E-09
    3C8VH-11   I51P 0.000000137
    3C8VH-12   N52L 2.15E-08
    3C8VH-13   G53H 2.13E-09
    3C8VH-14   G53Q 1.76E-09
    3C8VH-15   S54F 2.7565E-09
    3C8VH-16   S54I 1.09E-09
    3C8VH-17   S54P 1.59E-09
    3C8VH-18   G55E 9.285E-10
    3C8VH-19   D56E 7.99E-10
    3C8VH-20   D56Q 1.56E-09
    3C8VH-21   T57F 1.755E-09
    3C8VH-22   T57H 1.32E-09
    3C8VH-23   T57I 1.57E-09
    3C8VH-24   T57N 1.35E-09
    3C8VH-25   Y58E 1.43E-09
    3C8VH-26   Y58P 0.000000495
    3C8VH-27   Y59P 1.19E-09
    3C8VH-28   E61K 2.02E-09
    3C8VH-29   F63H 1.41E-09
    3C8VH-30   K64Q 6.96E-10
    3C8VH-31   D95I 1.22E-08
    3C8VH-32   D95T 1.5E-09
    3C8VH-33   R96F 3.92E-09
    3C8VH-34   R96G 1.39E-08
    3C8VH-35   R96Y 1.42E-09
    3C8VH-36   L97E 0.000000122
    3C8VH-37   L97S 4.28E-08
    3C8VH-triple01   A28K/E61A 1.31E-09
    3C8VH-triple02   I34V/G55E 1.03E-09
    3C8VH-triple03   T30V/F63A 9.51E-10
    3C8VH-triple04   T30K/F63S 1.03E-09
    3C8VH-triple05 3C8 (High) N31I/K64L 3.735E-10
    3C8VH-triple06   A28Q/F63Y 1.17E-09
    3C8VH-triple07   F29Y/G65S 1.25E-09
    3C8VH-triple08   T30L/Y59F 1.12E-09
    3C8VH-triple09   T30E/I51G 9.73E-10
    3C8VH-triple10   N31I/F63V 4.8125E-10
    3C8VL-01   H24P 4.78E-09
    3C8VL-02   H24R 1.03E-09
    3C8VL-03   H24S 1.38E-09
    3C8VL-04   H24V 8.72E-10
    3C8VL-05   A25L 1.03E-09
    3C8VL-06   A25P 1.63E-09
    3C8VL-07   A25V 1.71E-09
    3C8VL-08   S26G 9.51E-10
    3C8VL-09   S26P 4.46E-09
    3C8VL-10   Q27E 5.80E-10
    3C8VL-11   Q27P 1.63E-09
    3C8VL-12   Q27S 9.76E-10
    3C8VL-13   D28P 1.65E-09
    3C8VL-14   I29Q 2.47E-08
    3C8VL-15   I29Y 8.35E-09
    3C8VL-16   S30E 7.54E-10
    3C8VL-17   S30Q 1.09E-09
    3C8VL-18   S31E 9.63E-10
    3C8VL-19   S31Q 9.12E-10
    3C8VL-20   Y32L 2.13E-08
    3C8VL-21   Y32P 2.51E-09
    3C8VL-22   I33E 2.78E-09
    3C8VL-23   I33L 1.02E-09
    3C8VL-24   I33Y 1.83E-08
    3C8VL-27   H50N 6.76E-10
    3C8VL-28   G51P 5.01E-08
    3C8VL-29   N53P 3.50E-09
    3C8VL-30   L54D 4.06E-08
    3C8VL-31   L54F 3.80E-08
    3C8VL-32   E55F 2.59E-08
    3C8VL-33   E55T 1.49E-08
    3C8VL-34   S56D 7.70E-10
    3C8VL-35   S56E 5.91E-10
    3C8VL-36   S56I 7.10E-10
    3C8VL-37   S56V 3.86E-10
    3C8VL-38   S56Y 7.88E-10
    3C8VL-39   V89G 1.43E-09
    3C8VL-40   V89K 6.87E-06
    3C8VL-41   H90F 2.68E-08
    3C8VL-42   H90Q 1.44E-09
    3C8VL-43   Y91E 2.70E-08
    3C8VL-44   Y91V 8.31E-09
    3C8VL-45   A92P 3.08E-08
    3C8VL-46   A92S 7.14E-10
    3C8VL-47   Q93P 7.41E-09
    3C8VL-48   F94G 1.18E-08
    3C8VL-49   F94Q 9.77E-09
    3C8VL-50   F94R 6.74E-10
    3C8VL-51   P95I 9.89E-09
    3C8VL-52 3C8 (Low) P95Y 5.45E-08
    3C8VL-53   Y96E 3.66E-09
    3C8VL-54   Y96L 8.43E-10
    3C8VL-triple01   D28S;T52S;F94L 9.38E-10
    3C8VL-triple02   S30Y;N53K;A92S 4.77E-10
    3C8VL-triple03   S30N;S56L;Y96L 6.02E-10
    3C8VL-triple04   I33V;G51S;Y96F 4.98333E-10
    3C8VL-triple05   V34S;S56V;V89G 9.315E-10
    3C8VL-triple06   S56E;Q93N 8.07E-10
    3C8VL-triple07   V34T;S56L;H90G 1.22E-09
    3C8VL-triple08   S30G;T52S;Y96L 1.0345E-09
    3C8VL-triple09   S31N;T52S;Q93S 1.01E-09
    3C8VL-triple10   I33L;S56E 4.79E-10
    3C8VL-YA     2.10567E-08


    [0520] Variants were selected for incorporation into r:Fv-IgG 3C8-1A7. Selected variants are designated as High or Low in Table 4 and Table 5 above. Variants of 1A7 and 3C8 with increased affinity (lower KD) are referred to as 1A7(High) and 3C8(High) respectively. Variants of 1A7 and 3C8 with reduced affinity (higher KD) are referred to as 1A7(Low) and 3C8(Low) respectively.

    [0521] DNAs encoding high and low affinity variants of r:Fv-IgG 3C8-1A7 were constructed as described above. All constructs comprised a human IgG1 heavy constant region and human Ckappa light constant region. Variants were expressed in HEK293 cells and purified as described above. Binding of variants to OX40 was tested using Biacore as described above. FIG. 39 shows a plot of the affinities of high and low affinity variant versions of IgG1 and r:Fv-IgG formats. Affinity differences of up to 2 logs in IgG1 format translated to more modest affinity differences in r:Fv-IgG format.

    [0522] Affinity variant r:Fv-IgGs were tested for OX40 agonist activity in the primary human CD4+ memory T cell assay and OX40+ Jurkat cell luciferase reporter assay. The data are shown in FIG. 40. Increased and/or reduced affinity in 1A7 and 3C8 Fv's resulted in modest differences in OX40 agonism activity, and overall a range of affinities for the independent binding units are permissive for enabling intrinsic agonism activity of tetravalent biepitopic formats.

    Example 18. Agonism of Death Receptor 5 Using Tetravalent Biepitopic Antibodies



    [0523] The multivalent biepitopic approach was explored to engineer agonism activity into antibodies targeting the TNFRSF member Death Receptor 5 (DR5), also known as TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) and TNFRSF10B. DR5 is a target of therapeutic interest due to its role in mediating mediating apoptosis of cancer cells (Ashkenazi 2015, J Clin Invest 125(2): 487-489). It has been established that antibodies against DR5 rely on FcγR-mediated crosslinking in vivo for activity (Wilson et al., 2011, Cancer Cell 19:101-13; Kim & Ashkenazi, 2013, J Exp Med 210:1647-51).

    [0524] A set of 16 mouse anti-DR5 monoclonal Abs were generated from classical hybridoma-based technology. DNAs encoding heavy and light chains were cloned from hybridomas and subcloned into the pRK vector as described above with mouse IgG2a heavy constant and mouse Ckappa constant chains. The humanized anti-DR5 human IgG1 antibody drozitumab (Kang et al., 2011, Clin Cancer Res 17(10): 3181-3192), which has been previously characterized pre-clinically and clinically, was also produced as a comparator. Antibodies were expressed using CHO cells with a double-knock out for pro-apoptotic factors Bax and Bak (Bax-Bak-) (Macaraeg NF et al., 2013, Biotechnol Prog 29:1050-8) and purified over a Protein A column. When needed, most active antibodies were further purified to remove aggregate species via chromatography with either a size exclusion column, an SP cation exchange column, or a hydrophobic interaction column.

