(19)
(11)EP 2 794 650 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
14.08.2019 Bulletin 2019/33

(21)Application number: 12808642.8

(22)Date of filing:  17.12.2012
(51)Int. Cl.: 
C07K 16/00  (2006.01)
(86)International application number:
PCT/US2012/070146
(87)International publication number:
WO 2013/096221 (27.06.2013 Gazette  2013/26)

(54)

VARIANT FC-POLYPEPTIDES WITH ENHANCED BINDING TO THE NEONATAL FC RECEPTOR

FC-POLYPEPTIDVARIANTEN MIT VERBESSERTER BINDUNG AN DEN NEONATALEN FC-REZEPTOR

VARIANTS POLYPEPTIDIQUES FC AYANT UNE LIAISON AMÉLIORÉE AU RÉCEPTEUR DE FC NÉONATAL


(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: 21.12.2011 US 201161578780 P
12.01.2012 US 201261585993 P
21.11.2012 US 201261729050 P

(43)Date of publication of application:
29.10.2014 Bulletin 2014/44

(73)Proprietor: Amgen Inc.
Thousand Oaks, California 91320-1799 (US)

(72)Inventors:
  • SUN, Jeonghoon
    San Diego, California 92116 (US)
  • HAN, Seog Joon
    Simi Valley, California 93063 (US)
  • HARRIS, Susie M.
    Newbury Park, California 91320 (US)
  • KETCHEM, Randal R.
    Snohomish, Washington 98296 (US)
  • LU, Ji
    Thousand Oaks, California 91320 (US)
  • MICHAELS, Mark L.
    Encino, California 91316 (US)
  • RETTER, Marc W.
    Simi Valley, California 93065 (US)
  • TSAI, Mei-Mei
    Thousand Oaks, California 91360 (US)

(74)Representative: Dörries, Hans Ulrich 
df-mp Dörries Frank-Molnia & Pohlman Patentanwälte Rechtsanwälte PartG mbB Theatinerstrasse 16
80333 München
80333 München (DE)


(56)References cited: : 
WO-A1-2008/119096
WO-A2-2006/036834
US-A1- 2008 181 887
WO-A2-2005/047327
WO-A2-2009/058492
  
      
    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

    PRIORITY



    [0001] This application claims the benefit of US Provisional Application Nos. 61/578,780, 61/585,993, and 61/729,050 filed December 21, 2011, January 12, 2012, and November 21, 2012, respectively, each of which are incorporated herein in their entirety.

    FIELD



    [0002] The invention relates to polypeptides comprising variant Fc-fragments that bind to the neonatal Fc receptor with higher affinity and/or greater binding activity compared to a control Fc-fragment. The invention further relates to methods of isolating, making, and using such polypeptides.

    BACKGROUND



    [0003] Therapeutic monoclonal antibodies have been used successfully as treatments for a variety of diseases. The relatively long in vivo half life of antibodies is mediated at least in part by the interaction of the Fc region of the antibody with the neonatal Fc receptor (FcRn). See, e.g., Ghetie et al. (1996), Eur. J. Immunol. 26: 690-96. FcRn binds to the Fc region of IgG antibodies with nanomolar affinity (KD ≈ 100 nM) at a pH of less than or equal to 6.0, but does not bind at the pH of blood, i.e., about pH 7.4. Tesar and Björkman (2001), Curr. Opin. Struct. Biol. 20(2): 226-233. Upon internalization of an antibody by a cell, for example by pinocytosis, an IgG can be bound by FcRn in the acidic environment of an endosome. Id. When bound by FcRn within the endosome, the IgG will be directed back to the cell surface, as opposed to entering a default catabolic pathway within the endosome. Id. In the generally physiologic pH environment at the cell surface, the IgG can dissociate from FcRn and re-enter the circulation. This process allows the antibody to return to the circulation following internalization within a cell, as opposed to being degraded in the cell within an endosome.

    [0004] International Publication Number WO 2005/047327 A2 (Biogen IDEC MA Inc.; Cambridge MA (US)) discloses methods of generating Fc variants which exhibit differential binding to FcRn at, for example, pH 6.0 compared to pH 7.4. Fc-containing polypeptides, (antibodies and fusion proteins containing an Fc region or a biologically active portion thereof) may be modified at one, two, or more amino acid residues (but generally no more than ten residues) adjacent to one or more of the Fc region positions identified as impacting FcRn binding by the Fc region.

    [0005] International Publication Number WO2008/119096 A1 (F-Star Biotechnologische Forschungs- und Enwicklungsges.m.b.H.; Vienna (AT)) discloses methods of producing IgG variants exhibiting higher affinity binding to FcRn at pH 5-7 than at pH 7.1 to 7.6. These variants comprise substitution, deletion and/or insertion of two or more amino acid residues, preferably point mutations or mutation of up to 20, 25 or 30 amino acid residues of selected Fc-region loops (with loop 5 being particularly preferred), in order to achieve prolongation of in vivo half-life with the retention of biological activity.

    [0006] International Publication Number WO 2009/058492 A2 (Xencor, Inc.; Monrovia CA, (US)) discloses Fc variants, engineering methods for their generation and optimization, and their application, particularly for therapeutic purposes. Preparation of variants exhibiting increased serum-retention in vivo is proposed: insertion or deletion of amino acid residues at selected Fc region loop locations increases binding affinity between the Fc polypeptide and FcRn at around pH 6.0, while maintaining lower affinity at pH 7.4.

    [0007] There is a need in the art for therapeutic antibodies with increased in vivo half lives so as to decrease dosing amounts and/or frequencies. Such antibodies are advantageous because of increased patient convenience, and therefore also possibly increased patient compliance, and/or decreased cost. In the current cost-conscious health care environment, cost can be a determining factor in the practical utility of a therapeutic product.

    SUMMARY



    [0008] The present invention is defined by the claims. Provided are variant Fc-fragments that bind to FcRn with higher affinity and/or higher binding activity than does a control Fc-fragment at a slightly acidic pH and that bind to FcRn with about the same affinity as or lower than a control Fc-fragment, that is, little or no binding activity, at a physiologic pH. Also provided are variant Fc-polypeptides, which contain a variant Fc-fragment as well as a binding region that binds to a target molecule. It is demonstrated herein that variant Fc-polypeptides containing variant Fc-fragments with the binding properties mentioned above also have longer in vivo half lives than control Fc-polypeptides. Further provided are nucleic acids that encode these Fc-fragments and Fc-polypeptides and methods of making these proteins using these nucleic acids. Also included are methods for extending the in vivo half life of an Fc-polypeptide and methods for identifying variant Fc-fragments that bind to FcRn with higher affinity at pH 5-6 and bind FcRn with comparable or lower affinity at physiologic pH as compared to a control Fc-fragment.

    [0009] Described here is a variant Fc-polypeptide comprising a human IgG1, IgG2, IgG3, or IgG4 variant Fc-fragment, wherein the variant Fc-fragment comprises an insertion of 3 to 20, 10 to 20, 20 to 40, 40 to 60, or 60 to 80 amino acids within or adjacent to Loop 5, 8, and/or 10 of the variant Fc-fragment, wherein the variant Fc-polypeptide binds to a human neonatal Fc receptor (hFcRn) with higher affinity and/or higher binding activity at a pH from about 5.0 to about 6.0 and/or at a pH of about 5.0, 5.2, 5.5, 5.7, or 6.0 than a control Fc-polypeptide that has the same amino acid sequence as the variant Fc-polypeptide except that it does not contain the insertion within or adjacent to Loop 5, 8, and/or 10 and wherein the variant Fc-polypeptide binds to the human neonatal Fc receptor (hFcRn) with approximately the same or lower affinity or binding activity as compared to the control Fc-polypeptide, that is, little or no binding activity, at a physiologic pH and/or at a pH of about 7.4, 7.5, or 7.6. The insertion within or adjacent to the variant Fc-fragment can be at least six amino acids long, not more than 25 amino acids long, from 6 to 16 amino acids long, and/or at least 12 amino acids long. In some embodiments, the insertion does not contain methionine and/or tryptophan residues. The insertion within or adjacent to the variant Fc-fragment can comprise at least one cysteine among the first four inserted amino acids and at least one cysteine among the last four inserted amino acids. Optionally, the insertion can lack cysteine residues other than any cysteine residues occurring in the first or last four amino acids of the insertion. In some embodiments, the insertion is within or adjacent to Loop 10 of the variant Fc-fragment, optionally between amino acids 384 and 385 of the variant Fc-fragment using the EU numbering system shown in Table 1. The insertion can be within amino acids 383 to 387 using the EU numbering system. In some embodiments, the insertion in the variant human Fc-fragment can be between amino acids 382 and 383, 383 and 384, 385 and 386, 386 and 387, 387 and 388, 388 and 389, 389 and 390, or 390 and 391 using the EU numbering system shown in Table 1. Alternatively, amino acids 384-386 can be deleted, and the insertion can be between amino acids 383 and 387 using the EU numbering system shown in Table 1. As described herein, the insertion in the variant Fc-fragment can comprise the amino acid sequence of any one of SEQ ID NO:13, 14, 15,16, 17, 18, 19,20,21,22, 23, 24, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 90-356, and 359-379. Further, the insertion in the variant Fc-fragment can comprise the amino acid sequence of any one of the following: SEQ ID NOs:41-67; SEQ ID NOs:90-246; SEQ ID NOs:247-356; SEQ ID NOs:367, 369, 372, 373, and 375-379; amino acids 4-9 of any one of SEQ ID NOs:41-53, 67, 90-162, 90-163, 165-215, 164-214, 217-247, 216-246, 359-379 and 392-399, 401-409, 411-426, 428-439, 441-447, 449-453, 456, 459, 461, 464, and 470, 475, 477-489, 491-496; amino acids 4-10 of SEQ ID NO:164, 252, and 490, and amino acids 4-8 of SEQ ID NO:215 or 216; amino acids 4-11 of any one of SEQ ID NOs: 248-288, 290-306, 342, 400, 410, 427, 440, 448, 454, 455, 457, 458, 460, 465-469, 471-474, and 476; amino acids 4-12 of SEQ ID NO:289 or 307; and amino acids 4-13 of any one of SEQ ID NOs:308-341 and 343-356.

    [0010] Further described herein is a variant Fc-polypeptide comprising a human IgG1, IgG2, IgG3 or IgG4 variant Fc-fragment, wherein the variant Fc-fragment comprises an insertion of 3 to 20, 10 to 20, 20 to 40, 40 to 60, or 60 to 80 amino acids within or adjacent to Loop 10, wherein the variant Fc-polypeptide binds to a hFcRn with higher affinity and/or higher binding activity at a pH in a range from about 5.0 to about 6.0 and/or at a pH of about 5.0, 5.2, 5.5, 5.7, or 6.0 than a control Fc-polypeptide that is the same as the variant Fc-polypeptide except that it does not contain the insertion within or adjacent to Loop 10 and wherein the variant Fc-polypeptide binds to the hFcRn with approximately the same or lower affinity as compared to the control Fc-polypeptide, that is, with little or no binding activity, at a physiologic pH and/or at a pH of about 7.4, 7.5, or 7.6. The insertion can be at least six amino acids long, can be not more than 25 amino acids long, can be from 6 to 16 amino acids long, can be at least 12 amino acids long, and/or can contain at least one cysteine among the first four amino acids of the insertion and at least one cysteine among the last four amino acids of the insertion. The insertion can lack cysteine residues at other positions within the insertion. The insertion can lack methionine and/or tryptophan residues. The insertion in the variant Fc-fragment can be within amino acids 383-387 according to the EU numbering system. The insertion in the variant Fc-fragment can be between amino acids 382 and 383, 383 and 384, 384 and 385, 385 and 386, 386 and 387, 387 and 388, 388 and 389, 389 and 390, or 390 and 391 using the EU numbering system shown in Table 1. Alternatively amino acids 384-386 can be deleted, and the insertion can occur between amino acids 383 and 387, using the EU numbering system. The insertion in the variant Fc-fragment can comprise the amino acid sequence of any one of SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 90-356, 359-379, and 392-496. Further, the insertion in the variant Fc-fragment can comprise the amino acid sequence of any one of the following: SEQ ID NOs:41-67; SEQ ID NOs:90-246; SEQ ID NOs:247-356; SEQ ID NOs:367, 369, 372, 373, and 375-379; amino acids 4-9 of any one of SEQ ID NOs:41-53, 67, 90-162, 90-163, 165-215, 164-214, 217-247, 216-246, 359-379 and 392-399, 401-409, 411-426, 428-439, 441-447, 449-453, 456, 459, 461, 464, and 470, 475, 477-489, 491-496; amino acids 4-10 of SEQ ID NO:164, 252, and 490, and amino acids 4-8 of SEQ ID NO:215 or 216; amino acids 4-11 of any one of SEQ ID NOs: 248-288, 290-306, 342, 400, 410, 427, 440, 448, 454, 455, 457, 458, 460, 465-469, 471-474, and 476; amino acids 4-12 of SEQ ID NO:289 or 307; and amino acids 4-13 of any one of SEQ ID NOs:308-341 and 343-356. The variant Fc-polypeptide can be a variant Fc fusion protein comprising a non-antibody polypeptide. In particular aspects, a control Fc fusion protein for the variant Fc fusion protein can be alefacept, rilonacept, aflibercept, etanercept, romiplostim, or abatacept. In other aspects, the variant Fc-polypeptide can comprise a heavy chain variable region (VH) and/or a light chain variable region (VL) of any antibody and may also comprise a first heavy chain constant region (CH1) and a light chain constant region (CL). In some aspects, the variant Fc-polypeptide can comprise (a) a heavy chain comprising a VH region, a first heavy chain constant region (CH1), a hinge region, a CH2 region, and a CH3 region, and (b) a light chain comprising a VL region and a light chain constant region (CL). The variant Fc-polypeptide can be monovalent. The variant Fc-polypeptide can be a dimer or can be a tetramer.

    [0011] In further aspects, described herein are nucleic acids encoding the variant Fc-polypeptides or the variant Fc-fragments described herein, as well as the insertions within or adjacent to the loops of the variant Fc-fragments. Also described are host cells containing such nucleic acids. Also contemplated are methods of making a variant Fc-polypeptide or variant Fc-fragment comprising (a) introducing a nucleic acid encoding the variant Fc-polypeptide or Fc-fragment into a host cell, (b) culturing the host cell comprising the nucleic acid under conditions such that the nucleic acid is expressed, and (c) recovering the expressed variant Fc-polypeptide or Fc-fragment from the culture medium or the cell mass, wherein the variant Fc-fragment, or the variant Fc-polypeptide that contains a variant Fc-fragment, comprises an insertion of 3 to 20, 10 to 20, 20 to 40, 40 to 60, or 60 to 80 amino acids within or adjacent to Loop 5, 8, and/or 10 of the variant Fc-fragment, and wherein the variant Fc-polypeptide or Fc-fragment binds to a hFcRn with higher affinity and or higher binding activity at a pH in a range from about 5.0 to about 6.0 and/or at a pH of about 5.0, 5.2, 5.5, 5.7, or 6.0 than a control Fc-polypeptide or Fc-fragment and wherein the variant Fc-polypeptide or Fc-fragment binds to the human neonatal Fc receptor (hFcRn) with approximately the same or lower affinity or binding activity as compared to the control Fc-polypeptide or control Fc-fragment, that is, with little or no binding activity, at a physiologic pH and/or at a pH of about 7.4, 7.5, or 7.6..

    [0012] Also described are methods for making any of the variant Fc-fragments or Fc-polypeptides described herein comprising (a) introducing a nucleic acid encoding the variant Fc-polypeptide or Fc-fragment into a host cell, (b) culturing the host cell comprising the nucleic acid under conditions such that the nucleic acid is expressed, and (c) recovering the expressed variant Fc-polypeptide or Fc-fragment from the culture medium or the cell mass.

    [0013] In a further aspect, described herein is a method for extending the half life of an Fc-polypeptide comprising a human IgG Fc-fragment comprising the following steps: selecting a site within or adjacent to Loop 5, 8, and/or 10 for insertion; and inserting a peptide into the selected site, wherein the peptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:41-67; SEQ ID NOs:90-246; SEQ ID NOs:247-356; SEQ ID NOs:367, 369, 372, 373, and 375-379; amino acids 4-9 of any one of SEQ ID NOs:41-53, 67, 90-162, 90-163, 165-215, 164-214, 217-247, 216-246, 359-379 and 392-399, 401-409, 411-426, 428-439, 441-447, 449-453, 456, 459, 461, 464, and 470, 475, 477-489, 491-496; amino acids 4-10 of SEQ ID NO:164, 252, and 490, and amino acids 4-8 of SEQ ID NO:215 or 216; amino acids 4-11 of any one of SEQ ID NOs: 248-288, 290-306, 342, 400, 410, 427, 440, 448, 454, 455, 457, 458, 460, 465-469, 471-474, and 476; amino acids 4-12 of SEQ ID NO:289 or 307; and amino acids 4-13 of any one of SEQ ID NOs:308-341 and 343-356. The selected site can be within or adjacent to Loop 10, and the insertion site can be between amino acids 384 and 385, numbered according to the EU numbering system. The insertion site can be within amino acids 383 to 387 using the EU numbering system. The insertion in the variant Fc-fragment can be between amino acids 382 and 383, 383 and 384, 384 and 385, 385 and 386, 386 and 387, 387 and 388, 388 and 389, 389 and 390, or 390 and 391 using the EU numbering system shown in Table 1. Alternatively amino acids 384-386 can be deleted, and the insertion can occur between amino acids 383 and 387, using the EU numbering system.

    [0014] Further described herein is a method for identifying a human IgG variant Fc-fragment that confers a longer in vivo half life on a variant Fc-polypeptide that comprises the variant Fc-fragment, as compared to a control Fc-polypeptide, comprising the following steps: (a) creating a library of nucleic acids encoding Fc-fragments containing an insertion comprising 4-20, 10 to 20, 20-40, 40-60, or 60-80 randomized amino acids within or adjacent to Loop 10; (b) screening Fc-fragments encoded by the library to identify the variant Fc-fragments that (i) bind to human FcRn with higher affinity and/or higher binding activity at pH 5.5, and/or at a pH from about 5-6, than a control Fc-fragment and (ii) bind to human FcRn at the same or lower affinity or binding activity compared to the control Fc-fragment, that is, with little or no binding activity, at physiologic pH and/or at a pH of 7.4, 7.5, or 7.6; (c) constructing a nucleic acid encoding a variant Fc-polypeptide comprising a variant Fc-fragment identified in (b), wherein the concentration of a control Fc-polypeptide, which comprises a control Fc-fragment rather than the variant Fc-fragment, is known to decrease linearly over time when administered to an animal in vivo; (d) introducing the nucleic acid of (c) into a host cell and culturing the host cell under conditions such that the variant Fc-polypeptide encoded by the nucleic acid can be expressed; (e) recovering the variant Fc-polypeptide from the cell mass or cell culture medium; (f) administering the variant Fc-polypeptide to an animal and administering the control Fc-polypeptide to another animal; and (g) monitoring the concentrations of the variant and control Fc-polypeptides in peripheral blood over time subsequent to administration, thereby identifying a variant Fc-fragment that confers a longer in vivo half life on a variant Fc-polypeptide. The insertion of step (a) can be between positions 384 and 385 using the EU numbering system as illustrated in Table 1. The insertion site can be within amino acids 383 to 387 using the EU numbering system. The insertion in the variant Fc-fragment can be between amino acids 382 and 383, 383 and 384, 384 and 385, 385 and 386, 386 and 387, 387 and 388, 388 and 389, 389 and 390, or 390 and 391 using the EU numbering system shown in Table 1. Alternatively amino acids 384-386 can be deleted, and the insertion can occur between amino acids 383 and 387, using the EU numbering system.

    [0015] In another aspect, a method is provided for treating a chronic disease comprising administering to a patient in need thereof a variant Fc-polypeptide as described herein.

    BRIEF DESCRIPTION OF THE FIGURES



    [0016] 

    Figure 1: Ribbon diagram of the predicted three dimensional structure of portions of human FcRn (hFcRn) and a human IgG1 Fc-fragment that come in closest contact upon binding of FcRn to the Fc-fragment. At top is a ribbon diagram of a portion of the tertiary structure of hFcRn. Below is a ribbon diagram of a portion of a human IgG1 Fc-fragment that comes in closest contact with FcRn when FcRn is bound to it. Loops are shown as strings, whereas alpha helices and beta sheets are shown as ribbons. The six sites at which insertions were made are indicated by the names of the eight libraries that were constructed as described below, i.e., L1, L2A, L2B, L3, L4, L5, L6A, and L6B.

    Figure 2: Format of the insertion libraries. Above is shown the sequence of a human IgG1 Fc-fragment (SEQ ID NO:1). Amino acids within loops in which, or adjacent to which, insertions were made are indicated by underlining and boldface type. Library names, i.e., L1, L2A, etc., appear above the region into which insertions were made for that particular library. The format of the insertions is indicated below. At far left are the library names, i.e., L1, L2A, etc. Just to the right of the library names is the original sequence around the insertion site prior to the insertion of any amino acids. Italic letters indicate amino acids that are deleted prior to insertion. A vertical dashed line indicates the insertion site in those libraries in which no amino acids were deleted. At far right is the sequence around the insertion site with the insertion in place. The designations "(19R)5" or "(19R)6" means five or six, respectively, randomized amino acids, which can be any of the 19 amino acids other than cysteine.

    The designation "(20R)8" means eight randomized amino acids, which can be any of the twenty amino acids.

    Figure 3: Association and dissociation curves at pH 6 and pH 7.4, respectively, of variant Fc-fragments from library L5. The curves at left show the response detected using the ForteBio Octet® system as explained in Example 4 where the portions of the curves to the left of the central vertical line show the relative amounts of association of the various Fc-fragments to FcRn at pH 6 and the portions of the curves to the right of the central vertical line show the dissociation of the Fc-fragments from FcRn at pH 7.4. The table at right provides the maximal binding response detected at pH 6 for each variant and for a wild type Fc-fragment (FcWT).

    Figure 4: Average concentrations of antibodies containing variant and control Fc-fragments as a function of time post-injection in cynomolgus monkeys. The x axis indicates time in hours post-injection, and the y axis indicates the concentration of the antibody in nanograms per milliliter (ng/mL) in peripheral blood of cynomolgus monkeys that have been injected with an antibody.

    Figure 5: Sequences of variants of Loop 10. The amino acid sequences of wild type or variant versions of a loop in a human IgG1 Fc-fragment, plus three adjacent amino acids on either side, are shown. The amino acid sequence shown is from amino acid residues 166-178, in the numbering scheme of Figure 2, or amino acids 381-393 according to EU numbering (SEQ ID NO:563) as shown in this figure and in Table 1. The unaltered, wild type loop region sequence is designated "wild type sequence." The sequence of this loop region in Fc variant 5-1 (encoded by an isolate from library L5) is designated "5-1" (SEQ ID NO:564). Inserted amino acid sequences are shown in boldface with underlining. The sequence of this same loop region from various variants of 5-1, which have the same peptide inserted in different locations within or adjacent to this loop, in some cases with some loop amino acids deleted, are shown below.

    Figure 6: Association and dissociation curves at pH 6 and pH 7.4, respectively, of variant Fc-fragments having the same insertion at different positions of Loop 10. At left are shown the association curves at pH 6 (to the left of the central vertical line) and dissociation curves at pH 7.4 (to the right of the central vertical line) of the various variant Fc-fragments and a wild type Fc-fragment (FcWT). At right the maximal binding response detected for each variant at pH 6 is shown in tabular form.

    Figure 7: Association and dissociation curves at pH 6 and pH 7.4 of variant Fc-fragments from library L5 screened in yeast. At left are shown the association curves at pH 6 (to the left of the central vertical line) and dissociation curves at pH 7.4 (to the right of the central vertical line) of the various variant Fc-fragments and a wild type Fc-fragment (FcWT). At right the maximal binding response detected for each variant at pH 6 is shown in tabular form.

    Figure 8: Association and dissociation curves at pH 6 and pH 7.4 of variant Fc-fragments from library L-8 and L-10 screened in yeast. At left are shown the association curves at pH 6 (to the left of the central vertical line) and dissociation curves at pH 7.4 (to the right of the central vertical line) of the various variant Fc-fragments and a wild type Fc-fragment (FcWT). At right the maximal binding response detected for each variant at pH 6 is shown in tabular form.

    Figure 9: Average concentrations of antibodies containing variant and control Fc-fragments as a function of time post-injection in cynomolgus monkeys. The x axis indicates time in hours post-injection, and the y axis indicates the concentration of the antibody in nanograms per milliliter (ng/mL) in peripheral blood of cynomolgus monkeys that have been injected with an antibody.

    Figure 10: Average concentrations of antibodies containing variant and control Fc-fragments as a function of time post-injection in cynomolgus monkeys. The x axis indicates time in hours post-injection, and the y axis indicates the concentration of the antibody in nanograms per milliliter (ng/mL) in peripheral blood of cynomolgus monkeys that have been injected with an antibody. As indicated, filled circles represent individual data points for cynomolgus monkeys injected with Y-5-112, and the solid line represents the mean of these data. Similarly, triangles represent individual data points for cynomolgus monkeys injected with Antibody Y, and the dashed line represents the mean of these data.