    [0525] Anti-DR5 antibodies were classified into different epitope groups utilizing the array-based surface plasmon resonance imaging (SPRi) technique (Carterra USA, Wasatch Microfluidics). The epitope binning assay, analogous to a "classical" sandwich assay, utilized microfluidics technology to immobilize the anti-DR5 antibodies on a label-free biosensor, saturate all surface bound antibodies with purified recombinant flag-tagged DR5 extracellular domain (ECD) followed by the addition of each antibody to compete for binding to DR5. Antibodies were immobilized onto a gold surface biosensor using EDC/NHS chemistry in pre-defined locations. The immobilized antibodies were saturated with the extracellular domain of DR5 antigen followed by competition of the antibody/DR5 complex with a second antibody in the set. Blocking the binding of a solution antibody against an immobilized antibody/antigen complex indicated that antibodies belong to the same epitope group. Removal of antigen and solution antibody, re-saturation of the immobilized antibody with the DR5 antigen, and addition of a different antibody was repeated until all antibodies were tested. Once all antibodies were tested and placed into epitope groups, a network plot was generated to identify different epitopes that are recognized by the 16 mouse antibodies. The 16 mouse antibodies were distributed between three distinct epitope groups that were identified for the DR5 antigen represented as a network plot. Upon closer examination of the SPR sensorgrams, 3 mouse antibodies demonstrated low binding affinities with SPR responses at baseline. Removal of these antibodies defined the remaining 13 antibodies into their respective epitope groups. Epitope groups of the murine antibodies and drozitumab are provided in Table 6.
    Table 6. Summary of epitope bin and affinities of DR5-specific mouse monoclonal antibodies.
    Epitope GroupAntibodyKD (nM)Stdev (+/-)
    1 12A1 20.43 3.57
    1 13E11 52.23 2.35
    1 13E3 12.23 1.33
    2 11H12 0.72 0.18
    2 3F11 1.03 0.17
    2 3H3 0.97 0.24
    2 5C7 6.43 0.75
    2 7G4 52.37 4.48
    3 1B10 8.71 0.17
    3 14G8 4.46 1.99
    3 3C9 ND  
    3 3D5 2.63 0.16
    3 3H1 ND  
    3 4D9 0.33 0.12
    3 4H10 0.41 0.1
    3 6A5 57.83 1.51
    Drozitumab Drozitumab 3.35 0.7


    [0526] Binding affinities of antibodies to soluble DR5 antigen was measured using surface plasmon resonance (SPR). Binding experiments were carried out using SPR measurements on a Biacore T200 instrument (GE Healthcare) at 25 °C. Test proteins were immobilized at 1 ug/mL on an Protein-A coated sensor chip. Fivefold serial dilutions of the analyte, DR5-flag (100 to 0.16 nM) were injected in running buffer (HBS-P+ Buffer 1x; 0.01 M HEPES, 0.15 M NaCl, 0.05% (v/v) Surfactant-20 pH 7.4) at a flow rate of 30 µL/min and sensorgrams for association and disassociation phases were recorded. Analytes were injected for 180 s and allowed to dissociate for 600 s. Nonlinear regression analysis of the data fitted to a 1:1 Langmuir binding model provided association (kon), dissociation (koff) rate constants, and equilbrium dissociation constants (KD). For antibodies with fast association and dissociation rate constants, steady-state affinity analysis was also obtained. Affinities of mouse antibodies and drozitumab are shown in Table 6. KDs ranged from 0.1 to 100 nM for antibodies of epitope groups 2 and 3, while antibodies from group 1 displayed KD > 10 nM.

    [0527] Bivalent biepitopic and tetravalent monoepitopic and biepitopic versions of select anti-DR5 antibodies were engineered. DNAs encoding the VH and VL domains of select murine antibodies 13E3, 3H3, 4D9, and 11H12 were inserted into the pRK vector comprising human IgG1 heavy constant and human Ckappa light constant regions, as described above to create mouse Fv - human IgG1/Ckappa chimeric IgGs. Biepitopic versions of select combinations of 13E3, 3H3, 4D9, 11H12, and drozitumab antibodies were engineered by inserting VH regions into the appropriate heavy chain constructs with "knob" or "hole" variants (Ridgway JBB et al., 1996, Protein Eng 9:617-21). DNA encoding VH regions was subcloned into a variant human IgG1 comprising knob mutation T366W (EU numbering), with a C-terminal 6His tag, or subcloned into a variant human IgG1 comprising hole mutations T366S/L368A/Y407V (EU numbering), with a C-terminal Flag tag. Tetravalent biepitopic versions of select combinations of 13E3, 3H3, 4D9, 11H12, and drozitumab antibodies were engineered using the r:Fab-IgG version 2 (V2) format as described in Example 2 and illustrated FIG. 9. This format uses the knob/hole variants described above to promote heterodimer formation. DNA encoding VH regions was subcloned into a variant human IgG1 comprising knob mutation T366W (EU numbering) or subcloned into a variant human IgG1 comprising hole mutations T366S/L368A/Y407V (EU numbering).

    [0528] IgGs, Biepitopic IgGs, and r:Fab-IgGs were expressed the Bax-/Bak- CHO strain and purified as described above. Biepitopic IgGs and r:Fab-IgGs were produced using a method based on in vitro assembly of separate half-antibodies (Spiess et al., 2013, Nat Biotechnol 31(8):753-8). Knob/Hole variant half-antibodies were expressed as half-antibodies, assembled in vitro, and purified as described above.

    [0529] IgG, Biepitopic IgG, and r:Fab-IgG anti-DR5 antibodies were tested for their ability to promote caspase 8 activity. HT-29 and Colo205 cell lines were obtained from the American Type Cell Culture Collection. Cells were maintained in RPMI medium supplemented with L-glutamine (L-glut) and 10% fetal bovine serum (FBS) (Invitrogen Inc., Carlsbad, CA, USA) under 5% CO2 at 37°C. Antibodies were either tested at 50 nM, or dilutions of antibodies were prepared in growth media. 100 µL of each antibody sample was added to each well in a 96-white walled plate (Thermo Scientific). Adherent cells were trypsinized and detached from tissue culture flasks. 50 µL of a 1.0x106 cells/mL cell suspension was added to 100 µL of Ab in the 96-white walled plate. Cells were incubated for 4 hr for caspase-8 and 24 hr for cell viability at 37°C, 5% CO2. Caspase activity and cell viability were assessed using Caspase-Glo 8 and CellTiter-Glo (Cell Viability) luminescence assays from Promega. Experiments were done in triplicate and luminescence was read using Envision (PerkinElmer). Fold caspase 8 activity and % cell viability were calculated by normalizing to cells alone.

    [0530] FIG. 41 shows caspase 8 activity at 50 nM antibody concentration for IgG1, biepitopic IgG1, and biepitopic r:Fab-IgG versions of the anti-DR5 antibodies against human colorectal adenocarcinoma HT-29 cells. Neither the bivalent monoepitopic IgG1 nor bivalent biepitopic IgG1 antibodies mediated caspase 8 activity on their own, consistent with the requirement of extrinsic crosslinking for activity. Drozitumab IgG1 may have exhibited some modest level of activity in this experiment. In contrast, teravalent biepitopic r:Fab-IgG formats that paired drozitumab with 4D9 (Droz-4D9) and 11H12 with 4D9 (11H12-4D9) mediated intrinsic agonist activity against DR5 to promote caspase 8 signaling. Interestingly, the tetravalent monoepitopic r:Fab-IgG drozitumab-drozitumab format, wherein all 4 Fvs in the r:Fv-IgG are the drozitumab Fv, also mediated intrinsic agonist activity. Both tetravalent biepitopic r:Fab-IgG 13E3-3H3 and tetravalent monoepitopic r:Fab-IgG 4D9-4D9 mediated marginal to no DR5 agonist activity.

    [0531] The drozitumab-4D9 combinations were selected for further characterization. IgG, biepitopic IgG, and r:Fab-IgG antibodies were tested for caspase 8 activity and antiproliferative activity against both HT-29 cells and a human colorectal adenocarcinoma Colo205 cell line. FIGs. 42-43 show dose-response curves measuring caspase 8 activity (FIG. 42) and antiproliferative activity (FIG. 43) against HT-29 cells. FIGs. 44-45 show dose-response curves measuring caspase 8 activity (FIG. 44) and antiproliferative activity (FIG. 45) against Colo205 cells. Neither the IgG1 version of 4D9, the tetravalent monoepitopic r:Fab-IgG version of 4D9, nor the bivalent biepitopic combination of drozitumab with 4D9 mediated caspase 8 activity nor had any effect on cell viability of either cell line. IgG1 drozitumab mediated modest caspase 8 activity against both cell lines and anti-proliferative activity against Colo205 cells at higher concentrations. Tetravalent monoepitopic r:Fab-IgG drozitumab-drozitumab promoted superior caspase 8 activity to the IgG1 version for both cells lines, and mediated potent anti-proliferative activity against Colo205 cells. The tetravalent biepitopic combination of drozitumab and 4D9, r:Fab-IgG drozitumab-4D9, mediated the strongest level of agonist activity in terms of both potency and maximal activity relative to the other antibodies. r:Fab-IgG drozitumab-4D9 promoted profound caspase 8 activity in both cell lines and anti-proliferative activity against Colo205 cells. The lack of impact of these agents on HT-29 cell viability despite the strong caspase 8 activity suggests that this cell line is insensitive or weakly sensitive to signaling through the extrinsic death receptor-mediated pathway. This contrasts with the Colo205 cell line, where DR5 agonism and caspase 8 activation by r:Fab-IgG drozitumab-4D9, r:Fab-IgG drozitumab-drozitumab, and to a lesser extent IgG1 drozitumab leads to inhibition of cell proliferation. Nonetheless, altogether the data support the optimal targeting of multiple epitopes in multivalent antibody formats to agonize target receptors for therapeutic purposes.

    SEQUENCE LISTING



    [0532] 

    <110> Genentech, Inc. LAZAR, Greg YANG, Yanli CHRISTENSEN, Erin H. HANG, Julie KIM, Jeong HARRIS, Seth

    <120> MULTIVALENT AND MULTIEPITOPIC ANTIBODIES
    HAVING AGONISTIC ACTIVITY AND METHODS OF USE

    <130> 146392037740

    <140> Not Yet Assigned
    <141> Concurrently Herewith

    <150> 62/371,671 <151> 2016-08-05

    <160> 303

    <170> FastSEQ for Windows Version 4.0

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    <210> 129
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 129

    <210> 130
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 130

    <210> 131
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 131

    <210> 132
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 132

    <210> 133
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 133

    <210> 134
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 134



    <210> 135
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 135

    <210> 136
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 136

    <210> 137
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 137

    <210> 138
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 138

    <210> 139
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 139



    <210> 140
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 140

    <210> 141
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 141

    <210> 142
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 142

    <210> 143
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 143

    <210> 144
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 144



    <210> 145
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 145

    <210> 146
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 146



    <210> 147
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 147

    <210> 148
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 148

    <210> 149
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 149

    <210> 150
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 150

    <210> 151
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 151



    <210> 152
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 152

    <210> 153
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 153

    <210> 154
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 154

    <210> 155
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 155

    <210> 156
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 156



    <210> 157
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 157

    <210> 158
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 158

    <210> 159
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 159

    <210> 160
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 160

    <210> 161
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 161



    <210> 162
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 162

    <210> 163
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 163



    <210> 164
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 164

    <210> 165
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 165

    <210> 166
    <211> 113
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 166

    <210> 167
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 167

    <210> 168
    <211> 113
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 168



    <210> 169
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 169

    <210> 170
    <211> 113
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 170

    <210> 171
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 171

    <210> 172
    <211> 5
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <220>
    <221> VARIANT
    <222> 1
    <223> Xaa = Asp or Glu

    <220>
    <221> VARIANT
    <222> 2
    <223> Xaa = Ser or Ala

    <400> 172

    <210> 173
    <211> 17
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <220>
    <221> VARIANT
    <222> 6
    <223> Xaa = Asn or Ser

    <220>
    <221> VARIANT
    <222> 7
    <223> Xaa = Ala or Gly

    <220>
    <221> VARIANT
    <222> 8
    <223> Xaa = Asp or Ser

    <220>
    <221> VARIANT
    <222> 9
    <223> Xaa = Ala or Ser

    <400> 173

    <210> 174
    <211> 8
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <220>
    <221> VARIANT
    <222> 5
    <223> Xaa = Tyr or Ala

    <220>
    <221> VARIANT
    <222> 6
    <223> Xaa = Ala or Phe

    <220>
    <221> VARIANT
    <222> 7
    <223> Xaa = Ser or Ala

    <220>
    <221> VARIANT
    <222> 8
    <223> Xaa = Ala or Val

    <400> 174

    <210> 175
    <211> 9
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <220>
    <221> VARIANT
    <222> 2
    <223> Xaa = Ala or Gln

    <220>
    <221> VARIANT
    <222> 3
    <223> Xaa = Ala or Gly

    <220>
    <221> VARIANT
    <222> 4
    <223> Xaa = Ala or His

    <220>
    <221> VARIANT
    <222> 5
    <223> Xaa = Ala or Thr

    <220>
    <221> VARIANT
    <222> 6
    <223> Xaa = Ala or Leu

    <220>
    <221> VARIANT
    <222> 7
    <223> Xaa = Ala or Pro

    <220>
    <221> VARIANT
    <222> 8
    <223> Xaa = Ala or Pro

    <400> 175

    <210> 176
    <211> 17
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <220>
    <221> VARIANT
    <222> 9
    <223> Xaa = Thr, Ala, or Gln

    <400> 176

    <210> 177
    <211> 7
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <220>
    <221> VARIANT
    <222> 7
    <223> Xaa = Ser, Glu, or Gln

    <400> 177

    <210> 178
    <211> 9
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <220>
    <221> VARIANT
    <222> 1
    <223> Xaa = Val or Ala

    <220>
    <221> VARIANT
    <222> 2
    <223> Xaa = His or Ala

    <220>
    <221> VARIANT
    <222> 9
    <223> Xaa = Tyr or Ala

    <400> 178

    <210> 179
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 179

    <210> 180
    <211> 117
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 180

    <210> 181
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 181

    <210> 182
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 182



    <210> 183
    <211> 117
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 183

    <210> 184
    <211> 117
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 184



    <210> 185
    <211> 451
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 185



    <210> 186
    <211> 219
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 186

    <210> 187
    <211> 219
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 187



    <210> 188
    <211> 450
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 188



    <210> 189
    <211> 214
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 189



    <210> 190
    <211> 118
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 190

    <210> 191
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 191

    <210> 192
    <211> 124
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 192

    <210> 193
    <211> 106
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 193

    <210> 194
    <211> 122
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 194



    <210> 195
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 195

    <210> 196
    <211> 120
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 196



    <210> 197
    <211> 111
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 197

    <210> 198
    <211> 469
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 198



    <210> 199
    <211> 233
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 199



    <210> 200
    <211> 119
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 200

    <210> 201
    <211> 108
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 201



    <210> 202
    <211> 121
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 202

    <210> 203
    <211> 108
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 203

    <210> 204
    <211> 119
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 204

    <210> 205
    <211> 108
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 205

    <210> 206
    <211> 108
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 206



    <210> 207
    <211> 119
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 207

    <210> 208
    <211> 119
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 208

    <210> 209
    <211> 121
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 209

    <210> 210
    <211> 108
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 210

    <210> 211
    <211> 108
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 211



    <210> 212
    <211> 121
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 212

    <210> 213
    <211> 121
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 213



    <210> 214
    <211> 138
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 214

    <210> 215
    <211> 126
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 215

    <210> 216
    <211> 17
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 216

    <210> 217
    <211> 8
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 217

    <210> 218
    <211> 9
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 218

    <210> 219
    <211> 19
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 219

    <210> 220
    <211> 4
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 220



    <210> 221
    <211> 4
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 221

    <210> 222
    <211> 5
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 222

    <210> 223
    <211> 5
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 223

    <210> 224
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 224

    <210> 225
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 225

    <210> 226
    <211> 7
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 226

    <210> 227
    <211> 7
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 227

    <210> 228
    <211> 5
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <220>
    <221> VARIANT
    <222> 1, 2, 3, 4, 5
    <223> can be present in repeat of at least 1 and
    up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20

    <400> 228

    <210> 229
    <211> 5
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <220>
    <221> VARIANT
    <222> 1, 2, 3, 4, 5
    <223> can be present in repeat of at least 1 and
    up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20

    <400> 229

    <210> 230
    <211> 447
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 230



    <210> 231
    <211> 214
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 231

    <210> 232
    <211> 444
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 232



    <210> 233
    <211> 214
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 233



    <210> 234
    <211> 220
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 234



    <210> 235
    <211> 217
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 235

    <210> 236
    <211> 214
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 236



    <210> 237
    <211> 214
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 237

    <210> 238
    <211> 226
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 238

    <210> 239
    <211> 229
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 239



    <210> 240
    <211> 571
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 240



    <210> 241
    <211> 331
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 241



    <210> 242
    <211> 571
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 242



    <210> 243
    <211> 331
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 243



    <210> 244
    <211> 675
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 244





    <210> 245
    <211> 214
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 245

    <210> 246
    <211> 214
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 246



    <210> 247
    <211> 675
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 247



    <210> 248
    <211> 214
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <210> 249
    <211> 214
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 249



    <210> 250
    <211> 567
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 250



    <210> 251
    <211> 326
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 251



    <210> 252
    <211> 575
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 252



    <210> 253
    <211> 334
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 253



    <210> 254
    <211> 567
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 254



    <210> 255
    <211> 327
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 255



    <210> 256
    <211> 574
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 256



    <210> 257
    <211> 334
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 257

    <210> 258
    <211> 571
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 258





    <210> 259
    <211> 331
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 259

    <210> 260
    <211> 571
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 260



    <210> 261
    <211> 331
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 261



    <210> 262
    <211> 571
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 262



    <210> 263
    <211> 331
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 263



    <210> 264
    <211> 571
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 264



    <210> 265
    <211> 331
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 265



    <210> 266
    <211> 571
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 266



    <210> 267
    <211> 331
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 267



    <210> 268
    <211> 5
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 268

    <210> 269
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 269

    <210> 270
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 270

    <210> 271
    <211> 5
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 271

    <210> 272
    <211> 10
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 272

    <210> 273
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 273

    <210> 274
    <211> 13
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 274

    <210> 275
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 275

    <210> 276
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 276

    <210> 277
    <211> 13
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 277

    <210> 278
    <211> 13
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 278

    <210> 279
    <211> 330
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 279



    <210> 280
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 280



    <210> 281
    <211> 277
    <212> PRT
    <213> Homo sapiens

    <400> 281

    <210> 282
    <211> 5
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 282

    <210> 283
    <211> 8
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 283

    <210> 284
    <211> 117
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 284

    <210> 285
    <211> 117
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 285

    <210> 286
    <211> 7
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 286

    <210> 287
    <211> 107
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 287

    <210> 288
    <211> 5
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 288

    <210> 289
    <211> 17
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 289



    <210> 290
    <211> 114
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 290