    BRIEF DESCRIPTION OF THE SEQUENCES
    SEQUENCE LISTING NUMBERDESCRIPTION
    SEQ ID NO:1 Amino acid sequence of a human IgG1 Fc region
    SEQ ID NO:2 Nucleotide sequence encoding a human IgG1 Fc region
    SEQ ID NO:3 Amino acid sequence of a human IgG2 Fc region
    SEQ ID NO:4 Nucleotide sequence encoding a human IgG2 Fc region
    SEQ ID NO:5 Amino acid sequence of a human IgG3 Fc region
    SEQ ID NO:6 Nucleotide sequence encoding a human IgG3 Fc region
    SEQ ID NO:7 Amino acid sequence of a human IgG4 Fc region
    SEQ ID NO:8 Nucleotide sequence encoding a human IgG4 Fc region
    SEQ ID NO:9 Amino acid sequence of human FcRn alpha chain
    SEQ ID NO:10 Amino acid sequence of human FcRn β-2-microglobulin chain
    SEQ ID NO:11 Amino acid sequence of the extracellular region of mature human p75 tumor necrosis factor receptor
    SEQ ID NO:12 Amino acid sequence of the extracellular region of mature human (CTLA4)
    SEQ ID NO:13 Amino acid sequence of randomized insertion sequence (CXXXXXXC)
    SEQ ID NO:14 Amino acid sequence of randomized insertion sequence (CXXXXXXXC)
    SEQ ID NO:15 Amino acid sequence of randomized insertion sequence (CXXXXXXXXC)
    SEQ ID NO:16 Amino acid sequence of randomized insertion sequence (GCXXXXXXCG)
    SEQ ID NO:17 Amino acid sequence of randomized insertion sequence (GCXXXXXXXCG)
    SEQ ID NO:18 Amino acid sequence of randomized insertion sequence (GCXXXXXXXXCG)
    SEQ ID NO:19 Amino acid sequence of randomized insertion sequence (GGCXXXXXXCGG)
    SEQ ID NO:20 Amino acid sequence of randomized insertion sequence (GGCXXXXXXXCGG)
    SEQ ID NO:21 Amino acid sequence of randomized insertion sequence (GGCXXXXXXXXCGG)
    SEQ ID NO:22 Amino acid sequence of randomized insertion sequence (GGGCXXXXXXCGGG)
    SEQ ID NO:23 Amino acid sequence of randomized insertion sequence (GGGCXXXXXXXCGGG)
    SEQ ID NO:24 Amino acid sequence of randomized insertion sequence (GGGCXXXXXXXXCGGG)
    SEQ ID NO:25 Nucleotide sequence of the Fc-fragment-encoding portion of library L1
    SEQ ID NO:26 Amino acid sequence encoded by SEQ ID NO:25
    SEQ ID NO:27 Nucleotide sequence of the Fc-fragment-encoding portion of library L2A
    SEQ ID NO:28 Amino acid sequence encoded by SEQ ID NO:27
    SEQ ID NO:29 Nucleotide sequence of the Fc-fragment-encoding portion of library L2B
    SEQ ID NO:30 Amino acid sequence encoded by SEQ ID NO:29
    SEQ ID NO:31 Nucleotide sequence of the Fc-fragment-encoding portion of library L3
    SEQ ID NO:32 Amino acid sequence encoded by SEQ ID NO:31
    SEQ ID NO:33 Nucleotide sequence of the Fc-fragment-encoding portion of library L4
    SEQ ID NO:34 Amino acid sequence encoded by SEQ ID NO:33
    SEQ ID NO:35 Nucleotide sequence of the Fc-fragment-encoding portion of library L5
    SEQ ID NO:36 Amino acid sequence encoded by SEQ ID NO:35
    SEQ ID NO:37 Nucleotide sequence of the Fc-fragment-encoding portion of library L6A
    SEQ ID NO:38 Amino acid sequence encoded by SEQ ID NO:37
    SEQ ID NO:39 Nucleotide sequence of the Fc-fragment-encoding portion of library L6B
    SEQ ID NQ:40 Amino acid sequence encoded by SEQ ID NO:39
    SEQ ID NO:41 Amino acid sequence of the insertion in variant Fc-fragment Fc-5-51
    SEQ ID NO:42 Amino acid sequence of the insertion in variant Fc-fragment Fc-5-69
    SEQ ID NO:43 Amino acid sequence of the insertion in variant Fc-fragment Fc-5-104
    SEQ ID NO:44 Amino acid sequence of the insertion in variant Fc-fragment Fc-5-106
    SEQ ID NO:45 Amino acid sequence of the insertion in variant Fc-fragment Fc-5-112
    SEQ ID NO:46 Amino acid sequence of the insertion in variant Fc-fragment Fc-5-55
    SEQ ID NO:47 Amino acid sequence of the insertion in variant Fc-fragment Fc-5-60
    SEQ ID NO:48 Amino acid sequence of the insertion in variant Fc-fragment Fc-5-70
    SEQ ID NO:49 Amino acid sequence of the insertion in variant Fc-fragment Fc-5-79
    SEQ ID NO:50 Amino acid sequence of the insertion in variant Fc-fragment Fc-5-85
    SEQ ID NO:51 Amino acid sequence of the insertion in variant Fc-fragment Fc-5-95
    SEQ ID NO:52 Amino acid sequence of the insertion in variant Fc-fragment Fc-5-97
    SEQ ID NO:53 Amino acid sequence of the insertion in variant Fc-fragment Fc-5-99
    SEQ ID NO:54 Amino acid sequence of the middle six amino acids of the insertion in Fc-5-51
    SEQ ID NO:55 Amino acid sequence of the middle six amino acids of the insertion in Fc-5-69
    SEQ ID NO:56 Amino acid sequence of the middle six amino acids of the insertion in Fc-5-104
    SEQ ID NO:57 Amino acid sequence of the middle six amino acids of the insertion in Fc-5-106
    SEQ ID NO:58 Amino acid sequence of the middle six amino acids of the insertion in Fc-5-112
    SEQ ID NO:59 Amino acid sequence of the middle six amino acids of the insertion in Fc-5-55
    SEQ ID NO:60 Amino acid sequence of the middle six amino acids of the insertion in Fc-5-60
    SEQ ID NO:61 Amino acid sequence of the middle six amino acids of the insertion in Fc-5-70
    SEQ ID NO:62 Amino acid sequence of the middle six amino acids of the insertion in Fc-5-79
    SEQ ID NO:63 Amino acid sequence of the middle six amino acids of the insertion in Fc-5-85
    SEQ ID NO:64 Amino acid sequence of the middle six amino acids of the insertion in Fc-5-95
    SEQ ID NO:65 Amino acid sequence of the middle six amino acids of the insertion in Fc-5-97
    SEQ ID NO:66 Amino acid sequence of the middle six amino acids of the insertion in Fc-5-99
    SEQ ID NO:67 Amino acid sequence of the insertion in variant Fc-fragment 5-1
    SEQ ID NO:68 Nucleotide sequence of the forward primer used to construct a vector encoding variant Fc-polypeptide 5-1-1
    SEQ ID NO:69 Nucleotide sequence of the reverse primer used to construct a vector encoding variant Fc-polypeptide 5-1-1
    SEQ ID NO:70 Nucleotide sequence of the forward primer used to construct a vector encoding variant Fc-polypeptide 5-1-2
    SEQ ID NO:71 Nucleotide sequence of the reverse primer used to construct a vector encoding variant Fc-polypeptide 5-1-2
    SEQ ID NO:72 Nucleotide sequence of the forward primer used to construct a vector encoding variant Fc-polypeptide 5-1-3
    SEQ ID NO:73 Nucleotide sequence of the reverse primer used to construct a vector encoding variant Fc-polypeptide 5-1-3
    SEQ ID NO:74 Nucleotide sequence of the forward primer used to construct a vector encoding variant Fc-polypeptide 5-1-4
    SEQ ID NO:75 Nucleotide sequence of the reverse primer used to construct a vector encoding variant Fc-polypeptide 5-1-4
    SEQ ID NO:76 Nucleotide sequence of the forward primer used to construct a vector encoding variant Fc-polypeptide 5-1-5
    SEQ ID NO:77 Nucleotide sequence of the reverse primer used to construct a vector encoding variant Fc-polypeptide 5-1-5
    SEQ ID NO:78 Nucleotide sequence of the forward primer used to construct a vector encoding variant Fc-polypeptide 5-1-6
    SEQ ID NO:79 Nucleotide sequence of the reverse primer used to construct a vector encoding variant Fc-polypeptide 5-1-6
    SEQ ID NO:80 Nucleotide sequence of the forward primer used to construct a vector encoding variant Fc-polypeptide 5-1-7
    SEQ ID NO:81 Nucleotide sequence of the reverse primer used to construct a vector encoding variant Fc-polypeptide 5-1-7
    SEQ ID NO:82 Nucleotide sequence of the forward primer used to construct a vector encoding variant Fc-polypeptide 5-1-8
    SEQ ID NO:83 Nucleotide sequence of the reverse primer used to construct a vector encoding variant Fc-polypeptide 5-1-8
    SEQ ID NO:84 Nucleotide sequence of the forward primer used to construct a vector encoding variant Fc-polypeptide 5-1-9
    SEQ ID NO:85 Nucleotide sequence of the reverse primer used to construct a vector encoding variant Fc-polypeptide 5-1-9
    SEQ ID NO:86 Nucleotide sequence of the forward primer used to construct a vector encoding variant Fc-polypeptide 5-1-10
    SEQ ID NO:87 Nucleotide sequence of the reverse primer used to construct a vector encoding variant Fc-polypeptide 5-1-10
    SEQ ID NO:88 Amino acid sequence of the insertion in library L-8
    SEQ ID NO:89 Amino acid sequence of the insertion in library L-10
    SEQ ID NO:90 Amino acid sequence of insertion in variant Fc-fragment 5y-1
    SEQ ID NO:91 Amino acid sequence of insertion in variant Fc-fragment 5y-2
    SEQ ID NO:92 Amino acid sequence of insertion in variant Fc-fragment 5y-3
    SEQ ID NO:93 Amino acid sequence of insertion in variant Fc-fragment 5y-4
    SEQ ID NO:94 Amino acid sequence of insertion in variant Fc-fragment 5y-5
    SEQ ID NO:95 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-6
    SEQ ID NO:96 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-7
    SEQ ID NO:97 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-8
    SEQ ID NO:98 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-9
    SEQ ID NO:99 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-10
    SEQ ID NO:100 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-11
    SEQ ID NO:101 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-12
    SEQ ID NO:102 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-13
    SEQ ID NO:103 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-14
    SEQ ID NO:104 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-15
    SEQ ID NO:105 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-16
    SEQ ID NO:106 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-17
    SEQ ID N0:107 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-18
    SEQ ID NO:108 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-19
    SEQ ID N0:109 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-20
    SEQ ID NO:110 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-21
    SEQ ID NO:111 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-22
    SEQ ID NO:112 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-23
    SEQ ID NO:113 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-24
    SEQ ID NO:114 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-25
    SEQ ID NO:115 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-26
    SEQ ID NO:116 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-27
    SEQ ID NO: 117 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-28
    SEQ ID NO:118 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-29
    SEQ ID NO:119 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-30
    SEQ ID NO:120 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-31
    SEQ ID NO:121 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-32
    SEQ ID NO:122 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-33
    SEQ ID NO:123 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-34
    SEQ ID NO:124 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-35
    SEQ ID NO:125 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-36
    SEQ ID NO:126 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-37
    SEQ ID NO:127 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-38
    SEQ ID NO:128 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-39
    SEQ ID NO:129 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-40
    SEQ ID NO:130 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-41
    SEQ ID NO:131 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-42
    SEQ ID NO:132 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-43
    SEQ ID NO:133 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-44
    SEQ ID NO:134 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-45
    SEQ ID NO:135 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-46
    SEQ ID NO:136 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-47
    SEQ ID NO:137 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-48
    SEQ ID NO:138 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-49
    SEQ ID NO:139 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-50
    SEQ ID NO:140 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-51
    SEQ ID NO:141 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-52
    SEQ ID NO:142 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-53
    SEQ ID NO:143 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-54
    SEQ ID NO:144 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-55
    SEQ ID NO:145 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-56
    SEQ ID NO:146 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-57
    SEQ ID NO:147 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-58
    SEQ ID NO:148 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-59
    SEQ ID NO:149 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-60
    SEQ ID NO:150 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-61
    SEQ ID NO:151 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-62
    SEQ ID NO:152 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-63
    SEQ ID NO:153 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-64
    SEQ ID NO:154 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-65
    SEQ ID NO:155 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-66
    SEQ ID NO:156 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-67
    SEQ ID NO:157 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-68
    SEQ ID NO:158 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-69
    SEQ ID NO:159 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-70
    SEQ ID NO:160 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-71
    SEQ ID NO:161 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-72
    SEQ ID NO:162 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-73
    SEQ ID NO:163 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-74
    SEQ ID NO:164 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-75
    SEQ ID NO:165 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-76
    SEQ ID NO:166 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-77
    SEQ ID NO:167 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-78
    SEQ ID NO:168 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-79
    SEQ ID NO:169 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-80
    SEQ ID NO:170 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-81
    SEQ ID NO:171 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-82
    SEQ ID NO:172 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-83
    SEQ ID NO:173 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-84
    SEQ ID NO:174 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-85
    SEQ ID NO:175 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-86
    SEQ ID NO:176 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-87
    SEQ ID NO:177 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-88
    SEQ ID NO:178 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-89
    SEQ ID NO:179 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-90
    SEQ ID NO:180 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-91
    SEQ ID NO:181 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-92
    SEQ ID NO:182 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-93
    SEQ ID NO:183 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-94
    SEQ ID NO:184 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-95
    SEQ ID NO:185 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-96
    SEQ ID NO:186 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-97
    SEQ ID NO:187 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-98
    SEQ ID NO:188 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-99
    SEQ ID NO:189 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-100
    SEQ ID NO:190 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-101
    SEQ ID NO:191 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-102
    SEQ ID NO:192 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-103
    SEQ ID NO:193 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-104
    SEQ ID NO:194 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-105
    SEQ ID NO:195 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-106
    SEQ ID NO:196 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-107
    SEQ ID NO:197 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-108
    SEQ ID NO:198 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-109
    SEQ ID NO:199 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-110
    SEQ ID NO:200 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-111
    SEQ ID NO:201 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-112
    SEQ ID NO:202 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-113
    SEQ ID NO:203 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-114
    SEQ ID NO:204 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-115
    SEQ ID NO:205 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-116
    SEQ ID NO:206 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-117
    SEQ ID NO:207 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-118
    SEQ ID NO:208 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-119
    SEQ ID NO:209 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-120
    SEQ ID NO:210 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-121
    SEQ ID NO:211 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-122
    SEQ ID NO:212 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-123
    SEQ ID NO:213 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-124
    SEQ ID NO:214 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-125
    SEQ ID NO:215 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-126
    SEQ ID NO:216 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-127
    SEQ ID NO:217 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-128
    SEQ ID NO:218 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-129
    SEQ ID NO:219 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-130
    SEQ ID NO:220 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-131
    SEQ ID NO:221 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-132
    SEQ ID NO:222 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-133
    SEQ ID NO:223 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-134
    SEQ ID NO:224 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-135
    SEQ ID NO:225 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-136
    SEQ ID NO:226 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-137
    SEQ ID NO:227 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-138
    SEQ ID NO:228 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-139
    SEQ ID NO:229 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-140
    SEQ ID NO:230 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-141
    SEQ ID NO:231 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-142
    SEQ ID NO:232 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-143
    SEQ ID NO:233 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-144
    SEQ ID NO:234 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-145
    SEQ ID NO:235 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-146
    SEQ ID NO:236 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-147
    SEQ ID NO:237 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-148
    SEQ ID NO:238 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-149
    SEQ ID NO:239 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-150
    SEQ ID NO:240 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-151
    SEQ ID NO:241 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-152
    SEQ ID NO:242 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-153
    SEQ ID NO:243 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-154
    SEQ ID NO:244 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-155
    SEQ ID NO:245 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-156
    SEQ ID NO:246 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-157
    SEQ ID NO:247 Amino acid sequence of sequence of insertion in variant Fc-fragment 5y-158
    SEQ ID NO:248 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-1
    SEQ ID NO:249 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-2
    SEQ ID NO:250 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-3
    SEQ ID NO:251 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-4
    SEQ ID NO:252 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-5
    SEQ ID NO:253 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-6
    SEQ ID NO:254 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-7
    SEQ ID NO:255 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-8
    SEQ ID NO:256 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-9
    SEQ ID NO:257 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-10
    SEQ ID NO:258 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-11
    SEQ ID NO:259 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-12
    SEQ ID NO:260 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-13
    SEQ ID NO:261 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-14
    SEQ ID NO:262 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-15
    SEQ ID NO:263 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-16
    SEQ ID NO:264 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-17
    SEQ ID NO:265 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-18
    SEQ ID NO:266 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-19
    SEQ ID NO:267 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-20
    SEQ ID NO:268 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-21
    SEQ ID NO:269 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-22
    SEQ ID NO:270 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-23
    SEQ ID NO:271 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-24
    SEQ ID NO:272 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-25
    SEQ ID NO:273 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-26
    SEQ ID NO:274 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-27
    SEQ ID NO:275 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-28
    SEQ ID NO:276 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-29
    SEQ ID NO:277 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-30
    SEQ ID NO:278 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-31
    SEQ ID NO:279 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-32
    SEQ ID NO:280 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-33
    SEQ ID NO:281 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-34
    SEQ ID NO:282 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-35
    SEQ ID NO:283 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-36
    SEQ ID NO:284 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-37
    SEQ ID NO:285 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-38
    SEQ ID NO:286 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-39
    SEQ ID NO:287 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-40
    SEQ ID NO:288 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-41
    SEQ ID NO:289 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-42
    SEQ ID NO:290 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-43
    SEQ ID NO:291 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-44
    SEQ ID NO:292 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-45
    SEQ ID NO:293 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-46
    SEQ ID NO:294 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-47
    SEQ ID NO:295 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-48
    SEQ ID NO:296 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-49
    SEQ ID NO:297 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-50
    SEQ ID NO:298 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-51
    SEQ ID NO:299 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-52
    SEQ ID NO:300 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-53
    SEQ ID NO:301 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-54
    SEQ ID NO:302 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-55
    SEQ ID NO:303 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-56
    SEQ ID NO:304 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-57
    SEQ ID NO:305 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-58
    SEQ ID NO:306 Amino acid sequence of sequence of insertion in variant Fc-fragment 8y-59
    SEQ ID NO:307 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-1
    SEQ ID NO:308 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-2
    SEQ ID NO:309 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-3
    SEQ ID NO:310 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-4
    SEQ ID NO:311 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-5
    SEQ ID NO:312 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-6
    SEQ ID NO:313 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-7
    SEQ ID NO:314 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-8
    SEQ ID NO:315 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-9
    SEQ ID NO:316 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-10
    SEQ ID NO:317 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-11
    SEQ ID NO:318 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-12
    SEQ ID NO:319 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-13
    SEQ ID NO:320 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-14
    SEQ ID NO:321 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-15
    SEQ ID NO:322 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-16
    SEQ ID NO:323 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-17
    SEQ ID NO:324 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-18
    SEQ ID NO:325 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-19
    SEQ ID NO:326 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-20
    SEQ ID NO:327 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-21
    SEQ ID NO:328 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-22
    SEQ ID NO:329 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-23
    SEQ ID NO:330 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-24
    SEQ ID NO:331 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-25
    SEQ ID NO:332 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-26
    SEQ ID NO:333 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-27
    SEQ ID NO:334 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-28
    SEQ ID NO:335 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-29
    SEQ ID NO:336 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-30
    SEQ ID NO:337 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-31
    SEQ ID NO:338 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-32
    SEQ ID NO:339 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-33
    SEQ ID NO:340 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-34
    SEQ ID NO:341 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-35
    SEQ ID NO:342 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-36
    SEQ ID NO:343 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-37
    SEQ ID NO:344 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-38
    SEQ ID NO:345 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-39
    SEQ ID NO:346 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-40
    SEQ ID NO:347 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-41
    SEQ ID NO:348 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-42
    SEQ ID NO:349 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-43
    SEQ ID NO:350 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-44
    SEQ ID NO:351 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-45
    SEQ ID NO:352 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-46
    SEQ ID NO:353 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-47
    SEQ ID NO:354 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-48
    SEQ ID NO:355 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-49
    SEQ ID NO:356 Amino acid sequence of sequence of insertion in variant Fc-fragment 10y-50
    SEQ ID NO:357 Nucleotide sequence of a primer
    SEQ ID NO:358 Nucleotide sequence of a primer
    SEQ ID NO:359 Amino acid sequence of the peptide insertion in variant Fc-fragment 5-57
    SEQ ID NO:360 Amino acid sequence of the peptide insertion in variant Fc-fragment 5-64
    SEQ ID NO:361 Amino acid sequence of the peptide insertion in variant Fc-fragment 5-66
    SEQ ID NO:362 Amino acid sequence of the peptide insertion in variant Fc-fragment 5-73
    SEQ ID NO:363 Amino acid sequence of the peptide insertion in variant Fc-fragment 5-92
    SEQ ID NO:364 Amino acid sequence of the peptide insertion in variant Fc-fragment 5-96
    SEQ ID NO:365 Amino acid sequence of the peptide insertion in variant Fc-fragment 5-110
    SEQ ID NO:366 Amino acid sequence of the peptide insertion in variant Fc-fragment 5-113
    SEQ ID NO:367 Amino acid sequence of the peptide insertion in variant Fc-fragment Fc-5-69- WIF
    SEQ ID NO:368 Amino acid sequence of the peptide insertion in variant Fc-fragment Fc-5-69- WIY
    SEQ ID NO:369 Amino acid sequence of the peptide insertion in variant Fc-fragment Fc-5-106- M4A
    SEQ ID NO:370 Amino acid sequence of the peptide insertion in variant Fc-fragment Fc-5-106- M4G
    SEQ ID NO:371 Amino acid sequence of the peptide insertion in variant Fc-fragment Fc-5-106- M4H
    SEQ ID NO:372 Amino acid sequence of the peptide insertion in variant Fc-fragment Fc-5-106-M4I
    SEQ ID NO:373 Amino acid sequence of the peptide insertion in variant Fc-fragment Fc-5-106-M4L
    SEQ ID NO:374 Amino acid sequence of the peptide insertion in variant Fc-fragment Fc-5-106- M4N
    SEQ ID NO:375 Amino acid sequence of the peptide insertion in variant Fc-fragment Fc-5-106- M4Q
    SEQ ID NO:376 Amino acid sequence of the peptide insertion in variant Fc-fragment Fc-5-106- M4S
    SEQ ID NO:377 Amino acid sequence of the peptide insertion in variant Fc-fragment Fc-5-106- M4T
    SEQ ID NO:378 Amino acid sequence of the peptide insertion in variant Fc-fragment Fc-5-106- M4V
    SEQ ID NO:379 Amino acid sequence of the peptide insertion in variant Fc-fragment Fc-5-110
    SEQ ID NO:380 Nucleotide sequence of forward PCR primer used to make Antibody X-5y-8
    SEQ ID NO:381 Nucleotide sequence of reverse PCR primer used to make Antibody X-5y-8
    SEQ ID NO:382 Nucleotide sequence of forward PCR primer used to make Antibody X-5y-132
    SEQ ID NO:383 Nucleotide sequence of reverse PCR primer used to make Antibody X-5y-132
    SEQ ID NO:384 Nucleotide sequence of forward PCR primer used to make Antibody X-5y-38
    SEQ ID NO:385 Nucleotide sequence of reverse PCR primer used to make Antibody X-5y-38
    SEQ ID NO:386 Nucleotide sequence of forward PCR primer used to make Antibody X-5y-91
    SEQ ID NO:387 Nucleotide sequence of reverse PCR primer used to make Antibody X-5y-91
    SEQ ID NO:388 Nucleotide sequence of forward PCR primer used to make Antibody X-5y-119
    SEQ ID NO:389 Nucleotide sequence of reverse PCR primer used to make Antibody X-5y-119
    SEQ ID NO:390 Nucleotide sequence of forward PCR primer used to make Antibody X-5y-127
    SEQ ID NO:391 Nucleotide sequence of reverse PCR primer used to make Antibody X-5y-127
    SEQ ID NO:392 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:393 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:394 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:395 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:396 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:397 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:398 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:399 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NQ:400 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:401 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:402 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:403 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:404 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:405 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:406 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:407 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:408 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:409 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:410 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:411 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:412 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:413 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:414 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:415 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:416 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:417 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:418 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:419 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:420 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:421 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:422 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:423 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:424 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:425 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:426 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:427 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:428 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:429 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:430 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:431 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:432 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:433 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:434 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:435 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:436 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:437 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:438 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:439 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:440 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:441 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:442 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:443 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:444 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:445 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:446 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:447 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:448 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:449 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:450 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:451 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:452 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:453 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:454 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:455 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:456 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:457 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:458 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:459 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:460 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:461 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:462 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:463 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:464 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:465 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:466 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:467 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:468 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:469 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:470 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:471 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:472 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:473 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:474 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:475 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:476 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:477 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:478 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:479 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:480 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:481 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:482 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:483 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:484 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:485 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:486 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:487 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:488 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:489 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:490 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:491 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:492 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:493 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:494 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:495 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:496 Amino acid sequence of a peptide insertion in a variant Fc polypeptide with a positive ELISA score
    SEQ ID NO:497 Nucleotide sequence of a forward PCR primer used to make Fc variant Fc-5-69-W1 F
    SEQ ID NO:498 Nucleotide sequence of a reverse PCR primer used to make Fc variant Fc-5-69-W1 F
    SEQ ID NO:499 Nucleotide sequence of a forward PCR primer used to make Fc variant Fc-5-69-W1Y
    SEQ ID NO:500 Nucleotide sequence of a reverse PCR primer used to make Fc variant Fc-5-69-W1Y
    SEQ ID NO:501 Nucleotide sequence of a forward PCR primer used to make Fc variant Fc-5-106-M4A
    SEQ ID NO:502 Nucleotide sequence of a reverse PCR primer used to make Fc variant Fc-5-106-M4A
    SEQ ID NO:503 Nucleotide sequence of a forward PCR primer used to make Fc variant Fc-5-106-M4G
    SEQ ID NO:504 Nucleotide sequence of a reverse PCR primer used to make Fc variant Fc-5-106-M4G
    SEQ ID NO:505 Nucleotide sequence of a forward PCR primer used to make Fc variant Fc-5-106-M4H
    SEQ ID NO:506 Nucleotide sequence of a reverse PCR primer used to make Fc variant Fc-5-106-M4H
    SEQ ID NO:507 Nucleotide sequence of a forward PCR primer used to make Fc variant Fc-5-106-M4I
    SEQ ID NO:508 Nucleotide sequence of a reverse PCR primer used to make Fc variant Fc-5-106-M4
    SEQ ID NO:509 Nucleotide sequence of a forward PCR primer used to make Fc variant Fc-5-106-M4L
    SEQ ID NO:510 Nucleotide sequence of a reverse PCR primer used to make Fc variant Fc-5-106-M4L
    SEQ ID NO:511 Nucleotide sequence of a forward PCR primer used to make Fc variant Fc-5-106-M4N
    SEQ ID NO:512 Nucleotide sequence of a reverse PCR primer used to make Fc variant Fc-5-106-M4N
    SEQ ID NO:513 Nucleotide sequence of a forward PCR primer used to make Fc variant Fc-5-106-M4Q
    SEQ ID NO:514 Nucleotide sequence of a reverse PCR primer used to make Fc variant Fc-5-106-M4Q
    SEQ ID NO:515 Nucleotide sequence of a forward PCR primer used to make Fc variant Fc-5-106-M4S
    SEQ ID NO:516 Nucleotide sequence of a reverse PCR primer used to make Fc variant Fc-5-106-M4S
    SEQ ID NO:517 Nucleotide sequence of a forward PCR primer used to make Fc variant Fc-5-106-M4T
    SEQ ID NO:518 Nucleotide sequence of a reverse PCR primer used to make Fc variant Fc-5-106-M4T
    SEQ ID NO:519 Nucleotide sequence of a forward PCR primer used to make Fc variant Fc-5-106-M4V
    SEQ ID NO:520 Nucleotide sequence of a reverse PCR primer used to make Fc variant Fc-5-106-M4V
    SEQ ID NO:521 Nucleotide sequence of a forward PCR primer used to construct libraries L1, L2A, L2B, L3, L4, and L5
    SEQ ID NO:522 Nucleotide sequence of a reverse PCR primer used to construct library L1
    SEQ ID NO:523 Nucleotide sequence of a reverse PCR primer used to construct libraries L2A and L2B
    SEQ ID NO:524 Nucleotide sequence of a reverse PCR primer used to construct library L3
    SEQ ID NO:525 Nucleotide sequence of a reverse PCR primer used to construct library L4
    SEQ ID NO:526 Nucleotide sequence of a reverse PCR primer used to construct library L5
    SEQ ID NO:527 Nucleotide sequence of a reverse PCR primer used to construct libraries L1, L2A, L2B, L3, L4, and L5
    SEQ ID NO:528* Nucleotide sequence of a forward PCR primer used to construct library L1
    SEQ ID NO:529* Nucleotide sequence of a forward PCR primer used to construct library L2A
    SEQ ID NO:530* Nucleotide sequence of a forward PCR primer used to construct library L2B
    SEQ ID NO:531* Nucleotide sequence of a forward PCR primer used to construct library L3
    SEQ ID NO:532* Nucleotide sequence of a forward PCR primer used to construct library L4
    SEQ ID NO:533* Nucleotide sequence of a forward PCR primer used to construct library L5
    SEQ ID NO:534 Nucleotide sequence of a forward PCR primer used to construct libraries L1, L2A, L2B, L3, L4, and L5
    SEQ ID NO:535 Nucleotide sequence of a reverse PCR primer used to construct libraries L1, L2A, L2B, L3, L4, and L5
    SEQ ID NO:536 Nucleotide sequence of a forward PCR primer used to construct library L6A
    SEQ ID NO:537# Nucleotide sequence of a forward PCR primer used to construct library L6B
    SEQ ID NO:538 Nucleotide sequence of a reverse PCR primer used to construct libraries L6A and L6B
    SEQ ID NO:539 Nucleotide sequence of forward PCR primer used to construct Antibody X-5-51
    SEQ ID NO:540 Nucleotide sequence of reverse PCR primer used to construct Antibody X-5-51
    SEQ ID NO:541 Nucleotide sequence of forward PCR primer used to construct Antibody X-5-69
    SEQ ID NO:542 Nucleotide sequence of reverse PCR primer used to construct Antibody X-5-69
    SEQ ID NO:543 Nucleotide sequence of forward PCR primer used to construct Antibody X-5-104
    SEQ ID NO:544 Nucleotide sequence of reverse PCR primer used to construct Antibody X-5-104
    SEQ ID NO:545 Nucleotide sequence of forward PCR primer used to construct Antibody X-5-106
    SEQ ID NO:546 Nucleotide sequence of reverse PCR primer used to construct Antibody X-5-106
    SEQ ID NO:547 Nucleotide sequence of forward PCR primer used to construct Antibody X-5-112
    SEQ ID NO:548 Nucleotide sequence of reverse PCR primer used to construct Antibody X-5-112
    SEQ ID NO:549 Amino acid sequence of amino acids 36-41 of SEQ ID NO:1, which correspond to amino acids 251-256 in the EU numbering scheme
    SEQ ID NO:550 Amino acid sequence from amino acid 251-256 in the EU numbering scheme in the Fc fragments encoded by Library L1
    SEQ ID NO:551 Amino acid sequence of amino acids 92-99 of SEQ ID NO:1, which correspond to amino acids 307-314 in the EU numbering system
    SEQ ID NO:552 Amino acid sequence from amino acid 307-314 in the EU numbering scheme in the Fc fragments encoded by Library L2A
    SEQ ID NO:553 Amino acid sequence from amino acid 307-314 in the EU numbering scheme in the Fc fragments encoded by Library L2B
    SEQ ID NO:554 Amino acid sequence of amino acids 213-221 of SEQ ID NO:1, which correspond to 428-436 in the EU numbering scheme
    SEQ ID NO:555 Amino acid sequence from amino acid 428-436 in the EU numbering scheme in the Fc fragments encoded by Library L3
    SEQ ID NO:556 Amino acid sequence of amino acids 68-72 of SEQ ID NO:1, which correspond to amino acids 283-287 in the EU numbering scheme
    SEQ ID NO:557 Amino acid sequence from amino acid 283-287 in the EU numbering scheme in the Fc fragments encoded by Library L4
    SEQ ID NO:558 Amino acid sequence of amino acids 169-174 of SEQ ID NO:1, which correspond to amino acids 384-389 in the EU numbering system
    SEQ ID NO:559 Amino acid sequence from amino acid 384-389 in the EU numbering scheme in the Fc fragments encoded by Library L5
    SEQ ID NO:560 Amino acid sequence of amino acids 141-147 of SEQ ID NO:1, which correspond to amino acids 356-362 in the EU numbering system
    SEQ ID NO:561 Amino acid sequence from amino acid 356-362 in the EU numbering scheme in the Fc fragments encoded by Library L6A
    SEQ ID NO:562 Amino acid sequence from amino acid 356-362 in the EU numbering scheme in the Fc fragments encoded by Library L6B
    SEQ ID NO:563% Amino acid sequence of amino acids 166 to 178 of SEQ ID NO:1, corresponding to amino acids 381 to 393 in the EU numbering system
    SEQ ID NO:564% Amino acid sequence corresponding to amino acids 381 to 393 is the EU numbering system of variant Fc fragment 5-1
    SEQ ID NO:565% Amino acid sequence corresponding to amino acids 381 to 393 is the EU numbering system of variant Fc fragment 5-1-1
    SEQ ID NO:566% Amino acid sequence corresponding to amino acids 381 to 393 is the EU numbering system of variant Fc fragment 5-1-2
    SEQ ID NO:567% Amino acid sequence corresponding to amino acids 381 to 393 is the EU numbering system of variant Fc fragment 5-1-3
    SEQ ID NO:568% Amino acid sequence corresponding to amino acids 381 to 393 is the EU numbering system of variant Fc fragment 5-1-4
    SEQ ID NO:569% Amino acid sequence corresponding to amino acids 381 to 393 is the EU numbering system of variant Fc fragment 5-1-5
    SEQ ID NO:570% Amino acid sequence corresponding to amino acids 381 to 393 is the EU numbering system of variant Fc fragment 5-1-6
    SEQ ID NO:571% Amino acid sequence corresponding to amino acids 381 to 393 is the EU numbering system of variant Fc fragment 5-1-7
    SEQ ID NO:572% Amino acid sequence corresponding to amino acids 381 to 393 is the EU numbering system of variant Fc fragment 5-1-8
    SEQ ID NO:573% Amino acid sequence corresponding to amino acids 381 to 393 is the EU numbering system of variant Fc fragment 5-1-9
    SEQ ID NO:574% Amino acid sequence corresponding to amino acids 381 to 393 is the EU numbering system of variant Fc fragment 5-1-10