    <210> 291
    <211> 570
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 291



    <210> 292
    <211> 676
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 292





    <210> 293
    <211> 676
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 293



    <210> 294
    <211> 446
    <212> PRT
    <213> Artificial Sequence

    <220>
    <223> Synthetic Construct

    <400> 294

    <210> 295
    <211> 326
    <212> PRT
    <213> Homo sapiens

    <400> 295

    <210> 296
    <211> 377
    <212> PRT
    <213> Homo sapiens

    <400> 296



    <210> 297
    <211> 327
    <212> PRT
    <213> Homo sapiens

    <400> 297



    <210> 298
    <211> 107
    <212> PRT
    <213> Homo sapiens

    <400> 298

    <210> 299
    <211> 106
    <212> PRT
    <213> Homo sapiens

    <400> 299



    <210> 300
    <211> 106
    <212> PRT
    <213> Homo sapiens

    <400> 300

    <210> 301
    <211> 106
    <212> PRT
    <213> Homo sapiens

    <400> 301

    <210> 302
    <211> 106
    <212> PRT
    <213> Homo sapiens

    <400> 302



    <210> 303
    <211> 106
    <212> PRT
    <213> Homo sapiens

    <400> 303




    Claims

    1. A tetravalent antigen binding complex having agonist activity for OX40, the complex comprising four antigen binding domains that bind OX40, wherein each of the four antigen binding domains comprises an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, wherein (a) the complex comprises one or more antigen binding domains that bind a first epitope of OX40 and one or more antigen binding domains that bind a second epitope of OX40, and wherein the first and second epitopes of OX40 are different and/or (b) the antigen binding domains that bind the first epitope do not cross-compete for binding OX40 with the antigen binding domains that bind the second epitope; wherein the complex comprises two antibody heavy chain polypeptides and two antibody light chain polypeptides;
    wherein each of the antibody heavy chain polypeptides comprises:

            VH1-L1-VH2-L2-CH1-hinge-CH2-CH3     [I];

    wherein each of the antibody light chain polypeptides comprises:

            VL1-L3-VL2-L4-CL     [II];

    wherein each of the antibody heavy chain polypeptides associates with one antibody light chain polypeptide such that VH1 and VL1 form an antigen binding domain that binds the first epitope of OX40 and VH2 and VL2 form an antigen binding domain that binds the second epitope of OX40; and
    wherein VH1 is a first antibody heavy chain variable domain, VH2 is a second antibody heavy chain variable domain, VL1 is a first antibody light chain variable domain, VL2 is a second antibody light chain variable domain, CL is an antibody light chain constant domain, CH1 is an antibody first heavy chain constant domain, hinge is an antibody hinge region, CH2 is an antibody second heavy chain constant domain, CH3 is an antibody third heavy chain constant domain, and L1, L2, L3, and L4 are amino acid linkers.
     
    2. The complex of claim 1, wherein the first epitope comprises one or more amino acid residues selected from the group consisting of: 114-119, 124, 126, 127, 129, 130, 132, 140, and 142 of SEQ ID NO:281 and/or wherein the second epitope comprises one or more amino acid residues selected from the group consisting of: 68-71, 83-90, 95, and 98 of SEQ ID NO:281.
     
    3. The complex of claim 1 or claim 2, wherein the antigen binding domain that binds the first epitope of OX40 comprises: (a) a VH domain comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; and (b) a VL domain comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; and/or wherein the antigen binding domain that binds the second epitope of OX40 comprises: (c) a VH domain comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; and (d) a VL domain comprising: an HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.
     
    4. The complex of claim 3, wherein the VH domain of the antigen binding domain that binds the first epitope of OX40 comprises the amino acid sequence of SEQ ID NO:56, and wherein the VL domain of the antigen binding domain that binds the first epitope of OX40 comprises the amino acid sequence of SEQ ID NO:57; and/or wherein the VH domain of the antigen binding domain that binds the second epitope of OX40 comprises the amino acid sequence of SEQ ID NO:128, and wherein the VL domain of the antigen binding domain that binds the second epitope of OX40 comprises the amino acid sequence of SEQ ID NO: 129.
     
    5. The complex of any one of claims 1-4, wherein L2 and L4 are 0 amino acids in length and/or wherein L1 is between 0 and 20 amino acids in length.
     
    6. The complex of claim 5, wherein at least 90% of the amino acids of L1 are glycine and/or serine amino acids and/or L1 comprises an amino acid sequence selected from the group consisting of GGGGSG (SEQ ID NO:270), GGGGSGGGGS (SEQ ID NO:272), and GGGGSGGGGSGGGG (SEQ ID NO:273).
     
    7. The complex of any one of claims 1-6, wherein L3 comprises an amino acid sequence selected from the group consisting of GGSGG (SEQ ID NO:271), GGGGSGGGGS (SEQ ID NO:272), and GGSGGGGSGGGGS (SEQ ID NO:274).
     
    8. The complex of any one of claims 1-7, wherein L1 comprises the amino acid sequence GGGGSG (SEQ ID NO:270), and wherein L3 comprises the amino acid sequence GGSGG (SEQ ID NO:271) or wherein L1 and L3 both comprise the amino acid sequence GGGGSGGGGS (SEQ ID NO:272), or wherein L1 comprises the amino acid sequence GGGGSGGGGSGGGG (SEQ ID NO:273), and wherein L3 comprises the amino acid sequence GGSGGGGSGGGGS (SEQ ID NO:274).
     
    9. The complex of any one of claims 1-5, wherein L1 comprises an amino acid sequence found within a human antibody constant domain sequence.
     
    10. The complex of claim 9, wherein L1 comprises the amino acid sequence ASTKGP (SEQ ID NO:275) or ASTKGPSVFPLAP (SEQ ID NO:277) and/or L3 comprises the amino acid sequence RTVAAP (SEQ ID NO:276) or RTVAAPSVFIFPP (SEQ ID NO:278).
     
    11. The complex of any one of claims 1-5 or 9, wherein L1 comprises the amino acid sequence ASTKGP (SEQ ID NO:275), and wherein L3 comprises the amino acid sequence RTVAAP (SEQ ID NO:276), or
    wherein L1 comprises the amino acid sequence ASTKGPSVFPLAP (SEQ ID NO:277), and wherein L3 comprises the amino acid sequence RTVAAPSVFIFPP (SEQ ID NO:278).
     
    12. The complex of any one of claims 1-11, wherein VH1 comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; wherein VL1 comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:42; wherein VH2 comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; and wherein VL2 comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7; or
    wherein VH2 comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; wherein VL2 comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:42; wherein VH1 comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; and wherein VL1 comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.
     
    13. The complex of any one of claims 1-11, wherein VH1 comprises the amino acid sequence of SEQ ID NO:128, wherein VL1 comprises the amino acid sequence of SEQ ID NO:129, wherein VH2 comprises the amino acid sequence of SEQ ID NO:56, and wherein VL2 comprises the amino acid sequence of SEQ ID NO:57; or
    wherein VH2 comprises the amino acid sequence of SEQ ID NO:128, wherein VL2 comprises the amino acid sequence of SEQ ID NO: 129, wherein VH1 comprises the amino acid sequence of SEQ ID NO:56, and wherein VL1 comprises the amino acid sequence of SEQ ID NO:57.
     
    14. The complex of claim 1, wherein:

    (a) both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:240, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:241;

    (b) both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:242, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:243;

    (c) both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:250, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:251;

    (d) both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:252, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:253;

    (e) both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:254, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:255;

    (f) both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:256, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:257;

    (g) both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:258, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:259;

    (h) both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:260, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:261;

    (i) both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:262, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:263;

    (j) both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:264, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:265; or

    (k) both of the antibody heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO:266, and both of the antibody light chain polypeptides comprise the amino acid sequence of SEQ ID NO:267.


     
    15. The complex of any one of claims 1-14, wherein the complex comprises an antibody Fc region that comprises a modification for attenuating effector function, and/or wherein the complex comprises an antibody Fc region that comprises a modification for attenuating effector function that results in an aglycosylated Fc region.
     
    16. The complex of claim 15, wherein the complex comprises an antibody Fc region that comprises an amino acid substitution at one or more amino acid positions (EU numbering) selected from the group consisting of:

    (a) 297 in the Fc region of human IgG1,

    (b) 234 and 235 in the Fc region of human IgG1,

    (c) 234, 235 and 329 in the Fc region of human IgG1,

    (d) 234 and 237 in the Fc region of human IgG2,

    (e) 235, 237 and 318 in the Fc region of human IgG4,

    (f) 228 and 236 in the Fc region of human IgG4,

    (g) 268, 309, 330 and 331 in the Fc region of human IgG2,

    (h) 220, 226, 229 and 238 in the Fc region of human IgG1,

    (i) 226, 229, 233, 234 and 235 in the Fc region of human IgG1,

    (j) 234, 235 and 331 in the Fc region of human IgG1,

    (k) 226 and 230 in the Fc region of human IgG1, and

    (l) 267 and 328 in the Fc region of human IgG1.


     
    17. The complex of claim 15 or claim 16, wherein the complex comprises an antibody Fc region that comprises one or more amino acid substitutions (EU numbering) selected from the group consisting of:

    (a) N297A in the Fc region of human IgG1,

    (b) L234A and L235A in the Fc region of human IgG1,

    (c) L234A, L235A and P329G in the Fc region of human IgG1,

    (d) V234A and G237A in the Fc region of human IgG2,

    (e) L235A, G237A and E318A in the Fc region of human IgG4,

    (f) S228P and L236E in the Fc region of human IgG4,

    (g) one or more substitutions in the region spanning amino acid residues 118 to 260 in the Fc region of human IgG2 or in the region spanning amino acids 261 to 447 in the Fc region of human IgG4,

    (h) H268Q, V309L, A330S and A331S in the Fc region of human IgG2,

    (i) C220S, C226S, C229S and P238S in the Fc region of human IgG1,

    (j) C226S, C229S, E233P, L234V and L235A in the Fc region of human IgG1,

    (k) L234F, L235E and P331S in the Fc region of human IgG1,

    (l) C226S and P230S in the Fc region of human IgG1, and

    (m) S267E and L328F in the Fc region of human IgG1.


     
    18. One or more polynucleotides encoding one or more polypeptides of the complex of any one of claims 1-17.
     
    19. One or more vectors comprising the one or more polynucleotides of claim 18.
     
    20. One or more host cells comprising the one or more polynucleotides of claim 18 or the one or more vectors of claim 19.
     
    21. A pharmaceutical formulation comprising the complex of any one of claims 1-17 and a pharmaceutically acceptable carrier.
     
    22. The complex of any one of claims 1-17 for use in a method of treating an individual having cancer, said method comprising administering to the individual an effective amount of the complex.
     
    23. The complex for use in a method of claim 22, said method further comprising administering to the individual an additional therapeutic agent.
     
    24. The complex for use in method of claim 23, wherein the additional therapeutic agent comprises a chemotherapeutic agent or a PD-1 axis binding antagonist.
     
    25. The complex for use in a method of any one of claims 22-24, wherein the cancer is Urothelial carcinoma (uBC), melanoma, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), renal, or bladder cancer.
     


    Ansprüche

    1. Tetravalenter Antigenbindungskomplex mit Agonistenaktivität für OX40, wobei der Komplex vier Antigenbindungsdomänen umfasst, die OX40 binden, wobei jede der vier Antigenbindungsdomänen eine variable Domäne der schweren Kette (VH) des Antikörpers und eine variable Domäne der leichten Kette (VL) des Antikörpers umfasst, wobei (a) der Komplex eine oder mehrere Antigenbindungsdomäne(n), die ein erstes Epitop von OX40 bindet/binden, und eine oder mehrere Antigenbindungsdomäne(n), die ein zweites Epitop von OX40 bindet/binden, umfasst und wobei das erste und das zweite Epitop von OX40 verschieden sind und/oder (b) die Antigenbindungsdomänen, die das erste Epitop binden, nicht mit den Antigenbindungsdomänen, die das zweite Epitop binden, um die OX40-Bindung kreuzkonkurrieren; wobei der Komplex zwei Polypeptide der schweren Kette des Antikörpers und zwei Polypeptide der leichten Kette des Antikörpers umfassen;
    wobei jedes der Polypeptide der schweren Kette des Antikörpers Folgendes umfasst:

            VH1-L1-VH2-L2-CH1-Gelenk-CH2-CH3     [I];

    wobei jedes der Polypeptide der leichten Kette des Antikörpers Folgendes umfasst:

            VL1-L3-VL2-L4-CL     [II];

    wobei sich jedes der Polypeptide der schweren Kette des Antikörpers mit einem Polypeptid der leichten Kette des Antikörpers vereinigt, so dass VH1 und VL1 eine Antigenbindungsdomäne bilden, die das erste Epitop von OX40 bindet, und VH2 und VL2 eine Antigenbindungsdomäne bilden, die das zweite Epitop von OX40 bindet; und
    wobei VH1 eine erste variable Domäne der schweren Kette des Antikörpers ist, VH2 eine zweite variable Domäne der schweren Kette des Antikörpers ist, VL1 eine erste variable Domäne der leichten Kette des Antikörpers ist, VL2 eine zweite variable Domäne der leichten Kette des Antikörpers ist, CL eine konstante Domäne der leichten Kette des Antikörpers ist, CH1 eine erste konstante Domäne der schweren Kette des Antikörpers ist, Gelenk eine Antikörpergelenkregion ist, CH2 eine zweite konstante Domäne der schweren Kette des Antikörpers ist, CH3 eine dritte konstante Domäne der schweren Kette des Antikörpers ist und L1, L2, L3 und L4 Aminosäure-Linker sind.
     
    2. Komplex nach Anspruch 1, wobei das erste Epitop einen oder mehrere Aminosäurerest(e) umfasst, der/die aus der Gruppe ausgewählt ist/sind, bestehend aus: 114-119, 124, 126, 127, 129, 130, 132, 140 und 142 von SEQ ID NO:281; und/oder wobei das zweite Epitop einen oder mehrere Aminosäurerest(e) umfasst, der/die aus der Gruppe ausgewählt ist/sind, bestehend aus: 68-71, 83-90, 95 und 98 von SEQ ID NO:281.
     
    3. Komplex nach Anspruch 1 oder Anspruch 2, wobei die Antigenbindungsdomäne, die das erste Epitop von OX40 bindet, Folgendes umfasst: (a) eine VH-Domäne, die eine HVR-H1, umfassend die Aminosäuresequenz von SEQ ID NO:2, eine HVR-H2, umfassend die Aminosäuresequenz von SEQ ID NO:3, und eine HVR-H3, umfassend die Aminosäuresequenz von SEQ ID NO:4, umfasst; und (b) eine VL-Domäne, die eine HVR-L1, umfassend die Aminosäuresequenz von SEQ ID NO:5, eine HVR-L2, umfassend die Aminosäuresequenz von SEQ ID NO:6, und eine HVR-L3, umfassend die Aminosäuresequenz von SEQ ID NO:7, umfasst; und/oder wobei die Antigenbindungsdomäne, die das zweite Epitop von OX40 bindet, Folgendes umfasst: (c) eine VH-Domäne, die eine HVR-H1, umfassend die Aminosäuresequenz von SEQ ID NO:29, eine HVR-H2, umfassend die Aminosäuresequenz von SEQ ID NO:30, und eine HVR-H3, umfassend die Aminosäuresequenz von SEQ ID NO:33, umfasst; und (d) eine VL-Domäne, die eine HVR-L1, umfassend die Aminosäuresequenz von SEQ ID NO:37, eine HVR-L2, umfassend die Aminosäuresequenz von SEQ ID NO:39, und eine HVR-L3, umfassend die Aminosäuresequenz von SEQ ID NO:42, umfasst.
     
    4. Komplex nach Anspruch 3, wobei die VH-Domäne der Antigenbindungsdomäne, die das erste Epitop von OX40 bindet, die Aminosäuresequenz von SEQ ID NO:56 umfasst und wobei die VL-Domäne der Antigenbindungsdomäne, die das erste Epitop von OX40 bindet, die Aminosäuresequenz von SEQ ID NO:57 umfasst und/oder wobei die VH-Domäne der Antigenbindungsdomäne, die das zweite Epitop von OX40 bindet, die Aminosäuresequenz von SEQ ID NO:128 umfasst und wobei die VL-Domäne der Antigenbindungsdomäne, die das zweite Epitop von OX40 bindet, die Aminosäuresequenz von SEQ ID NO:129 umfasst.
     
    5. Komplex nach einem der Ansprüche 1-4, wobei L2 und L4 0 Aminosäuren lang sind und/oder wobei L1 zwischen 0 und 20 Aminosäuren lang ist.
     
    6. Komplex nach Anspruch 5, wobei mindestens 90 % der Aminosäuren von L1 Glycin- und/oder Serin-Aminosäuren sind und/oder L1 eine Aminosäuresequenz umfasst, die ausgewählt ist aus der Gruppe, bestehend aus GGGGSG (SEQ ID NO:270), GGGGSGGGGS (SEQ ID NO:272) und GGGGSGGGGSGGGG (SEQ ID NO:273).
     
    7. Komplex nach einem der Ansprüche 1-6, wobei L3 eine Aminosäuresequenz umfasst, die ausgewählt ist aus der Gruppe, bestehend aus GGSGG (SEQ ID NO:271), GGGGSGGGGS (SEQ ID NO:272) und GGSGGGGSGGGGS (SEQ ID NO:274).
     
    8. Komplex nach einem der Ansprüche 1-7, wobei L1 die Aminosäuresequenz GGGGSG (SEQ ID NO:270) umfasst und wobei L3 die Aminosäuresequenz GGSGG (SEQ ID NO:271) umfasst oder wobei L1 und L3 beide die Aminosäuresequenz GGGGSGGGGS (SEQ ID NO:272) umfassen oder wobei L1 die Aminosäuresequenz GGGGSGGGGSGGGG (SEQ ID NO:273) umfasst und wobei L3 die Aminosäuresequenz GGSGGGGSGGGGS (SEQ ID NO:274) umfasst.
     
    9. Komplex nach einem der Ansprüche 1-5, wobei L1 eine Aminosäuresequenz umfasst, die in einer Sequenz einer konstanten Domäne eines humanen Antikörpers zu finden ist.
     