    DETAILED DESCRIPTION



    [0017] Changes in a therapeutic protein that increase in vivo half-life can be useful because such proteins can be dosed in lower amounts and/or less frequently. The instant invention provides variant Fc-polypeptides comprising variant Fc-fragments that bind to FcRn with increased affinity and/or binding activity at slightly acidic pHs, such as, for example, pH 5.0-6.0, and do not bind well to FcRn at physiologic pHs of about 7.2 to 7.6. As demonstrated herein, such variant Fc-polypeptides containing the variant Fc-fragments can have longer in vivo half lives as compared to control Fc-polypeptides. Provided are variant Fc-polypeptides comprising variant human Fc-fragments, which contain at least one insertion within or adjacent to a loop, which bind to FcRn with enhanced affinity and/or binding activity at a pH of from about 5 to 6 as compared to control Fc-polypeptides that differ only in that they do not contain the insertion(s). Also provided are nucleic acids encoding proteins containing such variant Fc-fragments and variant Fc-polypeptides, host cells containing such nucleic acids, and methods of making variant Fc-polypeptides and variant Fc-fragments. Further, methods of extending the half life of an Fc-polypeptide are provided, and methods of identifying variant Fc-fragments that bind to FcRn with higher affinity and/or binding activity at pH 5-6 with lower or comparable affinity and/or binding activity at a physiologic pH as compared to control Fc-fragments are also provided.

    [0018] A variant Fc-fragment as described herein contains short insertions at selected sites in loops. FcRn interacts with an Fc-fragment. An immunoglobulin domain, such as the CH2 or CH3 domain, has a barrel-shaped structure comprising beta sheet portions alternating with loops. Hunkapiller and Hood (1989), Adv. Immunol. 44: 1-63; Williams and Barclay (1988), Ann. Rev. Immunol. 6: 381-405. Insertion sites in an Fc-fragment were chosen in loops that are part of the area that comes into relatively close contact with FcRn when it is bound to the Fc-fragment or in loops that are adjacent to these areas. Figure 1 shows ribbon diagrams of portions of FcRn (top) and an Fc-fragment (bottom) and indicates the positions in the Fc-fragment of the sites selected for insertions.

    [0019] As will be shown below, many insertions in library L5 have the desired properties of enhanced binding to FcRn at pH 5 to 6 and little or no binding to FcRn at physiologic pH. Other libraries, including libraries L1, L2B, L3, and L4 (see Figures 1 and 2), yielded no insertions that had the desired properties even though approximately the same number of variants were screened. Libraries L2A, L6A, and L6B yielded some insertions having the desired properties, although the numbers were much smaller than the numbers obtained from library L5. Surprisingly, the library that produced the most numerous insertions with the desired properties, i.e., library L5, lay in a loop region that is not part of the area that comes in to closest contact with FcRn. Figure 1.

    Definitions



    [0020] The term "affinity," as meant herein, refers to binding affinity measured as an EC50 measured using a BIAcore® T100 (or a similar instrument) as explained in Example 3. "Higher affinity" of a variant Fc-polypeptide (containing a variant Fc-fragment), as compared to an control Fc-polypeptide (containing a control Fc-fragment), for FcRn means that the variant Fc-polypeptide has an EC50 that is no more than 50% of the EC50 of the control Fc-polypeptide. Similarly, "lower affinity" of a variant Fc-polypeptide, as compared to a control Fc-polypeptide, means that the variant Fc-polypeptide has an EC50 that is more than 150% of that of a control Fc-polypeptide. Affinity of a variant Fc-polypeptide is considered "substantially the same" as that of a control Fc-polypeptide if its EC50 is 50-150% of the EC50 of the control Fc-polypeptide.

    [0021] The term "binding activity," as meant herein, refers to binding activity of an Fc-polypeptide to an FcRn measured using streptavidin biosensors coated with biotinylated human FcRn (hFcRn) in the Octet Red® system as described in Example 4 where association occurs at pH 6 and dissociation occurs at pH 7.4. Binding activity of a variant Fc-polypeptide at pH 6 is considered to be "higher than" that of a control Fc-polypeptide if the maximal response observed is at least 1.5, 1.75, or 2.0 times the maximal response observed for the control Fc-polypeptide. The very small amount of residual binding response, i.e., the binding response remaining after most of the protein has dissociated from hFcRn at pH 7.4, observed using a control Fc-polypeptide comprising a wild type Fc-fragment is referred to as "little or no binding activity." A variant Fc-polypeptide is also considered to have "little or no binding activity" at pH 7.4 if the residual binding response detected after some or all of the protein has dissociated at pH 7.4 is no more than 0.2 or 0.1 nanometers more than that detected using a control Fc-polypeptide using the ForteBio system as described in Example 4.

    [0022] An "amino acid," as meant herein, refers to any one of the twenty L-amino acids commonly found in human proteins, which are the following: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

    [0023] An "antibody," as meant herein, is a protein containing at least one heavy or light chain immunoglobulin variable region. For example, an scFv, a molecule comprising two or more scFv's, and the domain antibodies consisting essentially of a single immunoglobulin variable region (as described in,e.g., US Patent 7,563,443) are "antibodies" as meant herein. In many cases, an antibody includes a heavy and a light chain variable region plus a human IgG Fc region. Thus, the term "antibody" encompasses full length antibodies containing two full length heavy and two full length light chains, such as naturally-occurring IgG, IgA, IgD, IgD, or IgM antibodies found in mammals. Carayannopoulos and Capra, Ch. 9 in FUNDAMENTAL IMMUNOLOGY, 3rd ed., Paul, ed., Raven Press, New York, 1993, pp. 284-286); the portions of this reference that describe antibodies are incorporated herein by reference. Exemplary antibodies include adalimumab, (HUMIRA®, Abbott Laboratories), infliximab (REMICADE®, Centocor Ortho Biotech Inc.), ustekinumab (STELLARA®, Centocor), golimumab (SIMPONI®, Centocor Ortho Biotech), canakinumab (ILARIS®, Novartis Pharmceuticals Corporation), ofatumumab (ARZERRA®, Glaxo Group Ltd.), tocilizumab (ACTEMRA®, Chugai Seiyaku Kabushiki Corp., Japan), belimumab (BENLYSTA®, LYMPHOSTAT-B®, Human Genome Sciences, Inc.), bevacizumab (AVASTIN®, Genentech), cetuximab (ERBITUX®, ImClone Systems Inc.), efungumab (MYCOGRAB®, Novartis AB Corp.), efalizumab, (RAPTIVA®, Genentech Inc.), etaracizumab (ABEGRIN®, Medimmune LLC), gemtuzumab ozogamicin (MYLOTARG®, Wyeth), girentuximab (RENCAREX®, Wilex AG Corp., Germany), natalizumab (TYSABRI®, Elan Pharmceuticals, Inc.), omalizumab (XOLAIR®, Novartis AG Corp., Switzerland), oregovomab (OVAREX®,AltaRex Corp., Canada), palivizumab (SYNAGIS®, ABBOSYNAGIS®, Medimmune Inc.). panitumumab (VECTIBIX®, Amgen Inc.), ranibizumab (LUCENTIS®, Genentech Inc.), rituximab (MABTHERA®, RITUXAN®, Idec Pharmaceuticals Corp.) tefibazumab (AUREXIS®, Inhibitex Corp.), tositumomab, (BEXXAR®, GlaxoSmithKline Beecham Corp.), trastuzumab (HERCEPTIN®, Genentech Inc.) and denosumab (PROLIA® or XGEVA®, Amgen Inc.), among many others. Such IgG antibodies can be of the IgG1, IgG2, IgG3, or IgG4 isotype and can be chimeric, human or humanized antibodies. Further, the term "antibody" includes dimeric antibodies containing two heavy chains and no light chains, such as the naturally-occurring antibodies found in camels, other dromedary species, and sharks. See, e.g., Muyldermans et al., 2001, J. Biotechnol. 74:277-302; Desmyter et al., 2001, J. Biol. Chem. 276:26285-90; Streltsov et al. (2005), Protein Science 14: 2901-2909. An antibody can be monospecific (that is, binding to only one kind of antigen) or multispecific (that is, binding to more than one kind of antigen). In some embodiments, an antibody can be bispecific (that is, binding to two different kinds of antigen). Further, an antibody can be monovalent, bivalent, or multivalent, meaning that it can bind to one or two or more antigen molecules at once. Some of the possible formats for such antibodies include monospecific or bispecific full length antibodies, monospecific monovalent antibodies (as described in International Application WO 2009/089004 and US Application Publication 2007/0105199, and in US Application Publication 2005/0227324 ) that may inhibit or activate the molecule to which they bind, bivalent monospecific or bispecific dimeric Fv-Fc, scFv-Fc, or diabody Fc, monospecific monovalent scFv-Fc/Fc's, and the multispecific binding proteins and dual variable domain immunoglobulins described in US Publication 2009/0311253, among many other possible antibody formats.

    [0024] A "binding region," as meant herein, is a region included in an "Fc-polypeptide," a "control Fc-polypeptide," or "variant Fc-polypeptide," as described herein, that binds to a target molecule, such as, for example, a protein that is expressed at high levels on a cancer cell, a cell mediating an autoimmune or inflammatory condition, an infected cell, an infectious agent, or a cell mediating an immune effector function, for example, an NK cell. A binding region can contain a heavy and/or light chain immunoglobulin variable (VH and/or VL) region or a non-immunoglobulin polypeptide. Exemplary binding regions include, for example, an Fv (which comprises a VH and a VL region joined by a linker) or a soluble form of a human receptor that binds to a target molecule.

    [0025] An "Fc-fragment," as meant herein, is a polypeptide that consists of part or all of a hinge region plus the CH2 and CH3 regions of an antibody plus, optionally, regions found downstream from the CH3 region in some naturally occurring isotypes such as IgA or IgM antibodies. The antibody can be of the IgG isotype, including IgG1, IgG2, IgG3, or IgG4 isotypes, or of the IgM, IgE, IgD or IgA isotype. The antibody can be of human or animal origin. For example, the antibody can be from a mammal, such as a mouse, rat, hamster, rabbit, goat, or sheep, or from a camelid species or a shark. Sequences of human IgG1, IgG2, IgG3, and IgG4 Fc-fragments are disclosed in SEQ ID NOs: 1, 3, 5, and 7, respectively. These sequences are also shown in Table 1 below. In general, Fc-fragments form dimers via interactions between CH3 domains within two Fc-fragments, which are stabilized by disulfide bonds occurring between cysteine residues in the hinge regions. An Fc-fragment referred to herein as an "Fc region" is specifically defined as a dimeric Fc-fragment, although it is recognized that Fc-fragments will generally dimerize under physiologic conditions. Dimers can be dissociated in strongly reducing conditions.

    [0026] An "Fc-polypeptide," as meant herein, is a protein that comprises an Fc-fragment and a binding region. Fc-polypeptides include antibodies, Fc fusion proteins, and antibodies or fusion proteins that contain an additional "pharmacologically active moiety," which is a non-peptide organic moiety (i.e., "small molecule") or a peptide, which can act as a toxin and/or can mimic, antagonize, or agonize the activity of a biological pathway, covalently conjugated or fused to the Fc-polypeptide. Fc-polypeptides, like Fc-fragments, ordinarily form dimers that are stabilized by disulfide bonds between cysteine residues in the hinge regions of two Fc-polypeptides.

    [0027] A "control Fc-fragment," as meant herein, is an Fc-fragment that is the same as the "variant Fc-polypeptide" to which it is being compared except that it does not have an insertion in a loop as the variant Fc-fragment does. Thus, the "control Fc-fragment" has unmodified sequence found in a naturally-occurring Fc-fragment in the loop where the variant Fc-fragment has an insertion that increases the affinity of the variant Fc-fragment for FcRn at pH 5 to 6. A control Fc-fragment may contain modifications relative to a naturally-occurring Fc-fragment other than the insertion(s). For example, the heterodimerizing alterations or other minor modifications described below can be present in both the variant Fc-fragment and the control Fc-fragment to which it is being compared. Hence, the only difference between a "control Fc-fragment" and a "variant Fc-fragment" is the insertion within or adjacent to a loop in the variant Fc-fragment. A "control Fc region" is defined a dimer comprising two control Fc-fragments, although a control Fc-fragment would generally be expected to exist in a dimeric form.

    [0028] A "variant Fc-fragment," as meant herein is an Fc-fragment that includes an insertion of no more than 80, no more than 60, no more than 40, or no more than 20 amino acids within or adjacent to a loop of the Fc-fragment. The loops in a human IgG Fc-fragment are shown in Table 1, and the loops into which insertions were made are shown in Figure 2. Loops in other Fc-fragments are known in the art. Numerous sequences are known in the art. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, MD, 1991, portions of which relate to the sequences and tertiary structure of hinge, CH2, and CH3 regions. Kabat et al. provides alignments of numerous sequences. Given the alignments provided in Kabat et al., the highly conserved structure of immunoglobulin domains, and the locations of loops provided herein, as well as abundant further information available in the art, loops could be located in an Fc-fragment of any isotype from any species. For example, the Protein Data Bank website contains abundant information on tertiary structures of many proteins. In some aspects, the inserted amino acids can replace from one to ten amino acids that naturally occur in the loop. The inserted amino acids can be of a fewer, greater, or the same in number as the amino acids that are removed. In certain embodiments, all amino acids originally in the loop remain, and the inserted amino acids are simply added. In some aspects, a variant human IgG Fc-fragment can comprise other "additional minor modifications," that is, the insertion, deletion, or substitution of no more than sixteen amino acids at locations other than the loop in which the insertion occurs. In some aspects, there can be no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 insertions, deletions, or substitutions of a single amino acid at locations other than the loop in which the insertion occurs. In other embodiments, a variant Fc-fragment does not comprise any additional minor modifications. In some aspects, the additional minor modifications can be, for example the "heterodimerizing alterations" described below, which facilitate the formation of heterodimeric Fc regions. In some aspects, these additional minor modifications can include conservative amino acid substitutions.

    [0029] Conservative amino acid substitutions include the following: (1) valine, leucine or isoleucine for alanine; (2) lysine, glutamine, or asparagine for arginine; (3) glutamine, glutamate, or aspartate for asparagine; (4) glutamate, asparagine, or glutamine for aspartate; (5) serine or alanine for cysteine; (6) asparagine, glutamate, or aspartate for glutamine; (7) aspartate, gluatamine, or asparagine for glutamate; (8) proline or alanine for glycine; (9) asparagine, glutamine, lysine, or arginine for histidine; (10) leucine, valine, methionine, alanine, or phenylalanine for isoleucine; (11) isoleucine, valine, methionine, alanine, or phenylalanine for leucine; (12) arginine, asparagine, or glutamine for lysine; (13) leucine, phenylalanine, or isoleucine for methionine; (14) leucine, valine, isoleucine, alanine, or tyrosine for phenylalanine; (15) alanine for proline; (16) threonine, alanine, or cysteine for serine; (17) serine for threonine; (18) tyrosine or phenylalanine for tryptophan; (19) tryptophan, phenylalanine, threonine, or serine for tyrosine; and (20) isoleucine, methionine, leucine, phenylalanine, or alanine for valine.

    [0030] In addition, in some aspects, a variant Fc-fragment can contain an additional "pharmacologically active moiety," which is a non-peptide organic moiety (i.e., "small molecule") or a peptide, which can act as a toxin and/or can mimic, antagonize, or agonize the activity of a biological pathway, covalently conjugated or fused to the Fc-fragment. If a variant Fc-fragment contains such a pharmacologically active moiety, then its "control Fc-fragment" also contains such a moiety.

    [0031] In other aspects, the variant human IgG Fc-fragment comprises no alteration outside of those occurring in the loop at which the insertion occurs. Like an "Fc-fragment," a "variant Fc-fragment" can be of the IgG isotype, including IgG1, IgG2, IgG3, or IgG4 isotypes, or of the IgM, IgE, IgD or IgA isotype. It can be of human or animal origin. For example, an Fc-fragment can be from a mammal, such as a mouse, rat, hamster, rabbit, goat, or sheep, or from a camelid species or a shark can be modified to produce a variant Fc-fragment. As with other Fc-fragments, a variant Fc-fragment is normally dimeric, and a "variant Fc region" is specifically defined as a dimeric variant Fc-fragment.

    [0032] A "variant Fc-polypeptide," as meant herein, comprises a variant Fc-fragment and a binding region. Optionally, a variant Fc-polypeptide can contain multiple binding regions. A variant Fc-polypeptide can be a multimer, such as a dimer, trimer, tetramer, or higher order multimer. In many cases, the variant Fc-fragment portion of the Fc-polypeptide can form disulfide bonds so as to dimerize. Variant Fc-polypeptides include, for example, dimeric Fc fusion proteins, tetrameric full length antibodies (containing two heavy and two light chains), dimeric scFv-Fc's, etc. They can be heteromultimers or homomultimers, such as heterodimers or homodimers. A variant Fc-polypeptide can be an antibody, an Fc-fusion protein, or in some embodiments an antibody or an Fc-fusion protein comprising an additional, non-antibody pharmacologically active moiety. Such a "pharmacologically active moiety" can be covalently conjugated or fused to the Fc-polypeptide and can be a non-peptide organic moiety (i.e., "small molecule") or a peptide, which can act as a toxin and/or can mimic, antagonize, or agonize the activity of a biological pathway.

    [0033] An "Fc fusion protein," as meant herein, is a protein containing an Fc-fragment fused to another, non-antibody polypeptide. The non-antibody polypeptide comprises a binding region that binds to a target molecule and does not comprise a heavy or light chain variable region of an antibody. The binding region of an Fc fusion protein can comprise a non-antibody polypeptide such as a soluble portion of a receptor or one or more peptides that bind to a target molecule (such as, for example, a "monomer domain" as defined in US Patent 7,820,790 that binds to a target protein, which can be selected as discussed in US Patent 7,820,790), or other polypeptides. Portions of US Patent 7,820,790 describe monomer domains and how they are selected. In specific aspects, an Fc fusion protein can be etanercept (ENBREL® sold by Amgen Inc.), romiplostim (NPLATE® sold by Amgen Inc.), alefacept (AMEVIVE®, Biogen Corp.), abatacept (ORENCIA® sold by Bristol -Myers Squibb), rilonacept (ARCALYST®, sold by Regeneron), or aflibercept (EYLEA™, sold by Regeneron). Other polypeptides that can be part of a binding region of an Fc fusion protein include polypeptides comprising scaffold domains that have been randomized in certain positions and subjected to selection for binding to a certain target molecule. Such scaffold domains include, for example, T-lymphocyte associated protein-4 (CTLA-4; Nuttall et al. (1999), Proteins 36: 217-227), the Z domain of Staphylococcal protein 1 (Nord et al. (1995), Protein Eng. 8: 601-608), green fluorescent protein, and the tenth type III domain of human fibronectin (FN3; Koide et al. (1998), J. Mol. Biol. 284: 1141-1151; Karatan et al. (2004), Chem. & Biol. 11: 835-844). Portions of these references describe the scaffold domains and their use to generate binding domains. Fc fusion proteins, like other proteins containing Fc-fragments can form multimers, which can be dimers. These multimers can be heteromultimers or homomultimers. An Fc fusion protein can be heterodimeric or homodimeric and bispecific or monospecific.

    [0034] A "variant Fc fusion protein," as meant herein, is an Fc fusion protein that includes a variant Fc-fragment.

    [0035] A "control Fc fusion protein," as meant herein, is Fc fusion protein that has the same amino acid sequence as the variant Fc fusion protein to which it is being compared except for the insertion in the loop of the Fc-fragment portion of the variant Fc fusion protein.

    [0036] A "full length IgG antibody," as meant herein, is an IgG antibody comprising two complete heavy chains and two complete light chains.

    [0037] "Heterodimerizing alterations" generally refer to alterations in the two chains, that is, the A and B chains, of a dimeric Fc region that facilitate the formation of heterodimeric Fc regions, that is, Fc regions in which the A chain and the B chain of the Fc region do not have identical amino acid sequences. Heterodimerizing alterations can be asymmetric, that is, an A chain having a certain alteration can pair with a B chain having a different alteration. These alterations facilitate heterodimerization and disfavor homodimerization. Whether hetero- or homo-dimers have formed can be assessed by size differences as determined by polyacrylamide gel electrophoresis in situations where one polypeptide chain is an Fc-fragment (that is, lacking sequences other than the hinge, CH2 and CH3 regions) and the other is an scFv-Fc. One example of such paired heterodimerizing alterations are the so-called "knobs and holes" substitutions. See, e.g., US Patent 7,695,936 and US Patent Application Publication 2003/0078385, portions of which describe such mutations. As meant herein, an Fc region that contains one pair of knobs and holes substitutions, contains one substitution in one Fc-fragment (designated the A chain) and another in the other Fc-fragment (designated the B chain). For example, the following knobs and holes substitutions in the A and B chains of an IgG1 Fc region have been found to increase heterodimer formation as compared with that found with unmodified A and B chains: 1) Y407T in one chain and T366Y in the other; 2) Y407A in one chain and T366W in the other; 3) F405A in one chain and T394W in the other; 4) F405W in one chain and T394S in the other; 5) Y407T in one chain and T366Y in the other; 6) T366Y and F405A in one chain and T394W and Y407T in the other; 7) T366W and F405W in one chain and T394S and Y407A in the other; 8) F405W and Y407A in one chain and T366W and T394S in the other; and 9) T366W in one polypeptide of the Fc and T366S, L368A, and Y407V in the other. As meant herein, mutations are denoted in the following way. The amino acid (using the one letter code) normally present at a given position in the Fc-fragment using the EU numbering system (see Table 1 below) is followed by the EU position, which is followed by the alternate amino acid that is present at that position. For example, Y407T means that the tyrosine normally present at EU position 407 is replaced by a threonine. Alternatively or in addition to such alterations, substitutions creating new disulfide bridges can facilitate heterodimer formation. See, e.g., US Patent Application Publication 2003/0078385, portions of which describe such mutations. Such alterations in an IgG1 Fc region include, for example, the following substitutions: Y349C in one Fc-fragment and S354C in the other; Y349C in one Fc-fragment and E356C in the other; Y349C in one Fc-fragment and E357C in the other; L351C in one Fc-fragment and S354C in the other; T394C in one Fc-fragment and E397C in the other; or D399C in one Fc-fragment and K392C in the other. Similarly, substitutions changing the charge of a one or more residue, for example, in the CH3-CH3 interface, can enhance heterodimer formation as explained in WO 2009/089004, portions of which describe such substitutions. Such substitutions are referred to herein as "charge pair substitutions," and an Fc region containing one pair of charge pair substitutions contains one substitution in the A chain and a different substitution in the B chain. General examples of charge pair substitutions include the following: 1) K409D or K409E in one chain plus D399K or D399R in the other; 2) K392D or K392E in one chain plus D399K or D399R in the other; 3) K439D or K439E in one chain plus D356K or D356R in the other; and 4) K370D or K370E in one chain plus E357K or E357R in the other. In addition, the substitutions R355D, R355E, K360D, or K360R in both chains can stabilize heterodimers when used with other heterodimerizing alterations. Specific charge pair substitutions can be used either alone or with other charge pair substitutions. Specific examples of single pairs of charge pair substitutions and combinations thereof include the following: 1) K409E in one chain plus D399K in the other; 2) K409E in one chain plus D399R in the other; 3) K409D in one chain plus D399K in the other; 4) K409D in one chain plus D399R in the other; 5) K392E in one chain plus D399R in the other; 6) K392E in one chain plus D399K in the other; 7) K392D in one chain plus D399R in the other; 8) K392D in one chain plus D399K in the other; 9) K409D and K360D in one chain plus D399K and E356K in the other; 10) K409D and K370D in one chain plus D399K and E357K in the other; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other; 12) K409D and K392D on one chain and D399K on the other; 13) K409D and K392D on one chain plus D399K and E356K on the other; 14) K409D and K392D on one chain plus D399K and D357K on the other; 15) K409D and K370D on one chain plus D399K and D357K on the other; 16) D399K on one chain plus K409D and K360D on the other; and 17) K409D and K439D on one chain plus D399K and E356K on the other. Any of the these heterodimerizing alterations can be part of a variant Fc region as described herein, which can bind to FcRn with a higher affinity than does a control Fc region.

    [0038] A "loop," as meant herein, is a portion of a tertiary structure in an Fc-fragment that links beta strands that make up the beta sheets of the core immunoglobulin fold. A loop may contain no more than two consecutive residues designated as being involved in a beta sheet (as in Loops 10 and 11; see Table 1 below) and can also contain residues designated as a helix, a turn, an isolated β bridge, or a bend. The loop regions of human IgG Fc regions, as meant herein, are shown in Table 1.

    [0039] An "insertion within a loop" or "within" any stretch of amino acids, for example, an insertion within Loop 10, is an insertion of 3-35, 3-20, 6-20, 6-15, 6-12, 3-15, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids between two adjacent amino acids within a loop or between an amino acid within a loop and an adjacent amino acid that is not part of the loop. An "insertion within a loop" also encompasses situations where amino acids are removed from a loop, and other amino acids are inserted into the loop. The number of amino acids inserted into the loop can be 3-35, 3-22, 4-20, 6-18, 6-15, 6-12, 3-15, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, and the number of amino acids removed from the loop can be less than, the same as, or more than the number of amino acids inserted into the loop. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids can be removed from the loop. In addition, an "insertion within a loop" encompasses aspects in which more than one insertion occurs within the same loop, such as insertions between two different pairs of amino acids that are within or adjacent to a loop, as described above, or where one or more non-adjacent amino acids within the loop are deleted and replaced with fewer, the same number, or more amino acids than were deleted. Library L3 (as shown in Figure 2) is one example of such an insertion scheme.

    [0040] An "insertion within or adjacent to a loop," is like an "insertion within a loop," as described above, except that it also includes situations where the insertion is between a first amino acid that is adjacent to the loop and another amino acid that is adjacent to this first amino acid and is outside the loop.

    [0041] A "physiologic pH," as meant herein, is a pH from about 7.2-7.6.

    [0042] An "scFv -Fc/Fc" is a dimeric protein consisting essentially of an scFv-Fc plus an Fc-fragment that are linked by one or more disulfide bonds. Further, the Fc region formed by the Fc-fragment and the scFv-Fc can contain "heterodimerizing alterations" in the CH3 domains, such as one, two, three, or more pairs of charge pair substitutions, as described.

    [0043] A "peptide," as meant herein, is a short polypeptide. Generally, a peptide is from 2 to 80, 2 to 60, 2 to 50, 2 to 40, 2 to 30, or 2-20 amino acids in length.

    [0044] A "target molecule," as meant herein, is a molecule to which the binding region of an Fc-polypeptide, as described herein, binds. In some embodiments, a target molecule is, for example, a protein that is expressed at high levels on a cancer cell or is expressed on a cell mediating an autoimmune or inflammatory condition or on an infected cell, on an infectious agent, or on a cell mediating an immune effector function, for example, an NK cell.

    [0045] A "therapeutically effective amount" of a variant Fc-polypeptide that has higher affinity for FcRn than a control Fc-polypeptide, as described herein, is an amount that has the effect of, for example, reducing or eliminating the tumor burden of a cancer patient or reducing or eliminating the symptoms of any disease condition that the protein is used to treat. A therapeutically effective amount need not completely eliminate all symptoms of the condition, but may reduce severity of one or more symptoms or delay the onset of more serious symptoms or a more serious disease that can occur with some frequency following the treated condition. Alternatively, a therapeutically effective amount of a protein containing a modified Fc-polypeptide is sufficient to detectably affect the expression of a relevant biomarker when it is administered to a patient in need of treatment in vivo.

    [0046] "Treatment" of any disease mentioned herein encompasses an alleviation of at least one symptom of the disease, a reduction in the severity of the disease, or the delay or prevention of disease progression to more serious symptoms that may, in some cases, accompany the disease or lead to at least one other disease. Treatment need not mean that the disease is totally cured. A useful therapeutic agent needs only to reduce the severity of a disease, reduce the severity of one or more symptoms associated with the disease or its treatment, or delay the onset of more serious symptoms or a more serious disease that can occur with some frequency following the treated condition.

    Variant Fc-polypeptides



    [0047] Provided are variant Fc-polypeptides that bind to FcRn with greater affinity and/or binding activity at pHs from 5.0 to 6.0 than do control Fc-polypeptides. Such variant Fc-polypeptides, like control Fc-polypeptides, bind to FcRn with low affinity and/or binding activity, if at all, at pHs from about 7.2 to about 7.6. As described herein, these variant Fc-polypeptides can (before modification) be of human or animal origin and can be of an IgG isotype, including IgG1, IgG2, IgG3, or IgG4. The FcRn can also be of human or animal origin. These variant Fc-polypeptides further comprise a binding region that can bind to a target molecule, as described herein, and can be used to treat a condition that is mediated, at least in part, by the target molecule. In some embodiments, the target molecule may not directly mediate the condition treated, but binding to the target molecule can serve to localize the Fc-polypeptide or target molecule(s) it binds to. For example, a bispecific Fc-polypeptide could bridge two different cell types, e.g., a cancer cell and an immune system cell, each of which expresses a target molecule on its surface that the Fc-polypeptide can bind to. The variant Fc-polypeptides can be antibodies or Fc fusion proteins comprising a variant Fc-fragment. A variant Fc-polypeptide could, in addition, be an antibody that contains an additional, non-antibody binding region. Optionally, a variant Fc-polypeptide can contain a human IgG variant Fc-fragment.

    [0048] In Table 1 below, the amino acid sequences of human IgG1, IgG2, IgG3, and IgG4 Fc-polypeptides (SEQ ID NOs: 1, 3, 5, and 7, respectively) are aligned. Sequences are numbered according to the EU system of Edelman et al. (1969), Proc. Natl. Acad. Sci. 63: 78-85. As shown in Table 1, a single number can be used to refer to a position that is analogous in all four human IgG isotypes, even though a linear numbering scheme would force one to name a different number for this position in each isotype. For example, the asparagine at position 297 is a well-known and highly conserved glycosylation site present in all human IgG antibodies that is referred to as asparagine 297, regardless of the human IgG isotype under discussion.

    [0049] Human IgG3 antibodies have a much longer hinge region than other human IgG isotypes, and only the most carboxy-terminal portion of this hinge region is shown in Table 1. The human IgG1, IgG2, and IgG4 sequences shown in Table 1 include the entire hinge region. As is apparent in Table 1, the hinge region of a human IgG4 antibody is three amino acids shorter than that of an IgG1 antibody. Human IgG2 antibodies have a slightly shorter hinge region and a gap of one amino acid near the amino terminal end of the CH2 region as compared to human IgG1. Amino acids 216-447 shown in the IgG1 line and amino acids 224-447 in the IgG4 line of Table 1 correspond to amino acids 1-232 of SEQ ID NO:1 and 6-229 of SEQ ID NO:7, respectively. Amino acids 216 to 447 in the IgG3 line of Table 1 correspond to amino acids 48-279 of SEQ ID NO:5. Amino acids 237 to 447 in the IgG2 line of Table 1 correspond to amino acids 18 to 228 of SEQ ID NO:3.

    [0050] In Table 1, amino acids that are located in loops, as meant herein, are shown in boldface and are underlined. Each loop is labeled with a number, i.e., "Loop 1," "Loop 2," etc., beneath it. In more detail, loops occur the following locations: Loop 1, amino acids 29-43 of SEQ ID NO:1, amino acids 25-39 of SEQ ID NO:3, amino acid 76-90 of SEQ ID NO:5, and amino acids 26-40 of SEQ ID NO:7; Loop 2, amino acids 50-58 of SEQ ID NO:1, amino acids 46-54 of SEQ ID NO:3, amino acids 97-105 of SEQ ID NO:5, and amino acids 47-55 of SEQ ID NO:7; Loop 3, amino acids 70-72 of SEQ ID NO:1, amino acids 66-68 of SEQ ID NO:3, amino acids 117-119 of SEQ ID NO:5, and amino acids 67-69 of SEQ ID NO:7; Loop 4, amino acids 80-84 of SEQ ID NO:1, amino acids 76-80 of SEQ ID NO:3, amino acids 127-131 of SEQ ID NO:5, and amino acids 77-81 of SEQ ID NO:7; Loop 5, amino acids 92-103 of SEQ ID NO:1, amino acids 88-99 of SEQ ID NO:3, amino acids 139-150 of SEQ ID NO:5, and amino acids 89-100 of SEQ ID NO:7; Loop 6, amino acids 110-116 of SEQ ID NO:1, amino acids 106-112 of SEQ ID NO:3, amino acids 157-163 of SEQ ID NO:5, and amino acids 107-113 of SEQ ID NO:7; Loop 7, amino acids 122-131 of SEQ ID NO:1, amino acids 118-127 of SEQ ID NO:3, amino acids 169-178 of SEQ ID NO:5, and amino acids 119-128 of SEQ ID NO:7; Loop 8, amino acids 137-146 of SEQ ID NO:1, amino acids 133-142 of SEQ ID NO:3, amino acids 184-193 of SEQ ID NO:5, and amino acids 134-143 of SEQ ID NO:7; Loop 9, amino acids 158-162 of SEQ ID NO:1, amino acids 154-158 of SEQ ID NO:3, amino acids 205-209 of SEQ ID NO:5, and amino acids 155-159 of SEQ ID NO:7; Loop 10, amino acids 169-175 of SEQ ID NO:1, amino acids 165-171 of SEQ ID NO:3, amino acids 216-222 of SEQ ID NO:5, and amino acids 166-172 of SEQ ID NO:7; Loop 11, amino acids 179-188 of SEQ ID NO:1, amino acids 175-184 of SEQ ID NO:3, amino acids 226-235 of SEQ ID NO:5, and amino acids 176-185 of SEQ ID NO:7; Loop 12, amino acids 199-207 of SEQ ID NO:1, amino acids 195-203 of SEQ ID NO:3, amino acids 246-254 of SEQ ID NO:5, and amino acids 196-204 of SEQ ID NO:7; Loop 13, amino acids 214-221 of SEQ ID NO:1, amino acids 210-217 of SEQ ID NO:3, amino acids 261-268 of SEQ ID NO:5, and amino acids 211-218 of SEQ ID NO:7.



    [0051] The markings in the fifth line of each aligned group, designated "DSSP" (which stands for Dictionary of Protein Secondary Structure) were taken from the 1HZH (chain H) structure available from the Protein Data Bank, have the following meanings: "G" designates a 3-turn helix (310 helix), which has a minimum length 3 residues; "H" designates a 4-turn helix (α helix), which has a minimum length of 4 residues; "I" designates a 5-turn helix (π helix), which has a minimum length of 5 residues; "T" designates a hydrogen bonded turn (3, 4 or 5 turn); "E" designates an extended strand in parallel and/or anti-parallel β-sheet conformation, which has a minimum length of 2 residues; "B" designates a residue in an isolated β-bridge (single pair β-sheet hydrogen bond formation); "S" designates a bend (the only non-hydrogen-bond based assignment). Amino acid residues that are not in any of the above conformations are assigned a blank space in the "DSSP" line. These designations are standard in the art and available in the website for the Protein Data Bank (PDB).

    [0052] The hinge region of the human IgG Fc-fragments runs from amino acid 216 to 230 as numbered in Table 1. The CH2 region extends from amino acid 231 to 340, and the CH3 region extends from amino acid 341 to 447. It is apparent from the alignment of Table 1 that the sequences of the human IgG Fc-fragments are highly conserved. Most of the sequence differences occur in the hinge region, and very few sequence differences occur in the CH2 and CH3 regions. Thus, results obtained using variant Fc-polypeptides comprising a human IgG variant Fc-fragment comprising insertions in the CH2 or CH3 region of one human IgG subtype will likely be applicable to other human IgG subtypes.

    [0053] The insertions in the variant human IgG Fc-polypeptides described herein can occur at locations known to be in loops. The insertions can occur within or adjacent to any of Loops 1-13 (as shown in Table 1 above) of an Fc-fragment portion of the Fc-polypeptide. In some aspects, insertions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or 1-18, 6-16,10-16,12-14, 3-20, 20-30, 30-50, or 50-80 amino acids can be made between two adjacent amino acids, at least one of which is included in a loop of an Fc-fragment. Two cysteines may be non-randomly included in the insertion, one each among the first and last four amino acids of the insertion, so as to constrain the amino acids between the cysteines into a tight loop bounded by a disulfide bond; the first three amino acids of the insertion may be Gly-Gly-Cys, and the last three amino acids may be Cys-Gly-Gly. An insertion of an additional 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 10-30 amino acids can be inserted between these two cysteines. In still other aspects, 1,2, 3,4, 5, 6, 7, 8, 9, or 10 amino acids from the loop are deleted and replaced by an insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or 1-18, 6-16, 10-16, 12-14, 15-30, 30-50, or 50-80 amino acids and/or by an insertion that contains two cysteines among the first and last four amino acids of the insertion. The number of amino acids deleted can be the same, less, or greater in number than the amino acids inserted. In other aspects, more than one insertion can occur in a single loop. The variant Fc-polypeptides, which contain the insertions, can bind to human FcRn with a higher affinity at pH 5 to 6 than does a control Fc-polypeptide. The inserted amino acids can include any of all 20 amino acids or any of all amino acids other than cysteine. Further as described herein, a variant Fc-fragment can comprise an insertion in only one loop or in more than one loop, such as in 2, 3, or 4 loops.

    [0054] Insertions within or adjacent to Loops 5, 8, or 10 of an Fc-fragment can increase the binding affinity of such a variant Fc-fragment for human FcRn at pH 5 to 6. In specific aspects, an insertion can be made in a human IgG Fc-fragment within or adjacent to Loop 8 between amino acids 358 and 359 or, in certain embodiment, within or adjacent to Loop 10 between amino acids 384 and 385 (using the EU numbering scheme as illustrated in Table 1). The insertion, as disclosed and/or claimed herein, can contain from 4-20, 1-18, 6-16, 10-16, 12-14, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or amino acids. Optionally, the amino acids may be limited to all amino acids other than cysteine. In some embodiments, the insertion can contain a cysteine among the first and the last four amino acids of the insertion, and the inserted amino acids can otherwise be limited to amino acids other than cysteine. In some embodiments, the insertion can contain no cysteines. In some embodiments, the insertion can have a formula selected from the group consisting of CXXXXXXC (SEQ ID NO:13), CXXXXXXXC (SEQ ID NO:14), CXXXXXXXXC (SEQ ID NO:15), GCXXXXXXCG (SEQ ID NO:16), GCXXXXXXXCG (SEQ ID NO:17), GCXXXXXXXXCG (SEQ ID NO:18), GGCXXXXXXCGG (SEQ ID NO:19), GGCXXXXXXXCGG (SEQ ID NO:20), and GGCXXXXXXXXCGG (SEQ ID NO:21), GGGCXXXXXXCGGG (SEQE ID NO:22), GGGCXXXXXXXCGGG (SEQ ID NO:23), and GGGCXXXXXXXXCGGG (SEQ ID NO:24), where X represents any amino acid except cysteine. Since the actual insertion results from a screening process as described in the examples below, it is the randomized amino acids that likely play a dominant role in the observed changes in the properties of the Fc-fragment. As such, it is contemplated that a variant Fc-fragment having an insertion containing only the randomized portions of insertions which, like those above, contain some non-random amino acids can also have the desired properties. Examples of such sequence include the middle six amino acids from SEQ ID NOs: 41-53, which are shown as SEQ ID NOs: 54-66.

    [0055] In another aspect, amino acids 308 and 309 (using the EU numbering as shown in Table 1) within or adjacent to Loop 5 are deleted and replaced from 4-20, 1-18, 6-16, 10-16, 12-14, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13,14, 15, 16, 15-20, 20-40, 40-60, or 60-80 amino acids, which may be limited to all amino acids other than cysteine. In some aspects, the insertion can contain a cysteine among the first and the last four amino acids of the insertion, and the inserted amino acids can otherwise be limited to amino acids other than cysteine. The insertion can have a formula selected from the group consisting of SEQ ID NO:13-24.

    [0056] These variant Fc-fragments, as well as Fc-polypeptides that contain them, comprising the amino acid insertions described above and below, can bind to human FcRn at pH 5 to 6 with higher affinity and/or binding activity as compared to a control Fc-fragment and can bind to human FcRn at physiologic pH with an affinity and/or binding activity comparable to or lower than that of control Fc-fragments. Further, these variant Fc-fragments can be further altered and screened for alterations that confer even higher affinity and/or binding activity for human FcRn at pH 5 to 6, lower affinity and/or binding activity for human FcRn at physiologic pH, or other desirable properties, such as stability upon storage. In the Examples below, insertions of particular amino acid sequences (e.g, SEQ ID NOs:41-66 and 90-246) at defined sites within an Fc-fragment are shown to be effective at increasing affinity and/or binding activity of the Fc-fragment for human FcRn at pH 5 to 6.

    [0057] Variant Fc-polypeptides comprising human IgG variant Fc-fragments also comprise a binding region that binds to a target molecule. Binding of such variant Fc-polypeptides to the target molecule can modulate, optionally antagonize or agonize, the biological activity of the target molecule and/or can serve to localize the Fc-polypeptide to a location where the target molecule is expressed. An Fc-polypeptide can include more than one binding region, such as two, three, or four binding regions in an Fc-polypeptide. Since an Fc-polypeptide can multimerize forming, in some cases, dimers, trimers, or tetramers, multimerization of different Fc-polypeptides can form multimeric Fc-polypeptides that can bind to more than one target molecule. If there are plural binding regions in an Fc-polypeptide, they can bind to the same or different target molecules or portions of a target molecule. Modulation of the activity of the target molecule can affect the course of a disease or condition that is mediated at least in part by the target molecule. Human IgG variant Fc-polypeptides can comprise a binding region that includes one or more antibody variable regions that bind to one or more target molecules. Such human IgG variant Fc-polypeptides can, for example, be full length antibodies, such as human, humanized, or chimeric IgG antibodies, scFc-Fv's, monovalent forms of antibodies such as those described in International Application Publication WO 2005/063816 and US Application Publication 2007/0105199, which comprise a dimeric Fc region, as well as VH, CH1, VL, CL and hinge regions. Further forms are described, for example, in Figure 2 of US Application Publication 2010/0286374, along with the text describing and/or referring to Figure 2, and in US Patent 5,837,821, which describes "minibodies" comprising an scFv linked to an Fc region. Alternatively, the binding region can comprise all or part of a non-antibody protein, optionally a human protein, that binds to the target molecule. For example, the binding region could comprise an extracellular portion a receptor such as, for example, the extracellular region of the human p75 tumor necrosis factor receptor (SEQ ID NO:13) or the human T-lymphocyte associated protein-4 (CTLA4) protein (SEQ ID NO:14).

    [0058] The binding region can comprise one or more peptides that bind to a target molecule (such as, for example, a "monomer domain" as defined in US Patent 7,820,790 that binds to a target protein, which can be selected as discussed in US Patent 7,820,790), or other peptides. Portions of US Patent 7,820,79 describe monomer domains and how they are selected. One example of such a peptide is the binding portion of the Fc fusion protein romiplostim (NPLATE®, Amgen Inc., Thousand Oaks, California). Prescribing Information, NPLATE®, Amgen Inc., 2008-2011. Other polypeptides that can be part of a binding region of an Fc fusion protein include polypeptides comprising scaffold domains that have been randomized in certain positions and subjected to selection for binding to a certain target molecule. Such scaffold domains include, for example, CTLA-4 (Nuttall et al. (1999), Proteins 36: 217-227), the Z domain of Staphylococcal protein 1 (Nord et al. (1995), Protein Eng. 8: 601-608), green fluorescent protein, and the tenth type III domain of human fibronectin (FN3; Koide et al. (1998), J. Mol. Biol. 284: 1141-1151; Karatan et al. (2004), Chem. & Biol. 11: 835-844). Portions of these references describe the scaffold domains and their use to generate binding domains.

    [0059] A target molecule, to which a binding region binds, can be a molecule where the modulation of the biological activity of the molecule can affect the course of a disease or condition or can be a molecule localized in a diseased area, for example a protein expressed on the surface of cancer cells. In an autoimmune or inflammatory condition, the target molecule can be a molecule acting in a pathway that plays a role in mediating the condition. A target molecule can be a molecule that is localized such that binding to the target molecule will place an Fc-polypeptide appropriately such that it can affect the course of a disease. For example, if an Fc-polypeptide includes a toxin, placing it near a cancer cell by means of a binding region that binds to a protein highly expressed on the cancer cells can be advantageous. Target molecules include, for example, general classes of proteins such as soluble ligands, receptor bound ligands, soluble receptors, membrane bound receptors, membrane channel proteins, soluble and membrane bound proteins, and non-protein antigens, including extracellular and intracellular target molecules. Exemplary target molecules include, without limitation, the following human proteins: tumor necrosis factor, tumor necrosis factor receptor, interleukin-1, interleukin-6 (IL-6), IL-6 receptor, CD80/86, CD20, CD33, CD52, interleukin-12, interleukin-23, interleukin-17, HER2, HER2 neu receptor, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), B cell activating factor (BAFF), RANK ligand, OR51E2, claudins, CDH3, CD22, complement factors, and sclerostin, among many others.

    [0060] Depending in part on the host cell in which a variant Fc-polypeptide is produced and/or the purification methods used, the variant Fc-polypeptide can further comprise additional amino acid sequences such as, for example, a signal sequence facilitating secretion in eukaryotic cells (which is removed in mature forms of the protein), an N-terminal methionine to facilitate translation in bacteria, and/or a tag sequence (for example, a polyhistidine tag or a FLAG® tag) to facilitate protein purification or identification.

    Nucleic Acids Encoding Libraries of Variant Fc-fragments



    [0061] Also described herein are libraries of nucleic acids encoding Fc-fragments having insertions, which are at least partially randomized, that occur within or adjacent to loops of the Fc-fragments. Figure 2 illustrates the format for each of the eight libraries that were made, as described below in the Examples. The sequences of these libraries and the amino acid sequences encoded by them are disclosed in SEQ ID NOs:25-40. Such libraries are useful for panning in order to select individual nucleic acids that encode variant human IgG Fc-fragments that bind with enhanced affinity to FcRn at pH 5.0 to 6.0 as compared to a control Fc-fragment. Such variant human IgG Fc-polypeptides may also bind to FcRn poorly if at all at pH 7.2 to 7.6, as does a control Fc-fragment.

    [0062] In some aspects, these libraries of nucleic acids encoding Fc-fragments with insertions in regions encoding loop regions (as set forth in Table 1) can be of human or animal origin, including nucleic acids encoding mouse, rat, rabbit, or monkey Fc-fragments or Fc-fragments from a camelid species. As described herein, the encoded Fc-fragment may be an IgG Fc-fragment, such as an IgG1, IgG2, IgG3, or IgG4 Fc-fragment, or an Fc-fragment of an IgA, IgE, IgD, or IgM isotype. The insertions in the portions of the nucleic acids encoding loops can be within or adjacent to the nucleotide sequences encoding Loop 1, Loop 2, Loop 3, Loop 4, Loop 5, Loop 6, Loop 7, Loop 8, Loop 9, Loop 10, Loop 11, Loop 12, or Loop 13, as shown in Table 1.

    [0063] The size of the library as compared to the number of different variants in the library is an important consideration. In a best case, the panning procedure to which the library is subjected will have a high probability of including all different variants that are included in the library. The number of different variants in the library depends on the number of randomized positions in the library and the number of different amino acids that can be used at each randomized position. For example, a library that has six randomized positions that can contain any of 19 different amino acids can theoretically have 196 ≈ 4.7 x 107 different variants. If, for example, 109 independent phage or bacteria expressing these variants are panned, there is a very high probability that all isolates with the properties selected for in the screen will be recovered. See, e.g., Grossman and Turner, Mathematics for the Biological Sciences, Chapter 2, Section 2.9, pp. 97-104, Macmillan Publishing Co., Inc., New York and Collier Macmillan Publishers, London, 1974. Libraries of a size that make it possible to screen enough isolates to detect rare isolates with the desired properties are contemplated. Thus, libraries encoding from 105 to 1013, 106 to 1012, or 107 to 1010 different variants are contemplated.

    [0064] Libraries with randomized insertions within or adjacent to nucleotide sequences encoding Loops 5, 8, and 10 of the Fc-fragment-encoding portion can be advantageous. Such libraries can encode Fc-fragments with insertions within or adjacent to Loops 5, 8, or 10 that contain 20-40, 4-20, 1-18, 6-16, 10-16, 12-14, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 randomized amino acids. These randomized amino acids can be flanked by cysteine residues. As described herein, additional non-randomized amino acids can also be inserted before or after the randomized amino acids; the encoded randomized amino acids may be preceded by a Gly-Gly-Cys and followed by a Cys-Gly-Gly.

    Nucleic Acids Encoding Variant Fc-polypeptides, Fc-fragments, and Insertions



    [0065] Also described are nucleic acids encoding specific variant Fc-fragments, which can be part of a variant Fc-polypeptide, and nucleic acids encoding insertions within or adjacent to loops of variant Fc-fragments. Nucleotide sequences encoding Fc-fragments are known in the art (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, MD, 1991), and sequences encoding human IgG1, IgG2, IgG3, and IgG4 Fc-fragments (SEQ ID NOs: 2, 4, 6, and 8, respectively) are reported herein. The locations of loops within Fc-fragments are delineated (Table 1), and various kind of insertions into these regions are described herein. Sequences encoding amino acid insertions that increase binding to FcRn at pHs in the range of 5 to 6 are also reported (see, e.g., SEQ ID NOs:41-53).

    [0066] More generally, described herein are nucleic acids encoding a variant Fc-fragment having an insertion within or adjacent to a loop, wherein the Fc-fragment can bind to human FcRn with higher affinity at pH 5-6 than a control Fc-fragment. The positions at which such insertions occur in include Loops 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 within the Fc-polypeptide.