    10. Komplex nach Anspruch 9, wobei L1 die Aminosäuresequenz ASTKGP (SEQ ID NO:275) oder ASTKGPSVFPLAP (SEQ ID NO:277) umfasst und/oder L3 die Aminosäuresequenz RTVAAP (SEQ ID NO:276) oder RTVAAPSVFIFPP (SEQ ID NO:278) umfasst.
     
    11. Komplex nach einem der Ansprüche 1-5 oder 9, wobei L1 die Aminosäuresequenz ASTKGP (SEQ ID NO:275) umfasst und wobei L3 die Aminosäuresequenz RTVAAP (SEQ IDNO:276) umfasst oder
    wobei L1 die Aminosäuresequenz ASTKGPSVFPLAP (SEQ ID NO:277) umfasst und wobei L3 die Aminosäuresequenz RTVAAPSVFIFPP (SEQ ID NO:278) umfasst.
     
    12. Komplex nach einem der Ansprüche 1-11, wobei VH1 eine HVR-H1, umfassend die Aminosäuresequenz von SEQ ID NO:29, eine HVR-H2, umfassend die Aminosäuresequenz von SEQ ID NO:30, und eine HVR-H3, umfassend die Aminosäuresequenz von SEQ ID NO:33, umfasst; wobei VL1 eine HVR-L1, umfassend die Aminosäuresequenz von SEQ ID NO:37, eine HVR-L2, umfassend die Aminosäuresequenz von SEQ ID NO:39, und eine HVR-L3, umfassend die Aminosäuresequenz von SEQ ID NO:42, umfasst; wobei VH2 eine HVR-H1, umfassend die Aminosäuresequenz von SEQ ID NO:2, eine HVR-H2, umfassend die Aminosäuresequenz von SEQ ID NO:3, und eine HVR-H3, umfassend die Aminosäuresequenz von SEQ ID NO:4, umfasst; und wobei VL2 eine HVR-L1, umfassend die Aminosäuresequenz von SEQ ID NO:5, eine HVR-L2, umfassend die Aminosäuresequenz von SEQ ID NO:6, und eine HVR-L3, umfassend die Aminosäuresequenz von SEQ ID NO:7, umfasst; oder
    wobei VH2 eine HVR-H1, umfassend die Aminosäuresequenz von SEQ ID NO:29, eine HVR-H2, umfassend die Aminosäuresequenz von SEQ ID NO:30, und eine HVR-H3, umfassend die Aminosäuresequenz von SEQ ID NO:33, umfasst; wobei VL2 eine HVR-L1, umfassend die Aminosäuresequenz von SEQ ID NO:37, eine HVR-L2, umfassend die Aminosäuresequenz von SEQ ID NO:39, und eine HVR-L3, umfassend die Aminosäuresequenz von SEQ ID NO:42, umfasst; wobei VH1 eine HVR-H1, umfassend die Aminosäuresequenz von SEQ ID NO:2, eine HVR-H2, umfassend die Aminosäuresequenz von SEQ ID NO:3, und eine HVR-H3, umfassend die Aminosäuresequenz von SEQ ID NO:4, umfasst; und wobei VL1 eine HVR-L1, umfassend die Aminosäuresequenz von SEQ ID NO:5, eine HVR-L2, umfassend die Aminosäuresequenz von SEQ ID NO:6, und eine HVR-L3, umfassend die Aminosäuresequenz von SEQ ID NO:7, umfasst.
     
    13. Komplex nach einem der Ansprüche 1-11, wobei VH1 die Aminosäuresequenz von SEQ ID NO:128 umfasst, wobei VL1 die Aminosäuresequenz von SEQ ID NO:129 umfasst, wobei VH2 die Aminosäuresequenz von SEQ ID NO:56 umfasst und wobei VL2 die Aminosäuresequenz von SEQ ID NO:57 umfasst oder
    wobei VH2 die Aminosäuresequenz von SEQ ID NO:128 umfasst, wobei VL2 die Aminosäuresequenz von SEQ ID NO:129 umfasst, wobei VH1 die Aminosäuresequenz von SEQ ID NO:56 umfasst und wobei VL1 die Aminosäuresequenz von SEQ ID NO:57 umfasst.
     
    14. Komplex nach Anspruch 1, wobei:

    (a) beide Polypeptide der schweren Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:240 umfassen und beide Polypeptide der leichten Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:241 umfassen;

    (b) beide Polypeptide der schweren Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:242 umfassen und beide Polypeptide der leichten Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:243 umfassen;

    (c) beide Polypeptide der schweren Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:250 umfassen und beide Polypeptide der leichten Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:251 umfassen;

    (d) beide Polypeptide der schweren Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:252 umfassen und beide Polypeptide der leichten Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:253 umfassen;

    (e) beide Polypeptide der schweren Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:254 umfassen und beide Polypeptide der leichten Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:255 umfassen;

    (f) beide Polypeptide der schweren Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:256 umfassen und beide Polypeptide der leichten Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:257 umfassen;

    (g) beide Polypeptide der schweren Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:258 umfassen und beide Polypeptide der leichten Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:259 umfassen;

    (h) beide Polypeptide der schweren Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:260 umfassen und beide Polypeptide der leichten Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:261 umfassen;

    (i) beide Polypeptide der schweren Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:262 umfassen und beide Polypeptide der leichten Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:263 umfassen;

    (j) beide Polypeptide der schweren Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:264 umfassen und beide Polypeptide der leichten Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:265 umfassen oder

    (k) beide Polypeptide der schweren Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:266 umfassen und beide Polypeptide der leichten Kette des Antikörpers die Aminosäuresequenz von SEQ ID NO:267 umfassen.


     
    15. Komplex nach einem der Ansprüche 1-14, wobei der Komplex eine Antikörper-Fc-Region umfasst, die eine Modifikation zum Reduzieren der Effektorfunktion umfasst, und/oder wobei der Komplex eine Antikörper-Fc-Region umfasst, die eine Modifikation zum Reduzieren der Effektorfunktion umfasst, die zu einer aglycosylierten Fc-Region führt.
     
    16. Komplex nach Anspruch 15, wobei der Komplex eine Antikörper-Fc-Region umfasst, die eine Aminosäuresubstitution an einer oder mehreren Aminosäureposition(en) (EU-Nummerierung) umfasst, die aus der Gruppe ausgewählt ist/sind, bestehend aus:

    (a) 297 in der Fc-Region von humanem IgG1,

    (b) 234 und 235 in der Fc-Region von humanem IgG1,

    (c) 234, 235 und 329 in der Fc-Region von humanem IgG1,

    (d) 234 und 237 in der Fc-Region von humanem IgG2,

    (e) 235, 237 und 318 in der Fc-Region von humanem IgG4,

    (f) 228 und 236 in der Fc-Region von humanem IgG4,

    (g) 268, 309, 330 und 331 in der Fc-Region von humanem IgG2,

    (h) 220, 226, 229 und 238 in der Fc-Region von humanem IgG1,

    (i) 226, 229, 233, 234 und 235 in der Fc-Region von humanem IgG1,

    (j) 234, 235 und 331 in der Fc-Region von humanem IgG1,

    (k) 226 und 230 in der Fc-Region von humanem IgG1 und

    (l) 267 und 328 in der Fc-Region von humanem IgG1.


     
    17. Komplex nach Anspruch 15 oder Anspruch 16, wobei der Komplex eine Antikörper-Fc-Region umfasst, die eine oder mehrere Aminosäuresubstitution(en) (EU-Nummerierung) umfasst, die aus der Gruppe ausgewählt ist/sind, bestehend aus:

    (a) N297A in der Fc-Region von humanem IgG1,

    (b) L234A und L235A in der Fc-Region von humanem IgG1,

    (c) L234A, L235A und P329G in der Fc-Region von humanem IgG1,

    (d) V234A und G237A in der Fc-Region von humanem IgG2,

    (e) L235A, G237A und E318A in der Fc-Region von humanem IgG4,

    (f) S228P und L236E in der Fc-Region von humanem IgG4,

    (g) einer oder mehreren Substitution(en) in der Region, die die Aminosäurereste 118 bis 260 in der Fc-Region von humanem IgG2 überspannt, oder in der Region, die die Aminosäuren 261 bis 447 in der Fc-Region von humanem IgG4 überspannt,

    (h) H268Q, V309L, A330S und A331S in der Fc-Region von humanem IgG2,

    (i) C220S, C226S, C229S und P238S in der Fc-Region von humanem IgG1,

    (j) C226S, C229S, E233P, L234V und L235A in der Fc-Region von humanem IgG1,

    (k) L234F, L235E und P331S in der Fc-Region von humanem IgG1,

    (l) C226S und P230S in der Fc-Region von humanem IgG1 und

    (m) S267E und L328F in der Fc-Region von humanem IgG1.


     
    18. Ein oder mehrere Polynukleotid(e), das/die für ein oder mehrere Polypeptid(e) des Komplexes nach einem der Ansprüche 1-17 kodiert/kodieren.
     
    19. Ein oder mehrere Vektor(en), umfassend das eine oder die mehreren Polynukleotid(e) nach Anspruch 18.
     
    20. Eine oder mehrere Wirtszelle(n), umfassend das eine oder die mehreren Polynukleotid(e) nach Anspruch 18 oder den einen oder die mehreren Vektor(en) nach Anspruch 19.
     