    [0067] Nucleic acids encoding variant Fc-polypeptides can be used to produce variant Fc-polypeptides by introducing the nucleic acids into host cells, including prokaryotic or eukaryotic host cells, and culturing the cells under conditions such that the protein(s) encoded by the nucleic acids will be expressed. The variant Fc-polypeptide can be recovered from the cell mass or the culture supernatant. Such polypeptides can be purified by methods well known in the art such as, for example, Protein A or Protein G affinity chromatography, size exclusion chromatography, ion exchange chromatography, and hydrophobic interaction chromatography, among other available methods.

    Methods of Isolating and Making Variant Fc-fragments and Fc-polypeptides



    [0068] Variant Fc-fragments can be isolated using screening procedures described in detail in the Examples below. In broad terms, a library of nucleic acids encoding Fc-fragments with insertions including randomized amino acids within or adjacent to a loop is prepared. This library is then introduced into a virus or a cell, which is cultured under conditions such that the nucleic acids from the library can be expressed, so that the virus (which can, for example, be a baculovirus or a filamentous phage) or cell (which can, for example, be a bacterial, yeast, or mammalian cell), can display the Fc-fragments containing the insertions. In an alternate aspect, the library can be translated in vitro in a ribosome display system such that Fc-fragments with the desired properties, and the nucleic acids encoding them, can be selected out from this mixture. Alternatively, a CIS display system can be used. See, e.g., Odegrip et al. (2004), Proc. Natl. Acad. Sci. 101(9): 2806-2810. In the methods described herein, variant Fc-fragments that can bind to human FcRn with higher affinity as compared to a control Fc-fragment within the pH range from 5 to 6 can be enriched for by panning with FcRn as described in the examples below and screened for using, for example, enzyme-linked immunosorbent assay (ELISA), which may be performed initially on groups of isolates or on single isolates. If ELISA is initially performed on groups of isolates, these isolates can be amplified and rescreened individually to obtain individual isolates expressing a variant Fc-fragment with enhanced affinity to human FcRn at pH 5-6 and with lower or about the same affinity for human FcRn at physiologic pH as compared to a control Fc-fragment.

    [0069] In further steps, nucleic acids encoding the variant Fc-fragments can be isolated from phage or cells, such as bacteria, yeast or mammalian cells, or amplified from RNA in the case of ribosome display. These nucleic acids can be sequenced to determine the exact position and nature of the insertion. Using this information, nucleic acids encoding a desired variant Fc-polypeptide or variant Fc-fragment can be constructed using methods well-known in the art. Further modification of the nucleic acid encoding the variant Fc-fragment or Fc-polypeptide can be undertaken if desired.

    [0070] Such nucleic acids can be inserted into vectors appropriate for the host cell in which the Fc-polypeptide or Fc-fragment will be expressed. These nucleic acids can be introduced into the host cells by any of the methods well-known in the art. Host cells that could be used include bacteria, including Escherichia coli, yeast, including Saccharomyces cerevisiae or Pichia pastoris, insect cells, plant cells, and mammalian cells, including Chinese hamster ovary (CHO) cells, 293 cells, and baby hamster kidney (BHK) cells, among many others. These host cells can be cultured under conditions such that the introduced nucleic acids will be expressed, and the Fc-polypeptide or Fc-fragment can be recovered from the culture supernatant or the cell mass.

    [0071] Generally, the procedure used to introduce the nucleic acids into the host cells may depend upon the host cell into which the nucleic acids are to be introduced. Methods of introducing nucleic acids into bacteria are well-known in the art. For example, electroporation or calcium choride transformation are commonly used. Methods for introduction of nucleic acids into yeast are well-known in the art and include, for example, transformation methods using lithium acetate and polyethylene glycol. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

    [0072] Expression vectors used in any of the host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.

    Therapeutic Uses of Variant Fc-polypeptides



    [0073] Variant Fc-polypeptides described herein can be used as human therapeutics for a variety of conditions. Which variant Fc-polypeptide is appropriate for which condition can be determined by the binding region(s) of the Fc-polypeptide, as well as possibly other aspects of its structure such as, for example, an attached toxic moiety. Variant Fc-polypeptides can have increased in vivo half lives as compared to control Fc-polypeptides and may therefore require lower and/or less frequent dosing than control Fc-polypeptides. Thus, variant Fc-polypeptides might be well suited to treat chronic conditions, where less frequent dosing is particularly desirable. However, a variant Fc-polypeptide can be used as a treatment of most or all conditions that a control Fc-polypeptide (which has the same amino acid sequence as the variant Fc-polypeptide except that it does not have the insertion in a loop) can be used to treat. The variant Fc-polypeptides described herein can also be used concurrently with other medications used for treating the condition being treated.

    [0074] For example, an Fc fusion protein that is a variant Fc-polypeptide which contains an insertion in a loop can be used as a treatment for the same conditions that a control Fc-polypeptide is used. However, dosing amount and/or frequency of the variant Fc-polypeptide may be different because of its increased half-life. For example, a variant Fc-polypeptide could be made starting from a therapeutic Fc fusion protein such as etanercept (which is indicated for moderate to severe rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, and moderate to severe plaque psoriasis), abatacept (which is indicated for moderate to severe rheumatoid arthritis, moderate to severe polyarticular juvenile idiopathic arthritis), or romiplostim (which is indicated for chronic immune thrombocytopenic purpura) by inserting one of the insertions that enhance FcRn binding disclosed herein, e.g., SEQ ID NOs: 41-53, in a loop in the Fc-fragment portion of each of these molecules. The insertion could, for example, be made within or adjacent to Loops 5, 8, or 10 of the Fc-fragment portion of the Fc fusion protein. Such variant forms of etanercept , abatacept, or romiplostim could be used to treat the same diseases as unaltered etanercept, abatacept, or romiplostim can be used to treat.

    [0075] Similarly, if the variant Fc-polypeptide is a therapeutic antibody, such as, for example, adalimumab, ustikinumab, golimumab, natalizumab, infliximab, or denosumab, such a variant form of the antibody comprising a variant Fc-fragment can be used to treat the same diseases that these antibodies, in an unaltered form, are used to treat. Thus, a variant Fc-fragment, as described herein, can change the dosage amount or frequency of treatment, but not the condition the Fc-polypeptide is used to treat.

    [0076] Generally, Fc-polypeptides are used to treat a wide variety of diseases including oncologic indications, autoimmune and inflammatory conditions, bone-related conditions, conditions, metabolic conditions, and neurologic conditions such as, for example, chronic pain, among many others.

    Pharmaceutical Compositions



    [0077] The invention also relates to pharmaceutical compositions comprising the variant Fc-fragments or variant Fc-polypeptides described herein. Such compositions can comprise a therapeutically effective amount of a variant Fc-polypeptide or variant Fc-fragment with one or more additional components such as a physiologically acceptable carrier, excipient, or diluent. Such additional components can include buffers, carbohydrates, polyols, amino acids, chelating agents, stabilizers, and/or preservatives, among many possibilities.

    Methods of Administration



    [0078] The variant Fc-polypeptides or variant Fc-fragments, or pharmaceutical compositions containing these molecules, can administered by any feasible method. Therapeutics that comprise a protein will ordinarily be administered by injection since oral administration, in the absence of some special formulation or circumstance, would lead to hydrolysis of the protein in the acid environment of the stomach. Subcutaneous, intramuscular, intravenous, intraarterial, intralesional, or peritoneal injection are possible routes of administration. Topical administration is also possible, especially for diseases involving the skin. Alternatively, variant Fc-polypeptides or variant Fc-fragments can be administered through contact with a mucus membrane, for example by intra-nasal, sublingual, vaginal, or rectal administration or as an inhalant. Alternatively, certain pharmaceutical compositions comprising a variant Fc-polypeptide or variant Fc-fragment can be administered orally.

    [0079] Having described subject matter relating to the invention in general terms above, the following examples are offered by way of illustration.

    EXAMPLES


    Example 1: Creation of a model of the tertiary structure of human FcRn:human IgG1 Fc



    [0080] To aid design of the libraries described below, a homology model of the tertiary structure a human FcRn in a complex with a human IgG1 Fc-fragment was created based on the structure of a complex of a rat FcRn and a rat IgG Fc-fragment available in Protein Data Bank (PDB Accession No. 1FRT). The Protein Data Bank (PDB) was searched with amino acid sequences of human IgG1 Fc-fragment (SEQ ID NO:1) and human FcRn α and β-2-microglobulin (β2m) chains, SEQ ID NOs:9 and 10, respectively. Several templates were obtained based on amino acid sequence homology and tertiary structure. FcRn α chain structures (1EXU (chain A), 1FRT (chain A), and 1I1A (chain A)), FcRn β2m chain structures (1CE6 (chain B), 1HSA (chain B), 1YPZ (chain B), 1HHG (chain B) and 1I1A (chain B)), and Fc structures 1HZH (chain H), 1I1A (chain C), 1OQO (chain A), 1T83 (chain A) and 2J6E (chain A)) were selected. The FcRn α structures 1EXU (chain A), 1FRT (chain A) and 1I1A (chain A) were superimposed and the resulting root mean square deviation (RMSD), that is, the average distance between analogous atoms in superimposed protein structures, values are 0.30 Å to 0.76 Å, with the mean value 0.59 Å. The RMSD values between the FcRn β2m domain structures 1CE6 (chain B), 1HSA (chain B), 1YPZ (chain B), 1HHG (chain B), and 1I1A (chain B) range from 0.30 Å to 0.76 Å with the mean value 0.59 Å. The RMSD values between the Fc structures 1HZH (chain H), 1I1A (chain C), 1OQO (chain A), 1T83 (chain A) and 2J6E (chain A) range from 0.30 Å to 0.76 Å with the mean value 0.59 Å. These RMSD values indicate that the template FcRn and Fc structures from different sources share very similar tertiary structures.

    [0081] Based on these template structures and the amino acid sequences of human FcRn (hFcRn) and human IgG1 Fc-fragment (hlgG1 Fc), a homology model of hFcRn:hlgG1 Fc complex was created using a linux-based molecular modeling module of the computational software Molecular Operating Environment (MOE; Chemical Computing Group, Montreal, Quebec, Canada). Figure 1 shows the portion of this modeled complex where the hlgG1 Fc (below) and the hFcRn (above) come into closest contact, as well as some adjacent areas of these proteins.

    Example 2: Construction of the insertion libraries



    [0082] Eight libraries encoding variant human IgG1 Fc regions, each library having different kinds of insertions, were constructed. Figure 2 shows the sequence of a human IgG1 Fc region and the positions and the format of the six insertion libraries. The positions of the loops into which insertion libraries described below were inserted are indicated in Figures 1 and 2 as L1, L2A, L2B, L3, L4, L5, L6A and L6B. Some insertion sites in the Fc were among the closest points of contact between hlgG1 Fc and hFcRn (such as L1, L2A, L2B, and L3), and others (L4, L5, L6A, and L6B) were at some distance from the closest points of contact between hFc and hFcRn.

    [0083] As shown in Figure 2, in some of the libraries, such as L1, L2A, L2B, and L3, amino acids within a loop were deleted and replaced with randomized amino acids, in most cases with a slightly greater number of amino acids than had been deleted. The randomized amino acids included all amino acids other than cysteine. In library L2B, six randomized amino acids were preceded by the sequence Gly-Gly-Cys and followed by the sequence Cys-Gly-Gly. Since the two cysteines would be expected to form a disulfide bridge given their proximity, it would be expected that the six randomized amino acids between the cysteines would form a spatially constrained loop. For example, in L2B, six randomized amino acids were preceded by the sequence Gly-Gly-Cys and followed by the sequence Cys-Gly-Gly, thus creating spatially constrained loops containing the randomized amino acids. In other libraries, i.e., L4 and L5, randomized amino acids were inserted without deleting any amino acids normally present.

    [0084] Two rounds of PCR were used to construct L1, L2A, L2B, L3, L4 and L5 libraries encoding a group of human IgG1 variant Fc-fragments. The template used in the first and second sets of PCR reactions in the first round of reactions was a phagemid vector into which DNA encoding a human IgG1 Fc-fragment (SEQ ID NO:2) had been inserted so as to allow its expression as part of a phage coat protein in appropriate bacterial strains (plgG1-Fc). The first set of PCR reactions in the first round of reactions used to generate the libraries utilized forward and reverse primers in order to create PCR fragments that contain an ApaLI restriction enzyme site at the upstream end. The following forward primer, which matched vector sequences upstream of the region encoding the Fc-fragment, was used for all of the first set of PCR reactions in the first round of reactions: 5'-GTTCCT TTC TAT TCTCAC-3' (SEQ ID NO:521). The reverse primers, which were within Fc-encoding sequences, were the following: 5'-GAG GGT GTC CTT GGG TTT TGG GGG-3' (SEQ ID NO:522; Library L1); 5'-GGT GAG GAC GCT GAC CAC ACG GTA-3' (SEQ ID NO:523; Libraries L2A and L2B); 5'-ATG CAT CAC GGA GCA TGA GAA GAC-3' (SEQ ID NO:524; Library L3); 5'-ATT ATG CAC CTC CAC GCC GTC CAC-3' (SEQ ID NO:525; Library 4); and 5'-ATT GCT CTC CCA CTC CAC GGC GAT-3' (SEQ ID NO:526; Library L5).

    [0085] A second set of PCR reactions in the first round of reactions also used plgG1-Fc as a template and used an oligonucleotide that matched the complement of vector sequences downstream from the Fc-encoding sequences as a reverse primer. This reverse primer had the following sequence: 5'-CCC ATT CAG ATC CTC TTC-3' (SEQ ID NO:527). The forward primers used for these reactions were as follows: Library L1, 5'-AAA CCC AAG GAC ACC CTC (TRIM)6 ACC CCT GAG GTC ACA TGC-3' (SEQ ID NO:528); Library L2A, 5'-GTG GTC AGC GTC CTC ACC (TRIM)6 CAC CAG GAC TGG CTG AAT-3' (SEQ ID NO:529); Library L2B, 5'-GTG GTC AGC GTC CTC ACC GGT GGT TGT (TRIM)6 TGT GGT GGT CAC CAG GAC TGG CTG AAT-3' (SEQ ID NO:530); Library L3: 5'-TCA TGC TCC GTG ATG CAT (TRIM)5 CAC (TRIM)1 CAC TAC ACG CAG AAG AGC-3' (SEQ ID NO:531); Library L4: 5'-GGC GTG GAG GTG CAT AAT GGT GGT TGT (TRIM)6 TGT GGT GGT GCC AAG ACA AAG CCG CGG-3' (SEQ ID NO:532); and Library L5: 5'-GTG GAG TGG GAG AGC AAT GGT GGT TGT (TRIM)6 TGT GGT GGT GGG CAG CCG GAG AAC AAC-3' (SEQ ID NO:533). In these oligonucleotides, "TRIM" represents a mixture of trinucleotides encoding all amino acids except cysteine (Trimer Phosphoramidite Mix 2, Glen Research Catalog No. 13-1992-95). The mixture is an equimolar mixture of the following trinucleotides: AAA, AAC, ACT, ATC, ATG, CAG, CAT, CCG, CGT, CTG, GAA, GAC, GCT, GGT, GTT, TAC, TCT, TGG, TTC. Hence, codons encoding all amino acids except cysteine were represented approximately equally in the TRIM mixture. Thus, "(TRIM)6" means that six random trinucleotides encoding any amino acid except cysteine are included in the oligo.

    [0086] In a second round of PCR reactions (one for each library), the products from the first and second sets of PCR reactions from the first round described above served as templates. The primers used were the same for all libraries and were the following: 5'-GTT CCT TTC TAT TCT CAC-3' (forward; SEQ ID NO:534) and 5'-CCC ATT CAG ATC CTC TTC-3' (reverse; SEQ ID NO:535).

    [0087] Libraries L6A and L6B were constructed in one round of PCR, using plgG1-Fc as a template. In the PCR reaction described below, an Xmal restriction site (5'-CCCGGG-3') in the coding region for the Fc-fragment was changed to a Xhol restriction site (5'-CTCGAG-3") due to the sequences of the primers. These alterations were silent mutations that did not encode different amino acids from the unmodified plgG1-Fc and were made because an Xhol plus Notl restriction digestion was more efficient than an Xmal plus Notl digestion. The forward primers used for these reactions were as follows: Library L6A: 5'-CTG CCC CCA TCT CGA GAT GAG CTG GGT TGT (TRIM)8 TGT GGT GGT ACC AAC CAG GTC AGC CTG ACC-3' (SEQ ID NO:536); and library L6B: 5'-CTG CCC CCA TCT CGA GAT GAG CTG GGT TGT (NNK)8 TGT GGT GGT ACC AAC CAG GTC AGC CTG ACC-3' (SEQ ID NO:537). The reverse primer sequence was 5'-GGC CCC GTG ATG GTG ATG ATG-3' (SEQ ID NO:538). In L6B a mixture of trinucleotides called NNK, which contains trinucleotides encoding all twenty amino acids in 32 degenerate codons, was used. In this trinucleotide mixture, "N" represents a randomized position that is either adenosine, guanidine, cytidine, or thymidine and "K" represents a partially constrained position that is either guanidine or thymidine.

    [0088] For all PCR reactions, PCR core kits were used (Roche, Catalog No. 11 578 553 001) with the following reaction conditions: 95°C for 5 minutes, followed by 30 cycles of 95°C for 45 seconds- 55°C for 45 seconds- 72°C for 90 seconds, and finally 72°C 10 minutes. DNA from the PCR reactions was purified using QIAquick PCR Purification Kits (QIAGEN, 28104). About 200 µg of phagemid vector DNA and 10 µg of purified PCR product (from the third round of PCR reactions) were digested with ApaLI and Notl (for Libraries L1-L5) and Xhol and Notl (for Libraries L6A and L6B). The digested DNA was gel purified with QIAquick Gel Purification Kits (QIAGEN, 28704).

    [0089] Digested vector DNA and library PCR products in a molar ratio of 1:2 were ligated using T4 DNA ligase (New England Biolabs). The mixture was incubated at 16°C overnight. DNA was purified by ethanol precipitation. A total of 25 µg DNA was electroporated into 1000 µl of electrocompetent XL1 blue E. coli cells (Stratagene) in a 2.5 kV field using 200 Ω resistance and 25 µF capacitance to obtain about 1 x 109 E. coli transformants. Exact sizes of libraries ranged from 7 x 108 (for L5) to 1.8 x 109 (for L4). Cells were inoculated in 1000 mL of 2x YT medium (which contains 16 g/L Bacto Tryptone, 10 g/L Bacto Yeast Extract, and 5 g/L NaCl at a pH of 7.0-7.2) containing 100 µg/mL ampicillin and 2% glucose and grown until the OD600 was about 0.5. About 3 x 109 plaque-forming units per milliliter (PFU/mL) of M13 helper phage were then added, and the culture was incubated at 37°C for 1 hr. Infected cells were then spun down and resuspended with 1000 mL of 2x YT medium containing 100 µg/ml ampicillin and 40µg/mL kanamycin. Cells were grown at 30°C overnight. The phage were then precipitated with PEG 6000.

    [0090] All libraries used in these transformations, other than L6A and L6B, contained six randomized codons, each of which encoded any of nineteen different amino acids, and therefore would be expected to encode about 4.7 x 107 (196) different variant Fc-fragments. Similarly, L6A would be expected to encode about 1.76 x 1010 (208) different variant Fc-fragments, since it contained eight randomized codons that encoded any of 20 different amino acids. For libraries other than Libraries L6A and L6B, given the expected number of different variants in each library, the library sizes of about 109 (ranging from 7 x 108 to 1.8 x 109) were 7-33 times the number of variants.

    Example 3: Screening of the Fc loop Libraries



    [0091] Two rounds of panning for phage expressing variant Fc-fragments that bind to FcRn with increased affinity as compared a wild type Fc-fragment at pH 6.0 (and having a low binding affinity at pH 7.4) were performed as follows. In the first round, phage were resuspended in a liquid containing 0.4 ml 20 mM MES, pH 6.0, 5% skim milk, 0.05% Tween 20. Each of four wells of a MAXISORP™ immunoplate (Nunc, Rochester, NY) was coated with 10 µg of hFcRn and blocked with 5% skim milk, and 100 µL of phage at 5 x 1011 PFU/mL were added. After a 1hr incubation at 37°C, wells were washed 3-10 times with 20 mM MES, pH 6.0, 0.2% Tween 20. The phage were eluted by adding 100 µL phosphate buffered saline (PBS), pH 7.4 to each well and incubating the plate for 1 hr at 37°C. These phage were used to reinfect an exponentially growing E. coli XL1-blue culture, which was cultured until it reached an OD600 of about 0.5. About 3 x 109 PFU/mL of M13 helper phage were then added, and the culture was incubated at 37°C for 1 hr. Infected cells were then spun down and resuspended with 1000 ml of 2x YT medium containing 100 µg/ml ampicillin and 40µg/ml kanamycin. Cells were grown at 30°C overnight. The phage were then precipitated with PEG6000.

    [0092] Using these phage, a second round of panning was performed using essentially the same methods used in the first round. Stringency of the panning was increased slightly by reducing the concentration of FcRn coated onto the microtiter plates slightly and increasing the number of washes. The eluted phage, after washing several times, were used to reinfect an exponentially growing E. coli XL1-blue culture at an OD600 of about 0.5, which was then cultured for an hour at 37°C and plated on plates containing about 100 µg/ml ampicillin and kanamycin to obtain colonies.

    [0093] Single colonies were inoculated into 96-well tissue culture plates containing 120 µL/well 2x YT medium containing 100 µg/mL ampicillin and 2% glucose. The cells were grown at 37°C on a shaker until an OD600 of about 0.5 was reached. Then 3 x 109/mL M13 helper phage were added to each well and incubated for 1 hr. The cells then were spun down and resuspended with 180 µL/well 2x YT medium containing 100 µg/mL ampicillin and 40 µg/mL kanamycin. The culture was then incubated at 30°C overnight on a shaker.

    [0094] Approximately 100 µL of biotinylated human FcRn at 2 µg/mL was added to the wells of MAXISORP™ plates coated with 10 µg/mL streptavidin. After washing with PBS plus 0.05% Tween 20 (PBST) five times, phage from the overnight cultures in MES buffer at pH 6.0 or pH 7.4 were added to the plates and incubated at room temperature for 1 hr. A horseradish peroxidase (HRP)-conjugated anti-M13 antibody was used to detect the binding phage, and the plates were scored by a microtiter plate reader using a wavelength in the visible range. Positives were selected based on a higher signal at pH 5.5 (approximately 150% of the signal generated by a control Fc-fragment) and a signal that was comparable to that of a control Fc-fragment or lower at pH 7.4. In a second round of screening in which a single 96 well microtiter plate of isolated colonies was tested for each library, no positives were detected for libraries L2B, L3, or L4. About 20% of the colonies picked for Library L2A were positive, as were about 10% of the colonies picked for libraries L6A and L6B. Library L5 was clearly distinguishable from all other libraries since about 90% of the colonies picked were positive. Hence, library L5 was, by a large margin, the library that produced the most positives. These data indicate that the site at which library 5 is inserted is particularly favorable for isolation of variants that increase binding to FcRn at pH 5-6, while maintaining the low affinity of the Fc-fragment for FcRn at physiologic pHs.

    [0095] We selected positives from libraries L2A, L5, L6A, and L6B, which were sequenced and further characterized as described below. Binding was assessed by a quantitative enzyme-linked immunoadsorbent assay (ELISA). The ELISA scores reflect binding affinity, with higher scores meaning higher affinity. Typically, scores higher than about 3 ∼ 5 indicate binding above background. The sequences of the insertions of these positives and their scores in an ELISA are shown in Table 2 below. For library L5, the insertions are between the N169 and G170 according to the numbering in Figure 2. For libraries 6A and 6B, the insertions are between L143 and T144 according to the numbering in Figure 2.
    Table 2: Sequences and ELISA scores of selected positives
    LibraryControlELISA score pH 6ELISA score pH 7.4Insertion sequence (SEQ ID NO:)Isolate identifier
    L5   95.4 2.8 GGCVFNMFNCGG (SEQ ID NO:44) 5-106
    L5   95.1 15.1 GGCPHMFPWCGG (SEQ ID NO:392)  
    L5   92.1 28.4 GGCGHGWIFCGG (SEQ ID NO:393)  
    L5   91.0 4.4 GGCVFNMFNCGG (SEQ ID NO:394)  
    L5   89.5 23.5 GGCILNFYGCGG (SEQ ID NO:395)  
    L5   88.6 8.9 GGCREPHPFCGG (SEQ ID NO:396)  
    L5   87.3 1.8 GGCPFEFTQCGG (SEQ ID NO:397)  
    L5   86.5 0.4 GGCQLGSMHCGG (SEQ ID NO:398)  
    L5   86.3 1.6 GGCYENKTLCGG (SEQ ID NO:399)  
    L5   82.5 0.4 GGCHLPFAVCGG (SEQ ID NO:41) 5-51
    L5   82.4 2.0 GGCGHEYMWCGG (SEQ ID NO:43) 5-104
    L6A   79.5 0.7 GGCRAGYGDASCGG (SEQ ID NO:400)  
    L5   79.2 0.6 GGCMVPFSMCGG (SEQ ID NO:401)  
    L5   76.7 0.6 GGCWPLQDYCGG (SEQ ID NO:42) 5-69
    L5   75.5 0.3 GGCELQERWCGG (SEQ ID NO:402)  
    L5   74.8 0.5 GGCPANWGTCGG (SEQ ID NO:403)  
    L5   74.2 2.3 GGCMMEFAQCGG (SEQ ID NO:404)  
    L5   73.4 0.4 GGCQHHIMQCGG (SEQ ID NO:405)  
    L5   73.1 0.4 GGCYQHHMECGG (SEQ ID NO:406)  
    L5   72.7 0.5 GGCMQMNKWCGG (SEQ ID NO:364) 5-96
    L5   72.3 0.6 GGCMVPFSMCGG (SEQ ID NO:407)  
    L5   72 0.4 GGCQKGWVFCGG (SEQ ID NO:408)  
    L5   71.2 0.7 GGCVYDVKKCGG (SEQ ID NO:409)  
    L6B   68.9 0.8 GGCLKGMHGSACGG (SEQ ID NO:410)  
    L5   68.6 0.6 GGCNMLWGSCGG (SEQ ID NO:411)  
    L5   67.4 0.4 GGCMQPWAFCGG (SEQ ID NO:412)  
    L5   65.6 0.5 GGCMTQYNWCGG (SEQ ID NO:413)  
    L5   65.2 0.3 GGCVNTWWSCGG (SEQ ID NO:414)  
    L5   64.1 0.5 GGCDGRTKYCGG (SEQ ID NO:363) 5-92
    L5   63.6 0.7 GGCYITQKLCGG (SEQ ID NO:415)  
    L5   63 0.5 GGCETHYTYCGG (SEQ ID NO:416)  
    L5   62.7 0.4 GGCALYPTNCGG (SEQ ID NO:45) 5-112
    L5   62.2 0.3 GGCTEQVMWCGG (SEQ ID NO:417)  
    L5   61.2 0.4 GGCITEFSHCGG (SEQ ID NO:418)  
    L5   61 0.3 GGCQNRSYWCGG (SEQ ID NO:419)  
    L5   61 0.5 GGCHGTKQFCGG (SEQ ID NO:420)  
    L5   60.9 0.3 GGCNPHRTPCGG (SEQ ID NO:421)  
    L5   60.3 0.3 GGCQHSPPLCGG (SEQ ID NO:422)  
    L5   60.2 0.4 GGCNHEETFCGG (SEQ ID NO:423)  
    L5   59.6 0.3 GGCQYPRKLCGG (SEQ ID NO:424)  
    L5   59.2 0.3 GGCIGPFWWCGG (SEQ ID NO:425)  
    L5   59.2 0.3 GGCMQPWINCGG (SEQ ID NO:426)  
    L6B   58.9 0.8 GGCVQHKMGVVCGG (SEQ ID NO:427)  
    L5   58.5 0.5 GGCEMENAWCGG (SEQ ID NO:428)  
    L5   58.3 0.3 GGCPPWPERCGG (SEQ ID NO:429)  
    L5   57.8 0.3 GGCGKHWHQCGG (SEQ ID NO:359) 5-57
    L5   57.7 0.4 GGCHDPEPFCGG (SEQ ID NO:430)  
    L5   57.7 0.5 GGCNEPKYVCGG (SEQ ID NO:431)  
    L5   57.7 0.5 GGCDRPVWFCGG (SEQ ID NO:432)  
    L5   57.2 0.4 GGCHSFKHFCGG (SEQ ID NO:360) 5-64
    L5   57.2 0.7 GGCEIPHSFCGG (SEQ ID NO:433)  
    L5   56.7 0.4 GGCMPYEMHCGG (SEQ ID NO:434)  
    L5   56.1 0.7 GGCQGMWTWCGG (SEQ ID NO:366) 5-113
    L5   55.6 0.7 GGCKRENPYCGG (SEQ ID NO:435)  
    L5   55.5 1.4 GGCAERQYYCGG (SEQ ID NO:436)  
    L5   55.4 0.3 GGCNVLDLFCGG (SEQ ID NO:437)  
    L5   55.2 0.2 GGCKSMISMCGG (SEQ ID NO:438)  
    L5   54.9 0.6 GGCHHKQDQCGG (SEQ ID NO:439)  
    L6B   54.9 0.6 GGCNATLSGYLCGG (SEQ ID NO:440)  
    L5   54.7 0.6 GGCEATMTWCGG (SEQ ID NO:441)  
    L5   54.6 0.3 GGCNVLDLFCGG (SEQ ID NO:442)  
    L5   54.5 0.3 GGCAQQWHHEYCGG (SEQ ID NO:362) 5-73
    L5   54.2 0.7 GGCSRVFKYCGG (SEQ ID NO:443)  
    L5   53.2 0.3 GGCHAPQWECGG (SEQ ID NO:444)  
    L5   53.2 0.4 GGCPLVRADCGG (SEQ ID NO:445)  
    L5   53.2 0.4 GGCMHNEEFCGG (SEQ ID NO:446)  
    L5   52.4 1.2 GGCMFETKWCGG (SEQ ID NO:447)  
    L6B   52.1 0.4 GGCNMNEWKSGCGG (SEQ ID NO:448)  
    L5   51.6 0.4 GGCLQNLYVCGG (SEQ ID NO:449)  
    L5   51.3 0.2 GGCQTSMKNCGG (SEQ ID NO:450)  
    L5   51.2 0.5 GGCERFHHACGG (SEQ ID NO:361) 5-66
    L5   51.1 0.4 GGCNLGHMPCGG (SEQ ID NO:451)  
    L5   50.5 1.0 GGCWMWAEECGG (SEQ ID NO:452)  
    L5   50.3 0.4 GGCVHNDKLCGG (SEQ ID NO:453)  
    L6B   50.1 0.4 GGCYGKAGMRDCGG (SEQ ID NO:454)  
    L6B   50 0.3 GGCVSAATSRTCGG (SEQ ID NO:455)  
    L5   49.8 0.6 GGCYPQKEICGG (SEQ ID NO:456)  
    L6B   49.2 0.4 GGCNQSSSREACGG (SEQ ID NO:457)  
    L6A   48.5 0.2 GGCNPVSTGAYCGG (SEQ ID NO:458)  
    L5   48.2 1.8 GGCPGHEFRCGG (SEQ ID NO:459)  
    L6B   47.2 0.4 GGCGEYNYVGGCGG (SEQ ID NO:460)  
    L5   47.2 0.9 GGCKWSMTKCGG (SEQ ID NO:461)  
    L5   47 1.4 GGCDWHRMKCGG (SEQ ID NO:462)  
    L5   46.8 3.6 GGCMHSPHACGG (SEQ ID NO:463)  
    L5   46.2 0.9 GGCMMWKVNCGG (SEQ ID NO:464)  
    L6A   45.2 0.3 GGCFTNYASEKCGG (SEQ ID NO:465)  
    L6A   44 0.2 GGCDRFQNVNVCGG (SEQ ID NO:466)  
    L6A   43.8 0.6 GGCERHFPALFCGG (SEQ ID NO:467)  
    L6B   43.6 0.2 GGCTLGSAPTLCGG (SEQ ID NO:468)  
    L6B   43.2 0.3 GGCEMMKNKSGCGG (SEQ ID NO:469)  
    L5   42.4 1.6 GGCEASGQICGG (SEQ ID NO:470)  
    L6B   42.3 0.2 GGCLRNFMKQSCGG (SEQ ID NO:471)  
    L6B   42.2 0.4 GGCPNDTVRDACGG (SEQ ID NO:472)  
    L6B   41.8 0.3 GGCSFSRHMGACGG (SEQ ID NO:473)  
    L6A   41.2 0.2 GGCAKDQHTGSCGG (SEQ ID NO:474)  
    L5   41.1 1.6 GGCLGLRQECGG (SEQ ID NO:475)  
    L6B   41 0.2 GGCNMNEWKSGCGG (SEQ ID NO:476)  
    L5   38.7 1.6 GGCQQIKEWCGG (SEQ ID NO:477)  
    L5   36.1 1 GGCDLPNEMCGG (SEQ ID NO:478)  
    L5   35.8 0.7 GGCMFSHPHCGG (SEQ ID NO:479)  
    L5   33.7 0.7 GGCAGPYWACGG (SEQ ID NO:480)  
    L5   31.1 0.8 GGCEQQFVTCGG (SEQ ID NO:481)  
    L5   29.5 1.2 GGCMGWWHLCGG (SEQ ID NO:482)  
    L5   28.4 0.5 GGCPQHGEMCGG (SEQ ID NO:483)  
    L5   27.7 1.0 GGCYASPHECGG (SEQ ID NO:484)  
    L5   27.5 2.1 GGCMPPQWMCGG (SEQ ID NO:485)  
    L5   26.9 1.4 GGCDTIGWFCGG (SEQ ID NO:486)  
    L5   26.3 0.7 GGCGIFESWCGG (SEQ ID NO:487)  
    L5   26.1 0.9 GGCGPYKTECGG (SEQ ID NO:488)  
    L5   25.2 0.7 GGCQPQASWCGG (SEQ ID NO:489)  
    L5   25 0.4 GGCDRQVTGFCGG (SEQ ID NO:490)  
    L5   24.9 1.2 GGSQRAPASCGG (SEQ ID NO:491)  
    L5   23.9 1.5 GGCMMREQGCGG (SEQ ID NO:492)  
    L5   22 1.0 GGCLLPNMFCGG (SEQ ID NO:493)  
    L5   21.7 0.3 GGCCPVYQHCGG (SEQ ID NO:494)  
    L5   21.5 1.0 GGCLMSQDLCGG (SEQ ID NO:495)  
    L5   21.3 1.4 GGCGGPYVFCGG (SEQ ID NO:496)  
      WT* 13-20 0.5-1.3    
      phg* 0.4-0.7 0.3-1.8    
      media* 0.4-0.7 0.5-0.6    
    *Since these control samples were measured multiple times, these results are reported as a range. "WT," "phg," and "media" mean, respectively, phage expressing a control Fc-fragment, phage expressing no Fc-fragment, and no phage.

    Example 4: Studies on binding association and dissociation rates



    [0096] To further characterize some of the variant Fc-fragments identified by the phage ELISA, binding of the variant Fc-fragments to FcRn at pH 6 and 7.4 was characterized for a subset of the identified variant Fc- fragments. DNA encoding selected variant Fc-fragment isolates, all from library L5, was introduced into a mammalian expression vector, which was used to transfect 293 6E cells using deacylated PEI essentially as described by Thomas et al. (Proc. Natl. Acad. Sci. 102(16): 5679-5684, 2005). Isolates having high ELISA scores for binding to FcRn were selected. The concentrations of variant Fc-fragments in conditioned media (CM) were measured using 1:2 and 1:10 diluted CM samples with Protein A Biosensors (ForteBio, Inc., catalog number 18-5010) on Octet Red® (ForteBio Inc., Menlo Park, California). Concentrations were calculated using a standard curve created with a purified Fc fusion protein.

    [0097] Biotinylated hFcRn at 100 nM was captured on streptavidin (SA) biosensors (FortéBio Inc., 18-5019) off-line at RT for 2 hours. Using these biotinylated hFcRn-coated SA biosensors in the Octet Red® system, association and dissociation of unlabeled proteins can be detected in real time via diffraction of light. Variant and control Fc-fragment CM samples were diluted to 10 µg/mL at pH 6 or pH 7.4. Three association and dissociation conditions were set up: (1) association at pH 6 and dissociation at pH 6; (2) association at pH 6 and dissociation at pH 7.4; and (3) association at pH 7.4 and dissociation at pH 7.4. SA biosensors coated with biotinylated hFcRN were dipped into buffer at a specific pH for 1 min and then soaked in the samples containing variant Fc-fragments or controls at a specific pH for 5 min to allow binding to FcRn. The Fc biosensors were then soaked in buffer at a specific pH for 5 minutes more to allow the bound Fc-fragments to dissociate from FcRn. Detection of binding and dissociation of the Fc-fragments was possible through the use of bio-layer interferometry as implemented using the Octet Red® system, so that binding and dissociation were detected in real time.

    [0098] Association and dissociation rates of the variant Fc-fragments at different pHs were compared to the controls. Higher binding at pH 6, slower off-rate at pH 6, very weak binding at pH 7.4, and faster off-rate at pH 7.4 as compared to the control Fc-fragment were the criteria used for selection of variant Fc-fragments for further characterization. A number of the 61 variant Fc-fragments tested showed more binding and slower dissociation at pH 6 and comparable or faster dissociation at pH 7.4 after binding at pH 6 relative to a control Fc-fragment. Figure 3 shows the binding at pH 6 (to the left of the center vertical line) and dissociation at pH 7.4 (to the right of the center vertical line) curves for 24 variant Fc-fragments tested that had the most favorable properties. To the right of the curves the name of each variant Fc-fragment tested is listed along with the maximal binding response (in nanometers (nm)) observed during the association phase of the experiment. The absolute number for maximal response can vary somewhat from experiment to experiment, and it is not directly proportional to binding constants such as kon, kdis, or KD (kdis/kon). In Figure 3, the lowest binding response was observed with a wild type Fc-fragment, and all variants shown had higher responses at pH 6. Most of the variant Fc-fragments dissociated rapidly at pH 7.4, as does a wild type Fc-fragment. However, variant 5-1 clearly dissociated much more slowly at pH 7.4 than any other variant, whereas all other variants tested dissociated rapidly at pH 7.4, as did the wild type Fc-fragment. Fc variant 5-85 stood out as the variant with the largest response at pH 6 that also dissociated rapidly at pH 7.4. A number of other variants, such as 5-106, 5-104, 5-112, 5-79, 5-51, and 5-69, among others, also had high responses at pH 6 and rapid dissociation at pH 7.4. Thus, many variant Fc-fragments with improved properties were isolated from library L5.

    Example 5: Production of variant Fc-fragments



    [0099] DNA encoding selected isolates, all from Library L5, was introduced into a mammalian expression vector, which was used to transfect 293 cells using deacylated PEI essentially as described by Thomas et al. (Proc. Natl. Acad. Sci. 102(16): 5679-5684, 2005). Mammalian cells were chosen due to the ease of expression and post-translational processing in this system. The protein expression and production methods used are described in Durocher et al. (Nucl. Acids Res. 30(2): e9, 2002). The secreted variant Fc-fragments were purified from the culture media using Protein A affinity chromatograpy and size exclusion chromatography. High performance liquid chromatography (HPLC) was used to check the purity of the Fc-fragments. For general guidance in purification methods, see Methods in Molecular Biology: Protein Purification Protocols v.244, Cultler, ed., Humana, New Jersey, 2004. Protein titers of the Fc variants were assessed by Coomassie blue staining of a polyacrylamide gel. All isolates were expressed at adequate levels for testing.

    Example 6: Thermal stability of variant Fc-fragments



    [0100] Differential Scanning Calorimetry (DSC) measures the enthalpy (ΔH) of unfolding due to heat denaturation. Protein (or other macro-) molecules in solution are in equilibrium between the native, folded populations and denatured, unfolded populations. The higher the thermal transition midpoint (Tm), when 50% of the protein molecules are unfolded, the more stable the molecule. DSC is also used to determine the change in heat capacity (ΔCp) of denaturation. DSC experiments were performed with MicroCal VP-Capillary DSC (GE Helathcare, Piscataway, NJ) in order to measure Tm of the various Fc-fragments. The concentration of purified control (Fc-WT) or variant Fc-fragment in each experiment was 0.5 mg/mL in 10 mM sodium acetate, 9% sucrose at pH 5. The samples were heated from 20°C to 95°C at a heating rate of 60°C/hour. The thermal transition midpoints (Tm) of individual domains were determined when 50% of the individual domain was unfolded. The Tm values of the variant Fc-fragments and the control wild type Fc-fragment are listed in Table 3 below.
    Table 3: Tm values of the variant Fc-fragments
    Sample IdentifierTm of CH2 Domain (°C)Tm of CH3 Domain (°C)
    Fc-WT 67.7 84.3
    Fc-5-55 67.7 77.5
    Fc-5-60 67.7 78.4
    Fc-5-64 67.4 79.2
    Fc-5-69 68.3 76.2
    Fc-5-70 67.3 77.7
    Fc-5-73 67.7 78.8
    Fc-5-79 67.3 76.5
    Fc-5-85 66.5 73.4
    Fc-5-91 67.4 77.6
    Fc-5-92 67.5 79.5
    Fc-5-95 67.1 77
    Fc-5-96 67.0 79.2
    Fc-5-97 67.4 78.2
    Fc-5-99 67.0 78.2
    Fc-5-101 67.0 78.6
    Fc-5-104 67.1 78.0
    Fc-5-106 66.6 77.4
    Fc-5-112 67.2 77.1
    Fc-5-113 68.1 77.9
    Fc-5-1 68.4 77.4
    Fc-5-51 67.4 77.4
    Fc-5-57 67.1 79.1
    Fc-5-66 67.5 79


    [0101] These data indicate that the Tm of the CH2 domain was not substantially affected by library L5 insertions, which are in the CH3 domain. Although Tm of the CH3 domain was slightly decreased in variant Fc-fragments as compared to the control Fc-fragment (Fc-WT), the Tm values of the CH3 domain in the variant Fc-fragments are still approximately 10 °C higher than the Tm values of the CH2 domain. Thus, the lower threshold of the thermal stability of the entire Fc domain, that is, the Tm of the CH2 domain, is not affected.

    Example 7: Binding of variant Fc-fragments to human or cynomolgus monkey FcRn



    [0102] Binding of variant Fc-fragments to human and cynomolgus monkey FcRn was tested using a BIAcore®T100 analysis system (GE Healthcare Bio-Sciences AB Private Limited Liability Company, Uppsala, Sweden) at both pH 5.5 and pH 7.4. The test consisted of incubating various concentrations of the variant Fc-fragments or control Fc-fragments with a fixed amount of human or cynomolgus monkey FcRn under binding conditions and subsequently measuring the amount of free, unbound FcRn in these mixtures by binding to a surface (in a flow cell of a CM5 (Biacore) chip) on which an Fc-fragment was immobilized. The Fc-fragment immobilized on the chip was sufficient in quantity to bind essentially all of the free, unbound FcRn in the mixture. Thus, the amount of unbound FcRn in the mixture was quantitatively ascertained by surface plasmon resonance through use of the BIAcore® T100 analysis system. From this information, an EC50, that is, the concentration of the variant or control Fc-fragment at which 50% of the FcRn in the mixture was bound, was calculated. Initially, a group of 23 variant-Fc-fragments (Fc-5-1 being considered a control) identified in the experiments described above were tested for binding to human FcRn. Based on these data, fourteen variant Fc-fragments were selected and further tested for binding to cynomolgus monkey FcRn.

    [0103] The experimental protocol is described in more detail below. The Fc-fragments to be tested were produced in 293 cells and purified as described above. A wild-type human control Fc-fragment (Fc-WT) and a variant Fc-fragment (Fc-5-1) were immobilized on the flow cells of a CM5 chip (Biacore) using amine coupling with density around 6000 resonance units (RU). Fc-5-1 was used because it, unlike Fc-WT, binds to FcRn well at pH 7.4. One flow cell with no immobilized protein bound to it was used as a background control. It was possible to use a single flow cell for successive samples by washing between samples with a solution at pH 7.4 to release FcRn bound to the Fc-fragment immobilized on the flow cell.

    [0104] To obtain a reasonable signal for the different molecules and conditions tested, different assay conditions were used. For assays at pH 5.5, 10 nM of human or cynomolgus monkey FcRn was mixed with serial dilutions of the variant and control Fc-fragments being tested (which ranged from 0.1 ∼ 2,000 nM) and incubated for 1 hour at room temperature in 10mM sodium acetate, pH 5.5, 150 mM NaCl, 0.005% P20, 0.1mg/mL BSA. As positive controls, 10 mM human and cynomolgus monkey FcRn were each incubated in the same solution, for the same time, and at the same temperature described immediately above, but without an added Fc-fragment. In these samples, presumably all FcRn was unbound. Binding of the free, unbound FcRn in each of these mixtures to the immobilized Fc-WT and Fc-5-1 was measured by injecting the mixtures over the surfaces on the flow cells and detecting the FcRn bound to the surfaces via surface plasmon resonance.

    [0105] For assays at pH 7.4, 10 nM human and cynomolgus monkey FcRn was mixed with serial dilutions of the variant and control Fc-fragments being tested (which ranged from 0.1 ∼ 2,000 nM) and incubated for 1 hour at room temperature in phosphate buffered saline (PBS) with 0.005% polysorbate 20, 0.1 mg/mL BSA. As positive controls, 10 mM human and cynomolgus monkey FcRn were each incubated in the same solution, for the same time, and at the same temperature described immediately above, but without an added Fc-fragment. Amounts of free, unbound FcRn in these mixtures were determined by binding to immobilized Fc-5-1 Fc-fragment, which was measured by injecting these mixtures over a flow cell surface coated with Fc-5-1 and detecting the FcRn bound to the surfaces via surface plasmon resonance. Fc-5-1 was used in these assays because it binds to FcRn with much greater affinity that Fc-WT at pH 7.4.

    [0106] A decreased FcRn binding response with increasing concentrations of Fc-fragment in the mixtures indicated that FcRn was bound to the Fc-fragment in solution, which blocked FcRn from binding to the immobilized Fc-fragment on the surfaces of the flow cells. Plotting the FcRn binding signal versus Fc-fragment concentrations, EC50's were calculated using nonlinear regression of one-site competition in GraphPad Prism 5™ software. These results are shown in Table 4.
    Table 4. EC50 of Fc-fragments for binding to FcRn at pH 5.5 and 7.4
     Human FcRn pH 5.5Human FcRn pH 7.4Cynomolgus FcRn pH 5.5Cynomolgus FcRn pH 7.4
    SampleEC50 (nM)95% CI (nM)EC50 (nM)95% CI (nM)EC50 (nM)95% CI (nM)EC50 (nM)95% CI (nM)
    Fc-WT 290 200∼430 >2000   270 200∼360 >2000  
    Fc-5-1 2.6 1.4∼4.7 45 33-61 2.3 1.2∼4.5 42 28-63
    Fc-5-51 16 13∼19 >2000   21 12∼36 >2000  
    Fc-5-55 49 35∼69 >2000   53 46∼62 >2000  
    Fc-5-57 180 110∼310 >2000   ND*   ND  
    Fc-5-60 67 48∼94 >2000   60 43∼85 >2000  
    Fc-5-64 210 113∼375 >2000   ND   ND  
    Fc-5-66 200 170∼230 >2000   ND   ND  
    Fc-5-69 14 12∼17 >2000   18 14∼23 >2000  
    Fc-5-70 26 20∼32 >2000   27 18∼39 >2000  
    Fc-5-73 190 120∼290 >2000   ND   ND  
    Fc-5-79 53 45∼64 >2000   63 48∼81 >2000  
    Fc-5-85 18 15∼21 >2000   25 14∼43 >2000  
    Fc-5-91 57 40∼110 >2000   ND   ND  
    Fc-5-92 75 54∼106 >2000   ND   ND  
    Fc-5-95 42 31∼56 >2000   50 37∼67 >2000  
    Fc-5-96 76 58∼110 >2000   ND   ND  
    Fc-5-97 25 19∼32 >2000   31 27∼36 >2000  
    Fc-5-99 15 11∼19 >2000   16 10∼25 >2000  
    Fc-5-101 100 60∼170 >2000   ND   ND  
    Fc-5-104 15 3∼67 >2000   16 14∼19 >2000  
    Fc-5-106 5.3 4.5∼6.4 >2000   3.6 2.4∼5.3 >2000  
    Fc-5-110 250 140∼460 >2000   ND   ND  
    Fc-5-112 46 38∼56 >2000   49 34∼72 >2000  
    Fc-5-113 20 12∼33 >2000   27 21∼35 >2000  
    * ND indicates "not determined"


    [0107] The data in Table 4 indicate that many of the variant Fc-fragments tested have substantially improved binding to human FcRn at pH 5.5 (i.e., have a substantially lower EC50 compared to Fc-WT) and maintain low binding at pH 7.4 (i.e., have a high EC50, like that of Fc-WT). However, five of them (Fc-5-57, Fc-5-64, Fc-5-66, Fc-5-73, and Fc-5-110) did not show much improved binding at pH 5.5. Eight of them (Fc-5-55, Fc-5-60, Fc-5-79, Fc-5-91, Fc-5-92, Fc-5-96, Fc-5-101, and Fc-5-112) showed only around 2∼5x improvement in binding at pH 5.5. These data further indicate that EC50's of the variant Fc-fragments for binding to human and cynomolgus monkey FcRn's are comparable. All variant Fc-fragments tested (other than Fc-5-1, which was considered as a control) maintained low binding to both human and cynomolgus monkey FcRn at pH 7.4.

    Example 8: Construction of further modifed variant Fc-fragments



    [0108] Further modified versions of variant Fc-fragments Fc-5-69 and Fc-5-106 were made. The variants of Fc-5-69 were made as follows. DNA encoding variant Fc-fragment Fc-5-69 was inserted into a mammalian expression vector that could also be propagated in E. coli, which was used as a template. For variant Fc-5-69-W1F, the following two primers were used: forward, GAG AGC AAT GGT GGT TGT TTC CCG CTG CAG GAC TAC (SEQ ID NO:497); and reverse, GTA GTC CTG CAG CGG GAA ACA ACC ACC ATT GCT CTC (SEQ ID NO:498). For Fc-5-69-W1Y, the following two primers were used: forward, GAG AGC AAT GGT GGT TGT TAC CCG CTG CAG GAC TAC (SEQ ID NO:499); and reverse, GTA GTC CTG CAG CGG GTA ACA ACC ACC ATT GCT CTC (SEQ ID NO:500). The Quikchange Site-Directed Mutagenesis Kit (Stratagene, 200518) protocol was used. The reaction mixture was 200 nM dNTPs, 100 nM primers, 1ng DNA template, 1µL DNA polymerase and water in a total volume of 50µL. The reaction was run at 95°C for 30 seconds, then 16 cycles of 95°C for 30 seconds, 55°C for 60 seconds, 68°C for 6 minutes, followed by 68°C for 10 minutes. Then 1µL of Dpnl was added, and the reaction was incubated at 37°C for 1 hour. Then 2µL of the mixture was used to transform 30µl of XL1-blue supercompetent cells (Stratagene) at 42°C for 45 seconds. Thereafter, 0.5 mL SOC was added, and the cells were incubated at 37°C for 1 hour on a shaker at 300 revolutions per minute (rpm). The transformed cells were spread on LB-ampicillin agar plates and incubated at 37°C overnight.

    [0109] Individual colonies were picked, and plasmid DNA was prepared and sequenced. The DNA sequences indicated that the variant Fc-fragments had the following inserted amino acid sequences, which differed by one amino acid from the insert in Fc-5-69: Fc-5-69-W1F, GGCFPLQDYCGG (SEQ ID NO:367); and Fc-5-69-W1Y, GGCYPLQDYCGG (SEQ ID NO:368). DNA encoding these variant Fc-fragments was introduced into 293 cells, and purified Fc-fragments were produced as described above for use in Biacore® binding assays performed as described above.

    [0110] Modified versions of Fc-5-106 were made by similar methods except that the template for the PCR reactions was a DNA encoding Fc-5-106 inserted into a mammalian expression vector. The primers used for the PCR reactions had the following sequences: Fc-5-106-M4A, forward, GGT GGT TGT GTT TTC AAC GCG TTC AAC TGT GGT GGT GGG (SEQ ID NO:501) and reverse, CCC ACC ACC ACA GTT GAA CGC GTT GAA AAC ACA ACC ACC (SEQ ID NO:502); Fc-5-106-M4G, forward, GGT GGT TGT GTT TTC AAC GGG TTC AAC TGT GGT GGT GGG (SEQ ID NO:503), and reverse, CCC ACC ACC ACA GTT GAA CCC GTT GAA AAC ACA ACC ACC (SEQ ID NO:504); Fc-5-106-M4H, forward, GGT GGT TGT GTT TTC AAC CAT TTC AAC TGT GGT GGT GGG (SEQ ID NO:505), and reverse, CCC ACC ACC ACA GTT GAA ATG GTT GAA AAC ACA ACC ACC (SEQ ID NO:506); Fc-5-106-M4I, forward, GGT GGT TGT GTT TTC AAC ATC TTC AAC TGT GGT GGT GGG (SEQ ID NO:507), and reverse, CCC ACC ACC ACA GTT GAA GAT GTT GAA AAC ACA ACC ACC (SEQ ID NO:508); Fc-5-106-M4L, forward, GGT GGT TGT GTT TTC AAC TTG TTC AAC TGT GGT GGT GGG (SEQ ID NO:509), and reverse, CCC ACC ACC ACA GTT GAA CAA GTT GAA AAC ACA ACC ACC (SEQ ID NO:510); Fc-5-106-M4N, forward, GGT GGT TGT GTT TTC AAC AAC TTC AAC TGT GGT GGT GGG (SEQ ID NO:511), and reverse, CCC ACC ACC ACA GTT GAA GTT GTT GAA AAC ACA ACC ACC (SEQ ID NO:512); Fc-5-106-M4Q, forward, GGT GGT TGT GTT TTC AAC CAG TTC AAC TGT GGT GGT GGG (SEQ ID NO:513), and reverse, CCC ACC ACC ACA GTT GAA CTG GTT GAA AAC ACA ACC ACC (SEQ ID NO:514); Fc-5-106-M4S, forward, GGT GGT TGT GTT TTC AAC TCG TTC AAC TGT GGT GGT GGG (SEQ ID NO:515), and reverse, CCC ACC ACC ACA GTT GAA CGA GTT GAA AAC ACA ACC ACC (SEQ ID NO:516); Fc-5-106-M4T, forward, GGT GGT TGT GTT TTC AAC ACG TTC AAC TGT GGT GGT GGG (SEQ ID NO:517), and reverse, CCC ACC ACC ACA GTT GAA CGT GTT GAA AAC ACA ACC ACC (SEQ ID NO:518); Fc-5-106-M4V, forward, GGT GGT TGT GTT TTC AAC GTG TTC AAC TGT GGT GGT GGG (SEQ ID NO:519), and reverse, CCC ACC ACC ACA GTT GAA CAC GTT GAA AAC ACA ACC ACC (SEQ ID NO:520).