    21. Pharmazeutische Formulierung, umfassend den Komplex nach einem der Ansprüche 1-17 und einen pharmazeutisch unbedenklichen Träger.
     
    22. Komplex nach einem der Ansprüche 1-17 zur Verwendung in einem Verfahren zur Behandlung eines Individuums mit Krebs, wobei das Verfahren das Verabreichen einer wirksamen Menge des Komplexes an das Individuum umfasst.
     
    23. Komplex zur Verwendung in einem Verfahren nach Anspruch 22, wobei das Verfahren ferner das Verabreichen eines zusätzlichen Therapeutikums an das Individuum umfasst.
     
    24. Komplex zur Verwendung in einem Verfahren nach Anspruch 23, wobei das zusätzliche Therapeutikum ein Chemotherapeutikum oder einen PD-1-Achsenbindungsantagonisten umfasst.
     
    25. Komplex zur Verwendung in einem Verfahren nach einem der Ansprüche 22-24, wobei der Krebs Urothelkarzinom (uBC), Melanom, nicht kleinzelliges Lungenkarzinom (NSCLC), dreifach negativer Brustkrebs (TNBC), Nieren- oder Blasenkrebs ist.
     


    Revendications

    1. Complexe tétravalent de liaison à l'antigène ayant une activité agoniste pour OX40, le complexe comprenant quatre domaines de liaison à l'antigène qui se lient à OX40, dans lequel chacun des quatre domaines de liaison à l'antigène comprend un domaine variable de chaîne lourde (VH) d'anticorps et un domaine variable de chaîne légère (VL) d'anticorps, dans lequel (a) le complexe comprend un ou plusieurs domaines de liaison à l'antigène qui se lient à un premier épitope de OX40 et un ou plusieurs domaines de liaison à l'antigène qui se lient à un second épitope de OX40, et dans lequel les premier et second épitopes de OX40 sont différents et/ou (b) les domaines de liaison à l'antigène qui se lient au premier épitope ne présentent pas de compétition croisée pour la liaison de OX40 aux domaines de liaison à l'antigène qui se lient au second épitope ; dans lequel le complexe comprend deux polypeptides de chaîne lourde d'anticorps et deux polypeptides de chaîne légère d'anticorps ;
    dans lequel chacun des polypeptides de chaîne lourde d'anticorps comprend :

            VH1-L1-VH2-L2-CH1-charnière-CH2-CH3     [I] ;

    dans lequel chacun des polypeptides de chaîne légère d'anticorps comprend :

            VL1-L3-VL2-L4-CL     [II] ;

    dans lequel chacun des polypeptides de chaîne lourde d'anticorps s'associe à un polypeptide de chaîne légère d'anticorps de sorte que VH1 et VL1 forment un domaine de liaison à l'antigène qui se lie au premier épitope de OX40 et VH2 et VL2 forment un domaine de liaison à l'antigène qui se lie au second épitope de OX40 ; et
    dans lequel VH1 représente un premier domaine variable de chaîne lourde d'anticorps, VH2 représente un second domaine variable de chaîne lourde d'anticorps, VL1 représente un premier domaine variable de chaîne légère d'anticorps, VL2 représente un second domaine variable de chaîne légère d'anticorps, CL représente un domaine constant de chaîne légère d'anticorps, CH1 représente un premier domaine constant de chaîne lourde d'anticorps, charnière représente une région charnière d'anticorps, CH2 représente un deuxième domaine constant de chaîne lourde d'anticorps, CH3 représente un troisième domaine constant de chaîne lourde d'anticorps et L1, L2, L3 et L4 représentent des lieurs d'acides aminés.
     
    2. Complexe selon la revendication 1, dans lequel le premier épitope comprend un ou plusieurs résidus d'acides aminés choisis dans le groupe constitué par : 114 à 119, 124, 126, 127, 129, 130, 132, 140 et 142 de SEQ ID NO : 281 et/ou dans lequel le second épitope comprend un ou plusieurs résidus d'acides aminés choisis dans le groupe constitué par : 68 à 71, 83 à 90, 95 et 98 de SEQ ID NO : 281.
     
    3. Complexe selon la revendication 1 ou la revendication 2, dans lequel le domaine de liaison à l'antigène qui se lie au premier épitope de OX40 comprend : (a) un domaine VH comprenant une HVR-H1 comprenant la séquence d'acides aminés de SEQ ID NO : 2, une HVR-H2 comprenant la séquence d'acides aminés de SEQ ID NO : 3 et une HVR-H3 comprenant la séquence d'acides aminés de SEQ ID NO : 4 ; et (b) un domaine VL comprenant une HVR-L1 comprenant la séquence d'acides aminés de SEQ ID NO : 5, une HVR-L2 comprenant la séquence d'acides aminés de SEQ ID NO : 6 et une HVR-L3 comprenant la séquence d'acides aminés de SEQ ID NO : 7 ; et/ou dans lequel le domaine de liaison à l'antigène qui se lie au second épitope de OX40 comprend : (c) un domaine VH comprenant une HVR-H1 comprenant la séquence d'acides aminés de SEQ ID NO : 29, une HVR-H2 comprenant la séquence d'acides aminés de SEQ ID NO : 30 et une HVR-H3 comprenant la séquence d'acides aminés de SEQ ID NO : 33 ; et (d) un domaine VL comprenant : une HVR-L1 comprenant la séquence d'acides aminés de SEQ ID NO : 37, une HVR-L2 comprenant la séquence d'acides aminés de SEQ ID NO : 39 et une HVR-L3 comprenant la séquence d'acides aminés de SEQ ID NO : 42.
     
    4. Complexe selon la revendication 3, dans lequel le domaine VH du domaine de liaison à l'antigène qui se lie au premier épitope de OX40 comprend la séquence d'acides aminés de SEQ ID NO : 56, et dans lequel le domaine VL du domaine de liaison à l'antigène qui se lie au premier épitope de OX40 comprend la séquence d'acides aminés de SEQ ID NO : 57 ; et/ou dans lequel le domaine VH du domaine de liaison à l'antigène qui se lie au second épitope de OX40 comprend la séquence d'acides aminés de SEQ ID NO : 128, et dans lequel le domaine VL du domaine de liaison à l'antigène qui se lie au second épitope de OX40 comprend la séquence d'acides aminés de SEQ ID NO : 129.
     
    5. Complexe selon l'une quelconque des revendications 1 à 4, dans lequel L2 et L4 ont une longueur de 0 acide aminé et/ou dans lequel L1 a une longueur comprise entre 0 et 20 acides aminés.
     
    6. Complexe selon la revendication 5, dans lequel au moins 90 % des acides aminés de L1 sont des acides aminés glycine et/ou sérine et/ou L1 comprend une séquence d'acides aminés choisie dans le groupe constitué par GGGGSG (SEQ ID NO : 270), GGGGSGGGGS (SEQ ID NO : 272) et GGGGSGGGGSGGGG (SEQ ID NO : 273).
     
    7. Complexe selon l'une quelconque des revendications 1 à 6, dans lequel L3 comprend une séquence d'acides aminés choisie dans le groupe constitué par GGSGG (SEQ ID NO : 271), GGGGSGGGGS (SEQ ID NO : 272) et GGSGGGGSGGGGS (SEQ ID NO : 274).
     
    8. Complexe selon l'une quelconque des revendications 1 à 7, dans lequel L1 comprend la séquence d'acides aminés GGGGSG (SEQ ID NO : 270) et dans lequel L3 comprend la séquence d'acides aminés GGSGG (SEQ ID NO : 271) ou dans lequel L1 et L3 comprennent tous deux la séquence d'acides aminés GGGGSGGGGS (SEQ ID NO : 272) ou dans lequel L1 comprend la séquence d'acides aminés GGGGSGGGGSGGGG (SEQ ID NO : 273) et dans lequel L3 comprend la séquence d'acides aminés GGSGGGGSGGGGS (SEQ ID NO : 274).
     
    9. Complexe selon l'une quelconque des revendications 1 à 5, dans lequel L1 comprend une séquence d'acides aminés se trouvant au sein d'une séquence de domaine constant d'anticorps humain.
     
    10. Complexe selon la revendication 9, dans lequel L1 comprend la séquence d'acides aminés ASTKGP (SEQ ID NO : 275) ou ASTKGPSVFPLAP (SEQ ID NO : 277) et/ou L3 comprend la séquence d'acides aminés RTVAAP (SEQ ID NO : 276) ou RTVAAPSVFIFPP (SEQ ID NO : 278).
     
    11. Complexe selon l'une quelconque des revendications 1 à 5 ou 9, dans lequel L1 comprend la séquence d'acides aminés ASTKGP (SEQ ID NO : 275) et dans lequel L3 comprend la séquence d'acides aminés RTVAAP (SEQ ID NO : 276), ou
    dans lequel L1 comprend la séquence d'acides aminés ASTKGPSVFPLAP (SEQ ID NO : 277) et dans lequel L3 comprend la séquence d'acides aminés RTVAAPSVFIFPP (SEQ ID NO : 278).
     