    [0111] PCR reactions were performed as described above and used to transform E. coli. Plasmid DNAs from individual colonies were sequenced. Isolates encoding Fc-fragments having the following inserted sequences were selected: Fc-5-106-M4A, GGCVFNAFNCGG (SEQ ID NO:369); Fc-5-106-M4G, GGCVFNGFNCGG (SEQ ID NO:370); Fc-5-106-M4H, GGCVFNHFNCGG (SEQ ID NO:371); Fc-5-106-M4I, GGCVFNIFNCGG (SEQ ID NO:372); Fc-5-106-M4L, GGCVFNLFNCGG (SEQ ID NO:373); Fc-5-106-M4N, GGCVFNNFNCGG (SEQ ID NO:374); Fc-5-106-M4Q, GGCVFNQFNCGG (SEQ ID NO:375); Fc-5-106-M4S, GGCVFNSFNCGG (SEQ ID NO:376); Fc-5-106-M4T, GGCVFNTFNCGG (SEQ ID NO:377); Fc-5-106-M4V, GGCVFNVFNCGG (SEQ ID NO:378).

    [0112] These derivatives of Fc-5-69 and Fc-5-106 were made and tested for relative binding affinity to human and cynomolgus monkey FcRn at pH 5.5 and 7.4 using the methods described in Example 7.
    Table 5: Binding of variants of 5-69 and Fc 5-106 to human and cynomolgus monkey FcRn
     Human FcRn pH 5.5Human FcRn pH 7.4Cynomolgus FcRn pH 5.5Cynomolgus FcRn pH 7.4
    SampleEC50 (nM)95% CI (nM)EC50 (nM)95% CI (nM)EC50 (nM)95% CI (nM)EC50 (nM)95% CI (nM)
    Fc-WT 270 170∼440 >2000   250 130∼470 >2000  
    Fc-5-69 20 13∼30 1100 960∼1400        
    Fc-5-69- WIF 57 36∼89 >2000          
    Fc-5-69- WIY 140 75∼240 >2000          
    Fc-5-106 5.4 3.7∼8.0 1400 1100∼1700 4.6 3.5∼6.1 >2000  
    Fc-5-106- M4A 27 16∼45 >2000          
    Fc-5-106- M4G 100 66∼160 >2000          
    Fc-5-106- M4H 100 48∼230 >2000          
    Fc-5-106-M4I 2.6 1.8∼3.6 900 760∼1100 3.2 2.1∼4.8 970 810∼1200
    Fc-5-106-M4L 2.3 1.5∼3.4 650 460∼920 2.8 1.6∼4.7 710 460∼1100
    Fc-5-106- M4N 100 52∼180 >2000          
    Fc-5-106- M4Q 34 17∼68 >2000          
    Fc-5-106- M4S 58 32∼106 >2000          
    Fc-5-106- M4T 10 4.4∼21 >2000   8.3 5.3∼13 >2000  
    Fc-5-106- M4V 6.2 3.6∼11 >2000   5.0 3.4∼7.3 >2000  


    [0113] Both Fc-5-69 derivatives showed weaker binding affinity than Fc-5-69 itself at pH 5.5. Two Fc-5-106 derivatives (Fc-5-106-M4I and Fc-5-106-M4L) showed higher affinity binding to both human and cynomolgus FcRn at pH 7.4 compared to Fc-5-106. Also, four of the Fc-5-106 derivatives including these two (Fc-5-106-M4I, Fc-5-106-M4L, Fc-5-106-M4T and Fc-5-106-M4V) showed improved or approximately the same binding activity at pH 5.5 compared to Fc-5-106 itself. These four were tested for binding affinity to cynomolgus monkey FcRn. All four had EC50's for binding to cynomolgus monkey FcRn that were similar to those for human FcRn at both pH 5.5. and 7.4.

    Example 9: In vivo Characterization of variant Fc-fragments



    [0114] To determine whether antibodies containing the variant Fc-fragments identified above have improved pharmacokinetic (PK) properties in vivo, an unmodified Antibody X (which is a human IgG2 anti-human IL-23 antibody) and variant versions of Antibody X containing variant Fc-fragments were tested in vivo in cynomolgus monkeys to define pharmacokinetic parameters. Antibody X was selected as an appropriate antibody in which to test the pharmacokinetic parameters of the variant Fc-fragments because it was known to have a linear pK profile and because IL-23 was known to be expressed at low levels in vivo. Thus, it was expected that target-related effects on PK parameters would be minimal, making it easier to detect pK effects due to the variant Fc-fragments.

    [0115] Five variant IgG2 antibodies called (X-5-51, X-5-69, X-5-104, X-5-106, and X-5-112) were made. These IgG2 antibodies had the same insertions at the same positions (according to the alignment in Table1) as the variant IgG1 Fc-fragments Fc-5-51, Fc-5-69, Fc-5-104, Fc-5-106, and Fc-5-112, respectively. More specifically, the insertions were between amino acids 384 and 385 (EU numbering as in Table 1) in the human IgG2 Fc-fragment. A plasmid containing DNA encoding the heavy chain of Antibody X was used as a template for five PCR reactions done using the following primers: for X-5-51, forward, 5'-GAG TGG GAG AGC AAT GGT GGT TGT CAT CTG CCG TTC GCT GTT TGT GGT GGT GGG CAG CCG GAG AAC-3' (SEQ ID NO:539), and reverse, 5'-GTT CTC CGG CTG CCC ACC ACC ACA AAC AGC GAA CGG CAG ATG ACA ACC ACC ATT GCT CTC CCA CTC-3' (SEQ ID NO:540); for X-5-69,forward, 5'-GAG TGG GAG AGC AAT GGT GGT TGT TGG CCG CTG CAG GAC TAC TGT GGT GGT GGG CAG CCG GAG AAC-3' (SEQ ID NO:541), and reverse, 5'-GTT CTC CGG CTG CCC ACC ACC ACA GTA GTC CTG CAG CGG CCA ACA ACC ACC ATT GCT CTC CCA CTC-3' (SEQ ID NO:542); for X-5-104, forward, 5'-GAG TGG GAG AGC AAT GGT GGT TGT GGT CAT GAA TAC ATG TGG TGT GGT GGG CAG CCG GAG AAC-3' (SEQ ID NO:543), and reverse, 5'-GTT CTC CGG CTG CCC ACC ACA CCA CAT GTA TTC ATG ACC ACA ACC ACC ATT GCT CTC CCA CTC-3' (SEQ ID NO:544); for X-5-106, forward, 5'-GAG TGG GAG AGC AAT GGT GGT TGT GTT TTC AAC ATG TTC AAC TGT GGT GGT GGG CAG CCG GAG AAC-3' (SEQ ID NO:545), and reverse, 5'-GTT CTC CGG CTG CCC ACC ACC ACA GTT GAA CAT GTT GAA AAC ACA ACC ACC ATT GCT CTC CCA CTC-3' (SEQ ID NO:546); and for X-5-112, forward,5'-GAG TGG GAG AGC AAT GGT GGT TGT GCT CTG TAC CCG ACT AAC TGT GGT GGT GGG CAG CCG GAG AAC-3' (SEQ ID NO:547), and reverse, 5'-GTT CTC CGG CTG CCC ACC ACC ACA GTT AGT CGG GTA CAG AGC ACA ACC ACC ATT GCT CTC CCA CTC-3' (SEQ ID NO:548). The Quikchange Site-Directed Mutagenesis Kit (Stratagene, 200518) protocol was used. The reaction mixture was 200 nM dNTPs, 100 nM primers, 1 ng DNA template, 1 µL DNA polymerase and water in a total volume of 50 µL. The reaction was run at 95°C for 30 seconds, then 16 cycles of 95°C for 30 seconds, 55°C for 60 seconds, 68°C for 6 minutes, followed by 68°C for 10 minutes. Then 1 µL of DpnI was added, and the reaction was incubated at 37°C for 1 hour. Then 2 µL of the mixture was used to transform 30 µl of XL1-blue supercompetent cells (Stratagene) at 42°C for 45 seconds. Thereafter, 0.5 mL SOC was added, and the cells were incubated at 37°C for 1 hour on a shaker at 300 rpm. The transformed cells were spread on LB-ampicillin agar plates and incubated at 37°C overnight. Individual colonies were picked, and plasmid DNA was prepared and sequenced to ensure that the isolates chosen had the expected DNA sequence.

    [0116] Antibodies were prepared in essentially the same way as described above for Fc-fragments, except that the mammalian host cells were transfected with DNAs encoding both the IgG2 heavy chain, including a portion encoding either a variant or a control Fc-fragment, and the light chain of Antibody X. Host cells were incubated under conditions appropriate for expression of the antibodies, and the antibodies were recovered from the culture medium, purified as described above, and used for the following experiments.

    [0117] Cynomolgus monkeys (n=2/group) received a single intravenous dose of an unmodified or a variant version of Antibody X at a dose of 1 mg/kg and were followed during an 8-week in-life phase. Samples of blood were collected at specified time points over the course of the experiment. Samples were collected at pre-dose, 0.25, 1, 4, 8, 12, 24, 48, 72, 168, 240, 336, 408, 504, 576, 672, 744, 840, 1008, 1176 and 1344 hours post-dose.

    [0118] An anti-human IgG sandwich ELISA was used to determine systemic concentrations of the injected antibodies by comparison to a standard curve derived from the same molecule. Specifically, a mouse anti-human Fc antibody was diluted in PBS, coated onto plates, and incubated for 2 hours at room temperature. The well contents were discarded, and PBS-Tween 20 (SuperBlock®, Thermo Scientific) was added to the wells as a blocking buffer. After incubation for one hour at room temperature, the plate wells were washed with PBS-Tween-20, and serum samples were added to the wells and incubated for 1 hour with shaking. Wells were again washed, and a horseradish peroxidase labeled mouse anti-human Fc antibody was added to the plates. Following a one hour incubation, wells were washed and developed for 10 minutes using a 3,3',5,5' tetramethylbenzidine (TMB) substrate. The resulting colorimetric reaction was quenched with phosphoric or sulfuric acid added to the plate. Optical densities (ODs) were determined at 450 nm and 650nm, and the OD at 650 nm was subtracted from the OD at 450 nm. The conversion of OD values into concentrations for the study samples was achieved through data regression using a logistic model with weighting set to 1/Y2 in Watson LIMS version 7.0.0.04.

    [0119] Pharmacokinetic analysis was performed using the PK analytical package provided within Watson LIMS, version 7.0.0.04 by. Exposure (area under the curve (AUC)) and clearance (mL/kg/hr) values were derived from this analysis. Concentration vs. time data for the last 5 sampling time points for each cynomolgus monkey was used to calculate half-life (T½) values.

    [0120] As shown in Figure 4, variant versions of Antibody X containing variant Fc-fragments X-5-51, X-5-69, X-5-104, X-5-106 and X-5-112 all demonstrated higher mAb concentrations in cynomolgus monkeys at a given sampling time point as compared to unmodified Antibody X. As shown in Table 6, increased exposure values and decreased clearance values were demonstrated for all variant versions of Antibody X tested compared to unmodified Antibody X. In addition, four of the five variant versions of Antibody X had increased half lives. The one variant that did not exhibit an increased half life (X-5-106), may represent a situation where anti-drug antibodies against the injected antibody may have developed in one monkey, since data from one of the two monkeys tested indicated a very short half life (48 hours), whereas data from the other monkey indicated an increased half life (538 hours). Further experimentation to measure the presence of anti-drug antibodies could clarify this issue.
    Table 6 : Mean values for half-life, exposure, and clearance
     Unmodified Antibody XX- 5-51X-5-69X-5-104X-5-106X-5-112
    T ½ (hours) 335 910 550 477 191 520
    AUC - exposure (ug*hr/mL) 3890 6640 4960 5745 6005 7810
    Clearance (mL/kg/hr) 0.247 0.109 0.173 0.153 0.154 0.108


    [0121] Generally, these data indicate that increased binding of a variant Fc-fragment to FcRn at pH 5.5-6.0 (relative to a control Fc-fragment) and rapid dissociation from FcRn at pH 7.4 correlate with a longer in vivo half-life of an antibody containing the variant Fc-fragment in cynomolgus monkeys. However, an exact quantitative relationship between the degree of improvement in binding at pH 5.5 or 6 of an IgG1 variant Fc-fragment and the degree of improvement in pharmacokinetic parameters for a full length IgG2 antibody having the same insertion as the variant Fc-fragment is not shown by these data. These data do, however, indicate that X-5-51, X-5-69, X-5-104, and X-5-112 have increased half lives relative to an unaltered Antibody X and suggest that the same is true for X-5-106. Moreover, all variant antibodies tested had lower clearance rates and higher exposure as compared to the control antibody.

    Example 10: Construction of variant Fc-fragments with insertions at alternate sites



    [0122] The fact that many variants from library L5 had desirable properties indicated that the position of these insertions was favorable. The following experiments were done to determine whether other sites within or adjacent to the same loop as the L5 insertion site might have better properties. To test this idea, one of the selected peptides was inserted at different locations in this loop, and the resulting variant Fc-fragments were tested for FcRn binding. The peptide insertion of variant Fc-fragment 5-1 was chosen as the peptide to insert. This peptide has the following amino acid sequence: GGCGMPIEFCGG (SEQ ID NO:67). As shown in Figure 5 (which uses the EU numbering system as exemplified in Table 1), this peptide was inserted at sites within or adjacent to the library L5 loop other than the library L5 insertion site. Binding of the resulting Fc-fragments to FcRn was assayed using SA biosensors (ForteBio Inc. 18-5019) coated biotinylated huFcRn as described in Example 4 above.

    [0123] In more detail, the DNA constructs encoding these variant Fc-fragments were made as follows. The Quikchange® Site-Directed Mutagenesis Kit (Stratagene, 200518) protocol was used. The reaction mixture was composed of 200 nM dNTPs, 100 nM primers, 1 ng DNA template, 1 µL DNA polymerase and water to a total volume of 50 µL. The primers used in these reactions for each variant are shown below in Table 7. The DNA template was a cDNA encoding a wild type human IgG1 Fc-polypeptide inserted into a vector. The reaction was run at 95°C for 30 seconds, then 16 cycles of 95 °C for 30 seconds, 55°C for 60 seconds, and 68°C for 6 minutes, followed by a final cycle of 68°C for 10 minutes. Then 1 µL of Dpnl was then added, and the reaction was incubated at 37°C for 1 hour. Then 2 µL of the mixture was used to transform 30 µL of XL1-blue supercompetent E. coli cells (Stratagene) at 42°C for 45 seconds. Thereafter, 0.5 mL Super Optimal Broth with Catabolite repression (SOC; containing 2% bacto-tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCI, 10 mM MgCl2, and 20 mM glucose) was added, and the cells were incubated at 37°C for 1 hour on a shaker at 300 revolutions per minute (rpm). The transformed cells were spread on LB-ampicillin agar plates and incubated at 37°C overnight.
    Table 7: Primers used in PCR reactions for construction variant Fc-fragment-encoding DNAs
    Variant Fc-fragment designationForward PrimerReverse Primer
    5-1-1

     


     
    5-1-2

     


     
    5-1-3

     


     
    5-1-4

     


     
    5-1-5

     


     
    5-1-6

     


     
     

     


     
    5-1-7

     


     
    5-1-8

     


     
    5-1-9

     


     
    5-1-10

     


     


    [0124] Individual colonies were picked, and plasmid DNA was prepared and sequenced. DNA encoding these variant Fc-fragments was introduced into 293-6E cells, and Fc-fragments in conditioned media (CM) from these cells were used in ForteBio binding assays performed as described above. See Example 4.

    [0125] The results for binding at pH 6 and dissociation at pH 7 are shown in Figure 6. Two of the variant Fc-fragments, 5-1-10 and 5-1-2, had a greater maximal response than variant 5-1 at pH 6, but also dissociated more slowly at pH 7.4. See Figure 6. All other constructs showed a somewhat lesser maximal response, as compared to variant 5-1, at pH 6, but all of these responses were comparable to or higher than that of a wild type Fc-fragment (designated FcWT in Figure 6). Further, dissociation of these constructs at pH 7.4, unlike that of 5-1, was comparable to that of FcWT. Thus, most of the Fc variants, other than 5-1-2 and 5-1-10 (both of which have an insertion between positions 383 and 384), with insertions within or adjacent to Loop 10 dissociated faster at pH 7.4 than variant 5-1. Also, variants 5-1-1, 5-1-2, 5-1-3, 5-1-9, and 5-1-10 clearly had higher maximal responses that did FcWT at pH 6 variant 5-1, whereas variants 5-1-6, 5-1-7, and 5-1-8 had maximal responses comparable to that of FcWT. Variants 5-1-4 and 5-1-5 had only marginally higher responses than did FcWT. These data indicate that insertion of the peptide had more favorable effects at certain sites within Loop 10. Specifically, insertion between positions 382 and 383, 383 and 384, 384 and 385, 385 and 386 (using the EU numbering system as illustrated in Table 1 and Figure 5) produced Fc variants that had a greater maximal response at pH 6 than did FcWT plus rapid dissociation at pH 7.4. In contrast, Fc variants having insertions between positions 388 and 389, 389 and 390, or 390 and 391 had properties similar to FcWT. Finally, Fc variants having insertions between 386 and 387 or 387 and 388 showed marginally higher responses at pH 6 than Fc WT and rapid dissociation at pH 7.4. In addition, removal of amino acids 384-386 and insertion of the peptide in their place, as was done in variants 5-1-9 and 5-1-10, also improved the properties of the these Fc variants as compared to FcWT. The slightly longer insertion of 5-1-10, which included three rather than the two glycine residues as in 5-1-9 at the beginning and end of the insertion, had a higher response than any other variant, including 5-1-2, which had an insertion (with no accompanying deletion) between positions 383 and 384. These data indicate that insertions of a peptide that can enhance binding to FcRn within the region between positions 382 and 387 can enhance binding of an Fc-fragment to FcRn at pH 6 while preserving its fast dissociation from FcRn at pH 7.4. On the other hand, insertion of such a peptide between positions 388 and 391 did not substantially enhance binding activity and/or affinity of an Fc-fragment to FcRn at pH 6 as compared to FcWT and also had little or no effect on dissociation at pH 7.4. Thus, these data point to a portion of Loop 10, that is, from position 382 to 386, 387, or 388 (EU numbering), as containing particularly favorable sites for making insertions that can enhance binding activity and/or affinity to FcRn at pH 6.

    Example 11: Selection of variant Fc-fragment by yeast display



    [0126] To obtain a different group of variant Fc-polypeptides, a selection was performed using yeast display. To make libraries for screening in yeast, three previously made libraries were used as starting points. One of these was library L5, which is described above. See Figure 2 and Example 2. The two other libraries had insertions at exactly the same site as library L5 and were made using the same general methods that were used to make library L5, but the format of the insertions differed in that library L5 encoded insertions with six randomized amino acids while libraries L-8 and L-10 encoded insertions with eight and ten randomized amino acids, respectively. Specifically, these libraries encoded insertions with the following formats: library L-8, GGC(19R)8CGG (SEQ ID NO:88); and library L-10, GGC(19R)10CGG (SEQ ID NO:89). These insertions, like those in library L5, are between the "N" at position 169 and the "G" at position 170 according to the numbering scheme in Figure 2. The "(19R)8" and "(19R)10" indicate eight and ten, respectively, randomized amino acids, which can be any amino acid other than cysteine.

    [0127] Libraries L5, L-8 and L-10 were digested with SaclI and NotI, and the resulting fragments, which encoded the variant Fc-fragments, were purified and ligated into a vector appropriate for expression in both Escherichia coli and Saccharomyces cerevisiae. The vector also encoded an HA tag (a short peptide sequence (YPYDVPDYA)(SEQ ID NO:575) from human influenza hemagglutinin) in frame with the insertion, which was used to confirm that the Fc regions were displayed on the surface of the yeast cells. These three new libraries were introduced into E. coli, specifically, into XL1-Blue supercompetent E. coli cells. DNA from about 5 x 108 E. coli transformants was used to transform about 1-2 x 109 S. cerevisiae cells using the standard lithium acetate method. An overnight culture of S. cerevisiae was diluted to an OD600 of 0.2-0.3 in 100 milliliters of yeast extract peptone dextrose medium (which contains 20 g/L bacto-peptone, 10 g/L yeast extract, 2% glucose (YPD)) and grown at 30°C shaking at 300 revolutions per minute (rpm) until the OD600 reached 1.0-2.0. The cells were then washed with 30 milliliters of water and with 30 milliliters of 100 mM LiOAc. For each library, a transformation mixture containing 1M LiOAc, 50% PEG, single stranded carrier DNA, water, and 25 ug of library DNA was added to cells. Each transformation was heat shocked at 42°C for 45 minutes. The cells were pelleted and then grown in 50 milliliters YPD medium at 30°C for 1 hour. Cells were pelleted again and washed with 30 milliliters SD-leu medium (14.7g/L sodium citrate, 4.29g/L citric acid, 2% dextrose, 6.7g/L yeast nitrogen base (YNB), 1.6 g/L Yeast Synthetic Drop-out Medium Supplements without Leucine (Sigma, Y1376)). The cells were then resuspended in 10 milliliters of SD-leu media. The 10 milliliters of transformed cells were then inoculated into 300 milliliters SD-leu media and grown overnight at 30°C.

    [0128] Induction of expression of the variant Fc-fragments in the yeast transformants was accomplished by inoculating an aliquot of an overnight culture (about 108 cells) into induction medium (5.4g/L Na2HPO4, 8.56 g/L NaH2PO4, 2% galactose, 6.7 g/L YNB, and 1.6 g/L Yeast Synthetic Drop-out Medium Supplements without Leucine (Sigma, Y1376)) and culturing the cells for about 48 hours at 20 °C.

    [0129] These induced cells were spun down and resuspended in phosphate buffered saline (PBS) buffer (+0.5% BSA) at pH 7.4. Then 0.5-1 x 108 induced cells were incubated for one hour with 1 µM biotinylated FcRn (biotin-FcRn). Cells were then washed and incubated with 2 µg/mL labeled streptavidin (streptavidin (SA)-Alexa Fluor® 647 (Catalog number S32357, Invitrogen)) for 15 minutes. Cells were then washed and incubated with anti-Cy5/Anti-Alexa Fluor® 647 Microbeads (Catalog number 130-091-395, MACS Miltenyi Biotec) for 15 minutes. Cells were washed and separated using LS Columns (Catalog number 130-042-401, MACS Miltenyi Biotec) and a QuadroMACS® Separation Unit (Catalog number 130-091-051, MACS Miltenyi Biotec). Aliquots of the pre-column, flow-thru, washes, and elution fractions were collected for FACS analysis. Flow-thru and washes were collected as our depleted population, which contained cells expressing variant Fc-polypeptides that bound to FcRn with very low affinity at pH 7.4.

    [0130] This depleted population of cells was then grown overnight at 30 °C in SD-leu medium. Cells were then induced for about 48 hours at 20 °C in induction medium as described above. The subsequent wash and incubation steps were performed in MES buffer (20 mM MES, 137 mM NaCl, 0.5% BSA) at pH 5.5. Induced cells were incubated on ice with 250 nM biotin-FcRn for one hour. Subsequently, SA-Alexa Fluor® 647 (2 µg/mL) and anti-HA-FITC (1 µg/mL) were added, and, after being washed and put through a cell strainer, the cells were subjected to FACS analysis at pH 5.5. Gates were set to collect cells that exhibited a robust signal for both FITC and Alexa Fluor®. These cells were then cultured overnight at 30 °C in SD-leu medium and then induced for about 48 hours at 20 °C in induction medium. Then, in a second round, the induced cells were incubated on ice with 25 nM biotin-FcRn for one hour, and the labeling, washing, straining, FACS, culturing, and inductions steps, as described above, were repeated. Finally, in a third round, the induced cells were incubated on ice with 5 nM biotin-FcRn for one hour, followed by the labeling, washing, straining, FACS and culturing steps described above. Hence, the end result was a population of cells expressing Fc-fragments that could bind FcRn with higher affinity than a wild type Fc-fragment could at pH 5.5.