    12. Complexe selon l'une quelconque des revendications 1 à 11, dans lequel VH1 comprend une HVR-H1 comprenant la séquence d'acides aminés de SEQ ID NO : 29, une HVR-H2 comprenant la séquence d'acides aminés de SEQ ID NO : 30 et une HVR-H3 comprenant la séquence d'acides aminés de SEQ ID NO : 33 ; dans lequel VL1 comprend une HVR-L1 comprenant la séquence d'acides aminés de SEQ ID NO : 37, une HVR-L2 comprenant la séquence d'acides aminés de SEQ ID NO : 39 et une HVR-L3 comprenant la séquence d'acides aminés de SEQ ID NO : 42 ; dans lequel VH2 comprend une HVR-H1 comprenant la séquence d'acides aminés de SEQ ID NO : 2, une HVR-H2 comprenant la séquence d'acides aminés de SEQ ID NO : 3 et une HVR-H3 comprenant la séquence d'acides aminés de SEQ ID NO : 4 ; et dans lequel VL2 comprend une HVR-L1 comprenant la séquence d'acides aminés de SEQ ID NO : 5, une HVR-L2 comprenant la séquence d'acides aminés de SEQ ID NO : 6 et une HVR-L3 comprenant la séquence d'acides aminés de SEQ ID NO : 7 ; ou
    dans lequel VH2 comprend une HVR-H1 comprenant la séquence d'acides aminés de SEQ ID NO : 29, une HVR-H2 comprenant la séquence d'acides aminés de SEQ ID NO : 30 et une HVR-H3 comprenant la séquence d'acides aminés de SEQ ID NO : 33 ; dans lequel VL2 comprend une HVR-L1 comprenant la séquence d'acides aminés de SEQ ID NO : 37, une HVR-L2 comprenant la séquence d'acides aminés de SEQ ID NO : 39 et une HVR-L3 comprenant la séquence d'acides aminés de SEQ ID NO : 42 ; dans lequel VH1 comprend une HVR-H1 comprenant la séquence d'acides aminés de SEQ ID NO : 2, une HVR-H2 comprenant la séquence d'acides aminés de SEQ ID NO : 3, et une HVR-H3 comprenant la séquence d'acides aminés de SEQ ID NO : 4 ; et dans lequel VL1 comprend une HVR-L1 comprenant la séquence d'acides aminés de SEQ ID NO : 5, une HVR-L2 comprenant la séquence d'acides aminés de SEQ ID NO : 6 et une HVR-L3 comprenant la séquence d'acides aminés de SEQ ID NO : 7.
     
    13. Complexe selon l'une quelconque des revendications 1 à 11, dans lequel VH1 comprend la séquence d'acides aminés de SEQ ID NO : 128, dans lequel VL1 comprend la séquence d'acides aminés de SEQ ID NO : 129, dans lequel VH2 comprend la séquence d'acides aminés de SEQ ID NO : 56 et dans lequel VL2 comprend la séquence d'acides aminés de SEQ ID NO : 57 ; ou
    dans lequel VH2 comprend la séquence d'acides aminés de SEQ ID NO : 128, dans lequel VL2 comprend la séquence d'acides aminés de SEQ ID NO : 129, dans lequel VH1 comprend la séquence d'acides aminés de SEQ ID NO : 56 et dans lequel VL1 comprend la séquence d'acides aminés de SEQ ID NO : 57.
     
    14. Complexe selon la revendication 1, dans lequel :

    (a) les deux polypeptides de chaîne lourde d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 240 et les deux polypeptides de chaîne légère d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 241 ;

    (b) les deux polypeptides de chaîne lourde d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 242 et les deux polypeptides de chaîne légère d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 243 ;

    (c) les deux polypeptides de chaîne lourde d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 250 et les deux polypeptides de chaîne légère d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 251 ;

    (d) les deux polypeptides de chaîne lourde d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 252 et les deux polypeptides de chaîne légère d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 253 ;

    (e) les deux polypeptides de chaîne lourde d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 254 et les deux polypeptides de chaîne légère d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 255 ;

    (f) les deux polypeptides de chaîne lourde d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 256 et les deux polypeptides de chaîne légère d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 257 ;

    (g) les deux polypeptides de chaîne lourde d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 258 et les deux polypeptides de chaîne légère d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 259 ;

    (h) les deux polypeptides de chaîne lourde d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 260 et les deux polypeptides de chaîne légère d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 261 ;

    (i) les deux polypeptides de chaîne lourde d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 262 et les deux polypeptides de chaîne légère d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 263 ;

    (j) les deux polypeptides de chaîne lourde d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 264 et les deux polypeptides de chaîne légère d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 265 ; ou

    (k) les deux polypeptides de chaîne lourde d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 266 et les deux polypeptides de chaîne légère d'anticorps comprennent la séquence d'acides aminés de SEQ ID NO : 267.


     
    15. Complexe selon l'une quelconque des revendications 1 à 14, dans lequel le complexe comprend une région Fc d'anticorps qui comprend une modification pour atténuer la fonction effectrice et/ou dans lequel le complexe comprend une région Fc d'anticorps qui comprend une modification pour atténuer la fonction effectrice qui résulte en une région Fc aglycosylée.
     
    16. Complexe selon la revendication 15, dans lequel le complexe comprend une région Fc d'anticorps qui comprend une substitution d'acide aminé en une ou plusieurs positions d'acides aminés (numérotation EU) choisies dans le groupe constitué par :

    (a) 297 dans la région Fc de l'IgGl humaine,

    (b) 234 et 235 dans la région Fc de l'IgGl humaine,

    (c) 234, 235 et 329 dans la région Fc de l'IgGl humaine,

    (d) 234 et 237 dans la région Fc de l'IgG2 humaine,

    (e) 235, 237 et 318 dans la région Fc de l'IgG4 humaine,

    (f) 228 et 236 dans la région Fc de l'IgG4 humaine,

    (g) 268, 309, 330 et 331 dans la région Fc de l'IgG2 humaine,

    (h) 220, 226, 229 et 238 dans la région Fc de l'IgGl humaine,

    (i) 226, 229, 233, 234 et 235 dans la région Fc de l'IgGl humaine,

    (j) 234, 235 et 331 dans la région Fc de l'IgGl humaine,

    (k) 226 et 230 dans la région Fc de l'IgGl humaine, et

    (l) 267 et 328 dans la région Fc de l'IgGl humaine.


     
    17. Complexe selon la revendication 15 ou la revendication 16, dans lequel le complexe comprend une région Fc d'anticorps qui comprend une ou plusieurs substitutions d'acides aminés (numérotation EU) choisies dans le groupe constitué par :

    (a) N297A dans la région Fc de l'IgGl humaine,

    (b) L234A et L235A dans la région Fc de l'IgGl humaine,

    (c) L234A, L235A et P329G dans la région Fc de l'IgGl humaine,

    (d) V234A et G237A dans la région Fc de l'IgG2 humaine,

    (e) L235A, G237A et E318A dans la région Fc de l'IgG4 humaine,

    (f) S228P et L236E dans la région Fc de l'IgG4 humaine,

    (g) une ou plusieurs substitutions dans la région couvrant les résidus d'acides aminés 118 à 260 dans la région Fc de l'IgG2 humaine ou dans la région couvrant les résidus d'acides aminés 261 à 447 dans la région Fc de l'IgG4 humaine,

    (h) H268Q, V309L, A330S et A331S dans la région Fc de l'IgG2 humaine,

    (i) C220S, C226S, C229S et P238S dans la région Fc de l'IgGl humaine,

    (j) C226S, C229S, E233P, L234V et L235A dans la région Fc de l'IgGl humaine,

    (k) L234F, L235E et P331S dans la région Fc de l'IgGl humaine,

    (l) C226S et P230S dans la région Fc de l'IgGl humaine, et

    (m)S267E et L328F dans la région Fc de l'IgGl humaine.


     
    18. Polynucléotide ou polynucléotides codant pour un ou plusieurs polypeptides du complexe selon l'une quelconque des revendications 1 à 17.
     
    19. Vecteur ou vecteurs comprenant le ou les polynucléotides selon la revendication 18.
     
    20. Cellule hôte ou cellules hôtes comprenant le ou les polynucléotides selon la revendication 18 ou le ou les vecteurs selon la revendication 19.
     
    21. Formulation pharmaceutique comprenant le complexe selon l'une quelconque des revendications 1 à 17 et un véhicule pharmaceutiquement acceptable.
     
    22. Complexe selon l'une quelconque des revendications 1 à 17 pour une utilisation dans une méthode de traitement d'un individu ayant un cancer, ladite méthode comprenant l'administration à l'individu d'une quantité efficace du complexe.
     
    23. Complexe pour une utilisation dans une méthode selon la revendication 22, ladite méthode comprenant en outre l'administration à l'individu d'un agent thérapeutique supplémentaire.
     
    24. Complexe pour une utilisation dans une méthode selon la revendication 23, dans lequel l'agent thérapeutique supplémentaire comprend un agent chimiothérapeutique ou un antagoniste se liant à l'axe PD-1.
     
    25. Complexe pour une utilisation dans une méthode selon l'une quelconque des revendications 22 à 24, dans lequel le cancer est un carcinome urothélial (uBC), un mélanome, un cancer du poumon non à petites cellules (CPNPC), un cancer du sein triple négatif (CSTN), un cancer du rein ou de la vessie.
     




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    Cited references

    REFERENCES CITED IN THE DESCRIPTION



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    Patent documents cited in the description




    Non-patent literature cited in the description