    [0131] This population of cells was plated, and 500 colonies from Library L5 and 400 colonies from each of Libraries L-8 and L-10 were selected for further analysis. Each of these individual colonies was cultured at 30 °C overnight in SD-leu medium as described above. The sequences of the insertions in each of these isolates was determined from yeast plasmid DNA. In all, 317, 265, and 313 unique sequences were found among these isolates in each of libraries L5, L-8, and L-10, respectively. Cells were then induced at 20 °C for 48 hours in the induction medium described above. The subsequent wash and incubation steps were performed in parallel in MES buffer (20mM MES, 137mM NaCl, 0.5% BSA) at pH 5.5 and in PBS buffer (+0.5% BSA) at pH 7.4. About 5 x 105 cells per sample were incubated with 250 nM biotin-FcRn and 1 µg/ml anti-HA-FITC for 1 hour. Then cells were washed and incubated with 2 µg/mL SA-Alexa Fluor® 647 for 15 minutes. Cells were washed and analyzed for binding via FACS to determine whether they bound FcRn strongly at pH 5.5 and weakly at pH 7.4. Individual cell lines showing the highest signals for binding to FcRn at pH 5.5, along with a low signal for binding to FcRn at pH 7.4 and few or no methionine or tryptophan residues, were chosen from each library for further analysis. These selected isolates included 158, 59, and 50 cell lines from L5, L-8, and L-10, respectively, and the sequences of the insertions in these isolates are shown in Table 8 below. (Note that the clone named 5y-37 was not included in the further analysis because it was not re-cloned successfully into the appropriate vector.)
    Table 8: Sequences of variant Fc-polypeptides that bind with high affinity at pH 5.5 and low affinity at pH 7.4
    LIBRARY L5LIBRARY L-8LIBRARY L-10
    CloneINSERTION SEQ (SEQ ID NO)CloneINSERTION SEQ (SEQ ID NO)CloneINSERTION SEQ (SEQ ID NO)
    5y-1 GGCHFDIMNCGG (SEQ ID NO:90) 8y-1 GGCKTWQLLIFCGG (SEQ ID NO:248) 10y-1 GGCAFEFSSAFNCGG (SEQ ID NO:307)
    5y-2 GGCVIDFFGCGG (SEQ ID NO:91) 8y-2 GGCEQNWTLYMCGG (SEQ ID NO:249) 10y-2 SGCQTMLTAEGEWCGG (SEQ ID NO:308)
    5y-3 GGCDIMIFECGG (SEQ ID NO:92) 8y-3 SGCWPSPYIFPCGG (SEQ ID NO:250) 10y-3 GGCVMDLWPDLEICGG (SEQ ID NO:309)
    5y-4 GGCMTEFAICGG (SEQ ID NO:93) 8y-4 GGCWEALQVNNCGG (SEQ ID NO:251) 10y-4 GGCQPLFDDHDTWCGG (SEQ ID NO:310)
    5y-5 GGCIQYWQFCGG (SEQ ID NO:94) 8y-5 CGCQAMVVEDLCG (SEQ ID NO:252) 10y-5 GGCPFELVMSDEQCGG (SEQ ID NO:311)
    5y-6 GGCPFSWAFCGG (SEQ ID NO:95) 8y-6 GGCPLEWPRISCGG (SEQ ID NO:253) 10y-6 SGCGHGMQMDSVFCGG (SEQ ID NO:312)
    5y-7 GGCGFAFMYCGG (SEQ ID NO:96) 8y-7 GGCEPWIMEANCGG (SEQ ID NO:254) 10y-7 SGCDETQSAIWYFCGG (SEQ ID NO:313)
    5y-8 GGCPVLLFNCGG (SEQ ID NO:97) 8y-8 GGCPWDQHINFCGG (SEQ ID NO:255) 10y-8 GGCREPEQYWTVWCGG (SEQ ID NO:314)
    5y-9 GGCPFTWTKCGG (SEQ ID NO:98) 8y-9 GGCPWYIQMDHCGG (SEQ ID NO:256) 10y-9 SGCQEKKDLYWEYCGG (SEQ ID NO:315)
    5y-10 GGCYLYWQFCGG (SEQ ID NO:99) 8y-10 SGCQPWEISYYCGG (SEQ ID NO:257) 10y-10 SGCGQDNDLPWEWCGG (SEQ ID NO:316)
    5y-11 GGCVINMFPCGG (SEQ ID NO:100) 8y-11 GGCPVMFLDPRCGG (SEQ ID NO:258) 10y-11 GGCVFQLSFSRSDCGG (SEQ ID NO:317)
    5y-12 GGCPFTWNTCGG (SEQ ID NO:101) 8y-12 GGCSSDVLMIFCGG (SEQ ID NO:259) 10y-12 SGCAFDMIWFEGVCGG (SEQ ID NO:318)
    5y-13 GGCPFQIGECGG (SEQ ID NO:102) 8y-13 GGCVDEMVIYHCGG (SEQ ID NO:260) 10y-13 SGCAFYWQPWEHSCGG (SEQ ID NO:319)
    5y-14 GGCLDIIWMCGG (SEQ ID NO:103) 8y-14 GGCPFMVNLYSCGG (SEQ ID NO: :261) 10y-14 GGCQLSIILTGLPCGG (SEQ ID NO:320)
    5y-15 GGCMFVFPACGG (SEQ ID NO:104) 8y-15 GGCESDTMWYFCGG (SEQ ID NO:262) 10y-15 GGCGMLEWSGLQFCGG (SEQ ID NO:321)
    5y-16 GGCVMEQLWCGG (SEQ ID NO:105) 8y-16 GGCRSDEIIFFCGG (SEQ ID NO:263) 10y-16 SGCHEKALTYWEFCGG (SEQ ID NO:322)
    5y-17 GGCFKEYTWCGG (SEQ ID NO:106) 8y-17 GGCPWDLLLPLCGG (SEQ ID NO:264) 10y-17 GGCFENMQVWYNECGG (SEQ ID NO:323)
    5y-18 GGCPKDYHICGG (SEQ ID NO:107) 8y-18 GGCPWAMELVHCGG (SEQ ID NO:265) 10y-18 GGCPEWENQILLFCGG (SEQ ID NO:324)
    5y-19 GGCEVMVFPCGG (SEQ ID NO:108) 8y-19 SGCTASMYWEYCGG (SEQ ID NO:266) 10y-19 SGCESWQRDMNYFCGG (SEQ ID NO:325)
    5y-20 GGCVFNTVFCGG (SEQ ID NO:109) 8y-20 GGCGLYMDPPYYGG (SEQ ID NO:267) 10y-20 SGCNDQFPMYYLFCGG (SEQ ID NO:326)
    5y-21 GGCNLPQEWCGG (SEQ ID NO:110) 8y-21 GGCPVMVMEPYCGG (SEQ ID NO:268) 10y-21 GGCFEDMALQPTQCGG (SEQ ID NO:327)
    5y-22 GGCMIAPMYCGG (SEQ ID NO:111) 8y-22 GGCQTEFILEFCGG (SEQ ID NO:269) 10y-22 SGCKGPWQFEFLVCGG (SEQ ID NO:328)
    5y-23 GGCMMLYPMCGG (SEQ ID NO:112) 8y-23 SGCAFQAHGAMCGG (SEQ ID NO:270) 10y-23 GGCEAFSMKFNDFCGG (SEQ ID NO:329)
    5y-24 GGCIIGPFLCGG (SEQ ID NO:113) 8y-24 GGCPDFMFRMNCGG (SEQ ID NO:271) 10y-24 GGCVQPAIAMWPFCGG (SEQ ID NO:330)
    5y-25 GGCTGMVFFCGG (SEQ ID NO:114) 8y-25 SGCSVWFDTISCGG (SEQ ID NO:272) 10y-25 GGCTDQGRFVLYECGG (SEQ ID NO:331)
    5y-26 GGCLMYKNVCGG (SEQ ID NO:115) 8y-26 GGCPWSMEISNCGG (SEQ ID NO:273) 10y-26 GGCPVQEFLWGVYCGG (SEQ ID NO:332)
    5y-27 GGCAFGIMWCGG (SEQ ID NO:116) 8y-27 GGCPTWNWEITCGG (SEQ ID NO:274) 10y-27 GGCSNSWEWTLYACGG (SEQ ID NO:333)
    5y-28 GGCRHRKKWCGG (SEQ ID NO:117) 8y-28 SGCPWDMHIVDCGG (SEQ ID NO:275) 10y-28 SGCHGLVEWGYMACGG (SEQ ID NO:334)
    5y-29 GGCFMGIWQCGG (SEQ ID NO:118) 8y-29 SGCFPWEPAYFCGG (SEQ ID NO:276) 10y-29 SGCEAFGLIFEDFCGG (SEQ ID NO:335)
    5y-30 GGCPSLPQFCGG (SEQ ID NO:119) 8y-30 GGCPFGWNVFHCGG (SEQ ID NO:277) 10y-30 GGCANPEFQMWYFCGG (SEQ ID NO:336)
    5y-31 GGCPSVFTWCGG (SEQ ID NO:120) 8y-31 GGCPWHMEVNECGG (SEQ ID NO:278) 10y-31 SGCGYEVPIPLFTCGG (SEQ ID NO:337)
    5y-32 GGCQEYWEFCGG (SEQ ID NO:121) 8y-32 GGCPFALGMGECGG (SEQ ID NO:279) 10y-32 GGCWFQQFAWRATCGG (SEQ ID NO:338)
    5y-33 GGCQWPTEFCGG (SEQ ID NO:122) 8y-33 GGCMFPFMLSNCGG (SEQ ID NO:280) 10y-33 GGCGFELNMISQYCGG (SEQ ID NO:339)
    5y-34 GGCIKFFDWCGG (SEQ ID NO:123) 8y-34 GGCAFQFMPAHCGG (SEQ ID NO:281) 10y-34 GGCEPFELRFYHEGCGG (SEQ ID NO:340)
    5y-35 GGCEMSFFLCGG SEQ ID NO:124) 8y-35 GGCQIQGFEFTCGG (SEQ ID NO:282) 10y-35 GGCPFQL VWSPAFCGG (SEQ ID NO:341)
    5y-36 GGCHSEVEYCGG (SEQ ID NO:125) 8y-36 GGCPMGIILDLCGG (SEQ ID NO:283) 10y-36 SGCAWEIKGIWCGG (SEQ ID NO:342)
    5y-37 GGCWEHPHYCGG (SEQ ID NO:126) 8y-37 GGCLMLEPTVTCGG (SEQ ID NO:284) 10y-37 SGCSSIQSWRLWLCGG (SEQ ID NO:343)
    5y-38 GGCETYWLFCGG (SEQ ID NO:127) 8y-38 GGCGKNEVAMFCGG (SEQ ID NO:285) 10y-38 GGCGVMQVLNRAHCGG (SEQ ID NO:344)
    5y-39 GGCRVPYPSCGG (SEQ ID NO:128) 8y-39 GGCSFLLEIANCGG (SEQ ID NO:286) 10y-39 RGCQVKYYMGEGDCGG (SEQ ID NO:345)
    5y-40 GGCGWPFVMCGG (SEQ ID NO:129) 8y-40 GGCDVEKIMIFCGG (SEQ ID NO:287) 10y-40 GGCPVWIPFHWEECGG (SEQ ID NO:346)
    5y-41 GGCMLFLESCGG (SEQ ID NO:130) 8y-41 GGCFPMTPWGLCGG (SEQ ID NO:288) 10y-41 SGCLLWQQSMLLFCGG (SEQ ID NO:347)
    5y-42 GGCFHVKRWCGG (SEQ ID NO:131) 8y-42 SGCDWYLEWSGNCGG (SEQ ID NO:289) 10y-42 SGCEQQWSWRLYLCGG (SEQ ID NO:348)
    5y-43 GGCVWEQEHCGG (SEQ ID NO:132) 8y-43 GGCGVEIMFHGCGG (SEQ ID NO:290) 10y-43 GGCSVQSTWQLWACGG (SEQ ID NO:349)
    5y-44 GGCILHFKDCGG (SEQ ID NO:133) 8y-44 GGCMDGLHLYFCGG (SEQ ID NO:291) 10y-44 SGCKYPIFWDTIDCGG (SEQ ID NO:350)
    5y-45 GGCHFEVFQCGG (SEQ ID NO:134) 8y-45 SGCPIFIFDYYCGG (SEQ ID NO:292) 10y-45 SGCVEYQYQMVYFCGG (SEQ ID NO:351)
    5y-46 GGCVFEVMQCGG (SEQ ID NO:135) 8y-46 GGCAVWIFSDACGG (SEQ ID NO:293) 10y-46 GGCTDQRWFVLYECGG (SEQ ID NO:352)
    5y-47 GGCMTEFSWCGG (SEQ ID NO:136) 8y-47 GGCPWSLHIQQCGG (SEQ ID NO:294) 10y-47 GGCPFWQEWHLSYCGG (SEQ ID NO:353)
    5y-48 GGCEGNMRFCGG (SEQ ID NO:137) 8y-48 SGCAFSMLFINCGG (SEQ ID NO:295) 10y-48 SGCYMGYMHLIAECGG (SEQ ID NO:354)
    5y-49 GGCKGHMWYCGG (SEQ ID NO:138) 8y-49 SGCLPWELYMFCGG (SEQ ID NO:296) 10y-49 GGCFMGSFSLVYGCGG (SEQ ID NO:355)
    5y-50 GGCEAYWQFCGG (SEQ ID NO:139) 8y-50 SGCPFTINFYTCGG (SEQ ID NO:297) 10y-50 SGCPWGFMFPISYCGG (SEQ ID NO:356)
    5y-51 GGCVFSRFWCGG (SEQ ID NO:140) 8y-51 GGCPIWFTWSTCGG (SEQ ID NO:298)    
    5y-52 GGCMMPFWPCGG (SEQ ID NO:141) 8y-52 GGCQIQVVNPYCGG (SEQ ID NO:299)    
    5y-53 GGCIFQFEMCGG (SEQ ID NO:142) 8y-53 GGCAFQIEFLMCGG (SEQ ID NO:300)    
    5y-54 GGCKRQMWYCGG (SEQ ID NO:143) 8y-54 GGCAWEIRILGCGG (SEQ ID NO:301)    
    5y-55 GGCKTPNPWCGG (SEQ ID NO:144) 8y-55 GGCPYQLVIMWCGG (SEQ ID NO:302)    
    5y-56 GGCKAFYPWCGG (SEQ ID NO:145) 8y-56 GGCMFAMHVFGCGG (SEQ ID NO:303)    
    5y-57 GGCKMYQYDCGG (SEQ ID NO:146) 8y-57 SGCTVMYTLQIFGG (SEQ ID NO:304)    
    5y-58 GGCYPDNMFCGG (SEQ ID NO:147) 8y-58 SGCAHQVYWAFCGG (SEQ ID NO:305)    
    5y-59 GGCQVKIFWCGG (SEQ ID NO:148) 8y-59 GGCPNFFNFWFCGG (SEQ ID NO:305)    
    5y-60 GGCSIPQEWCGG (SEQ ID NO:149)        
    5y-61 GGCKMYQATCGG (SEQ ID NO:150)        
    5y-62 GGCQYERWHCGG (SEQ ID NO:151)        
    5y-63 GGCRFQHQWCGG (SEQ ID NO:152)        
    5y-64 GGCQNMFWQCGG (SEQ ID NO:153)        
    5y-65 GGCVMEIVFCGG (SEQ ID NO:154)        
    5y-66 GGCILNFNMCGG (SEQ ID NO:155)        
    5y-67 GGCMHMDYFCGG (SEQ ID NO:156)        
    5y-68 GGCQVMVLPCGG (SEQ ID NO:157)        
    5y-69 GGCLFDWPSCGG (SEQ ID NO:158)        
    5y-70 GGCKMYHQTCGG (SEQ ID NO:159)        
    5y-71 GGCQWLYESCGG (SEQ ID NO:160)        
    5y-72 GGCFTNFWLCGG (SEQ ID NO:161)        
    5y-73 GGCWEPTHWCGG (SEQ ID NO:162)        
    5y-74 GGCAFAMLQCGG (SEQ ID NO:163)        
    5y-75 GGCMYQRQAWCGG (SEQ ID NO:164)        
    5y-76 GGCPFLWAECGG (SEQ ID NO:165)        
    5y-77 GGCMFDHKVCGG (SEQ ID NO:166)        
    5y-78 GGCMEIFNFCGG (SEQ ID NO:167)        
    5y-79 GGCVMERLWCGG (SEQ ID NO:168)        
    5y-80 GGCEYYWQFCGG (SEQ ID NO:169)        
    5y-81 GGCPFSWDQCGG (SEQ ID NO:170)        
    5y-82 GGCIEYFSWCGG (SEQ ID NO:171)        
    5y-83 GGCVFEIMKCGG (SEQ ID NO:172)        
    5y-84 GGCESPQYFCGG (SEQ ID NO:173)        
    5y-85 GGCHHDFEWCGG (SEQ ID NO:174)        
    5y-86 GGCMFPFSWCGG (SEQ ID NO:175)        
    5y-87 GGCNTVLQECGG (SEQ ID NO:176)        
    5y-88 GGCVFDIMLCGG (SEQ ID NO:177)        
    5y-89 GGCMYQQPWCGG (SEQ ID NO:178)        
    5y-90 GGCKKLYHYCGG (SEQ ID NO:179)        
    5y-91 GGCPHWPFECGG (SEQ ID NO:180)        
    5y-92 GGCPIFPMICGG (SEQ ID NO:181)        
    5y-93 GGCMSKDLWCGG (SEQ ID NO:182)        
    5y-94 GGCMFQMGVCGG (SEQ ID NO:183)        
    5y-95 GGCYEWPSYCGG (SEQ ID NO: 184)        
    5y-96 GGCQMLYMDCGG (SEQ ID NO:185)        
    5y-97 GGCTVQVFFCGG (SEQ ID NO:186)        
    5y-98 GGCITFPMMCGG (SEQ ID NO:187)        
    5y-99 GGCVMYWEYCGG (SEQ ID NO:188        
    5y-100 GGCMWEVLHCGG (SEQ ID NO:189)        
    5y-101 GGCMQERSWCGG (SEQ ID NO:190)        
    5y-102 GGCVFETIQCGG (SEQ ID NO:191)        
    5y-103 GGCQWANSYCGG (SEQ ID NO:192)        
    5y-104 GGCKFGQWYCGG (SEQ ID NO:193        
    5y-105 GGCVFDQMWCGG (SEQ ID NO:194)        
    5y-106 GGCEVMIFNCGG (SEQ ID NO:195)        
    5y-107 GGCESPMFVCGG (SEQ ID NO:196)        
    5y-108 GGCITMFQNCGG (SEQ ID NO:197)        
    5y-109 GGCVFERMFCGG (SEQ ID NO:198)        
    5y-110 GGCGFEIFMCG (SEQ ID NO:199)        
    5y-111 GGCLLQFTGCGG (SEQ ID NO:200)        
    5y-112 GGCHFQIFQCGG (SEQ ID NO:200)        
    5y-113 GGCPFDWDKCGG (SEQ ID NO:202)        
    5y-114 GGCVTPLPFCG (SEQ ID NO:203)        
    5y-115 GGCYMYMDYCGG (SEQ ID NO:204)        
    5y-116 GGCMFEWYVCGG (SEQ ID NO:205)        
    5y-117 GGCPFTWRICGG (SEQ ID NO:206)        
    5y-118 GGCENDWKMCGG (SEQ ID NO:207)        
    5y-119 GGCAFEFIYCGG (SEQ ID NO:208)        
    5y-120 GGCPVAVFMCGG (SEQ ID NO:209)        
    5y-121 GGCHFDIFDCGG (SEQ ID NO:210)        
    5y-122 GGCPPENMFCGG (SEQ ID NO:211)        
    5y-123 GGCPFQMGECGG (SEQ ID NO:212)        
    5y-124 GGCISGFFWCGG (SEQ ID NO:213)        
    5y-125 GGCPFHFQVCGG (SEQ ID NO:214)        
    5y-126 GGCMFQIINCGG (SEQ ID NO:215)        
    5y-127 GGCQYFLPCGG (SEQ ID NO:216)        
    5y-128 GGCHFAVLDCGG (SEQ ID NO:217)        
    5y-129 GGCWNVMGLCGG (SEQ ID NO:218)        
    5y-130 GGCYTTHELCGG (SEQ ID NO:219)        
    5y-131 GGCLYKQVDCGG (SEQ ID NO:220)        
    5y-132 GGCVFSALWCGG (SEQ ID NO:221)        
    5y-133 GGCPFQFQTCGG (SEQ ID NO:222)        
    5y-134 GGCAFLMMDCGG (SEQ ID NO:223)        
    5y-135 GGCEVWYEFCGG (SEQ ID NO:224)        
    5y-136 GGCAFDIGVCGG (SEQ ID NO:225)        
    5y-137 GGCLSPLMWCGG (SEQ ID NO:226)        
    5y-138 GGCPFSWVICGG (SEQ ID NO:227)        
    5y-139 GGCMLMFQGCGG (SEQ ID NO:228)        
    5y-140 GGCQPNHWLCGG (SEQ ID NO:229)        
    5y-141 GGCIDTYVWCGG (SEQ ID NO:230)        
    5y-142 GGCHFHLMFCGG (SEQ ID NO:231)        
    5y-143 GGCQMIFSTCGG (SEQ ID NO:232)        
    5y-144 GGCKMYQPDCGG (SEQ ID NO:233)        
    5y-145 GGCMWGVFKCGG (SEQ ID NO:234)        
    5y-146 GGCGLFGQSCGG (SEQ ID NO:235)        
    5y-147 GGCQFNFPWCGG (SEQ ID NO:236)        
    5y-148 GGCNIAYPWCGG (SEQ ID NO:237)        
    5y-149 GGCKTIPIFCGG (SEQ ID NO:238)        
    5y-150 GGCQMELFLCGG (SEQ ID NO:239)        
    5y-151 GGCLGAFYWCGG (SEQ ID NO:240)        
    5y-152 GGCPFNFASCGG (SEQ ID NO:241)        
    5y-153 GGCQFDILWCGG (SEQ ID NO:242)        
    5y-154 GGCYYTHELCGG (SEQ ID NO:243)        
    5y-155 GGCQQRWRYCGG (SEQ ID NO:244)        
    5y-156 GGCLWVDEYCGG (SEQ ID NO:245)        
    5y-157 GGCGMLGWFCGG (SEQ ID NO:246)        
    5y-158 GGCWEQHYLCGG (SEQ ID NO:247)        


    [0132] Selected individual clones from each library were pooled and DNA was isolated using the Zymoprep Yeast Plasmid Miniprep II Kit (D2004, Zymo Research). PCR was performed on the pooled DNA using forward primer (5' GGA AAA GTC GAC TAG ACC ACC ATG GA 3' (SEQ ID NO:357)) and reverse primer (5' CTT TGC GGC CGC TCA TTA TTT 3' (SEQ ID NO:358)). A PCR Core Kit (Roche, Catalog No. 11 578 553 001) was used with the following reaction conditions: 95 °C for 5 minutes, followed by 30 cycles of 95 °C for 45 seconds, 55 °C for 45 seconds, 72 °C for 90 seconds, and finally 72 °C 10 minutes. The PCR reaction and a mammalian expression vector were digested overnight at 37°C with Sall and Notl restriction enzymes (New England Biolabs). The digested DNA was gel purified using the QIAquick Gel Purification Kit (28704, Qiagen). The digested vector DNA and PCR products were ligated using T4 DNA ligase (New England Biolabs) and incubated at 16 °C overnight. The ligated DNA was used to transform XL10-gold Ultracompetent E. coli cells (#200315, Stratagene), and individual clones were selected. Plasmid DNA from an E. coli clone containing DNA encoding each selected variant Fc-polypeptide were introduced into 293-6E mammalian cells. Specifically, 293-6E cells were seeded at 5 x 104 cells/well in poly-D-lysine-coated, 96well flat bottom microtiter plates and incubated overnight at 37°C. Then 200 ng of plasmid DNA was transfected into the cells using the Fugene® HD transfection reagent (#E2312, Promega) and incubated overnight at 37°C. The following day the growth media was changed to serum free media containing 0.5% Tryptone. Conditioned media was collected after another 6 days of growth at 37°C. The conditioned media, which contained the variant Fc-fragments, was then tested for binding to FcRn using the ForteBio technology described in Example 4. Association and dissociation curves at pH 6 and 7.4, respectively, from variant Fc-fragments having the most favorable binding profiles are shown in Figure 7.

    [0133] Figure 7 shows association and dissociation profiles for FcRn binding at pH 6 and pH 7.4, respectively, of the 28 best variant Fc-fragments from yeast library L5, as well as a wild type Fc-fragment (FcWT), and Figure 8 shows FcRn binding profiles for the 18 best variant Fc-fragments selected from yeast libraries L-8 and L-10. All of the L5 variant Fc-fragments had a much higher maximal response at pH 6, that is, higher binding activity, than FcWT. At pH 7.4, all dissociated rapidly, that is, had little or no binding activity, similar to FcWT. All of the L-8 and L-10 variant Fc-fragments had a much higher maximal response at pH 6, that is, higher binding activity, than FcWT. At pH 7.4, all dissociated rapidly, although most showed low levels of residual binding not observed in FcWT. Thus, all of these variant Fc-fragments have favorable properties as compared to FcWT. Therefore, insertions of peptides with different lengths can be successfully used to enhance binding activity to FcRn at pH 6 while preserving rapid dissociation from FcRn at pH 7.4 with little or no residual binding.

    Example 12: In vivo Characterization of variant Fc-fragments selected in yeast



    [0134] To determine whether antibodies containing the variant Fc-fragments identified in the yeast screen described above have improved pharmacokinetic (PK) properties in vivo, an unmodified Antibody X (which is a human IgG2 anti-human IL-23 antibody) and variant versions of Antibody X containing variant Fc-fragments were tested in vivo in cynomolgus monkeys to define pharmacokinetic parameters. Antibody X was selected as an appropriate antibody in which to test the pharmacokinetic parameters of the variant Fc-fragments because it was known to have a linear pK profile and because IL-23 was known to be expressed at low levels in vivo. Thus, it was expected that target-related effects on PK parameters would be minimal, making it easier to detect pK effects due to the variant Fc-fragments.

    [0135] Six variant IgG2 antibodies called (X-5y-8, X-5y-132, X-5y-38, X-5y-91, X-5y-119, and X-5y-127) were made. These IgG2 antibodies had the same insertions at the same positions as the variant IgG1 Fc-fragments 5y-8, 5y-132, 5y-38, 5y-91, 5y-119, and 5y-127, respectively. A plasmid containing DNA encoding the heavy chain of Antibody X was used as a template for five PCR reactions done using the following primers: for X-5y-8, forward, 5'-GAG TGG GAG AGC AAT GGT GGT TGT CCG GTT CTG CTG TTC AAC TGT GGT GGT GGG CAG CCG GAG AAC-3' (SEQ ID NO:380), and reverse, 5'- GTT CTC CGG CTG CCC ACC ACC ACA GTT GAA CAG CAG AAC CGG ACA ACC ACC ATT GCT CTC CCA CTC -3'(SEQ ID NO:381); for X-5y-132,forward, 5'-GAG TGG GAG AGC AAT GGT GGT TGT GTT TTC TCT GCT CTG TGG TGT GGT GGT GGG CAG CCG GAG AAC-3'(SEQ ID NO:382), and reverse, 5'-GTT CTC CGG CTG CCC ACC ACC ACA CCA CAG AGC AGA GAA AAC ACA ACC ACC ATT GCT CTC CCA CTC-3' (SEQ ID NO:383); for X-5y-38, forward, 5'- GAG TGG GAG AGC AAT GGT GGT TGT GAA ACT TAC TGG TTG TTC TGT GGT GGT GGG CAG CCG GAG AAC-3' (SEQ ID NO:384), and reverse, 5'-GTT CTC CGG CTG CCC ACC ACC ACA GAA CAA CCA GTA AGT TTC ACA ACC ACC ATT GCT CTC CCA CTC-3' (SEQ ID NO:385); for X-5y-91, forward, 5'-GAG TGG GAG AGC AAT GGT GGT TGT CCG CAT TGG CCG TTC GAA TGT GGT GGT GGG CAG CCG GAG AAC-3' (SEQ ID NO:386), and reverse, 5'-GTT CTC CGG CTG CCC ACC ACC ACA TTC GAA CGG CCA ATG CGG ACA ACC ACC ATT GCT CTC CCA CTC-3' (SEQ ID NO:387); for X-5y-119, forward, 5'-GAG TGG GAG AGC AAT GGT GGT TGT GCT TTC GAA TTC ATC TAC TGT GGT GGT GGG CAG CCG GAG AAC-3' (SEQ ID NO:388), and reverse, 5'- GTT CTC CGG CTG CCC ACC ACC ACA GTA GAT GAA TTC GAA AGC ACA ACC ACC ATT GCT CTC CCA CTC -3' (SEQ ID NO:389); and for X-5y-127, forward, 5'-GAG TGG GAG AGC AAT GGT GGT TGT CAG TAC TTC TTG CCG TGT GGT GGT GGG CAG CCG GAG AAC-3' (SEQ ID NO:390), and reverse 5'-GTT CTC CGG CTG CCC ACC ACC ACA CGG CAA GAA GTA CTG ACA ACC ACC ATT GCT CTC CCA CTC-3' (SEQ ID NO:391). The Quikchange Site-Directed Mutagenesis Kit (Stratagene, 200518) protocol was used. The reaction mixture was 200 nM dNTPs, 100 nM primers, 1ng DNA template, 1 µL DNA polymerase and water in a total volume of 50 µL. The reaction was run at 95°C for 30 seconds, then 16 cycles of 95°C for 30 seconds, 55°C for 60 seconds, 68°C for 6 minutes, followed by 68°C for 10 minutes. Then 1 µL of Dpnl was added, and the reaction was incubated at 37°C for 1 hour. Then 2 µL of the mixture was used to transform 30 µl of XL1 -blue supercompetent cells (Stratagene) at 42°C for 45 seconds. Thereafter, 0.5 mL SOC was added, and the cells were incubated at 37°C for 1 hour on a shaker at 300 rpm. The transformed cells were spread on LB-ampicillin agar plates and incubated at 37°C overnight. Individual colonies were picked, and plasmid DNA was prepared and sequenced to ensure that the isolates chosen had the expected DNA sequence.

    [0136] Antibodies were prepared in essentially the same way as described above for Fc-fragments, except that the mammalian host cells were transfected with DNAs encoding both the IgG2 heavy chain, including a portion encoding either a variant or a control Fc-fragment, and the light chain of Antibody X. Host cells were incubated under conditions appropriate for expression of the antibodies, and the antibodies were recovered from the culture medium, purified as described above, and used for the following experiments.

    [0137] Cynomolgus monkeys (n=2/group) received a single intravenous dose of an unmodified or a variant version of Antibody X at a dose of 1 mg/kg and were followed during an 8-week in-life phase. Antibody X-5-112, which was previously tested, was used as a control, as well as Antibody X itself. Samples of blood were collected at specified time points over the course of the experiment. Samples were collected at pre-dose, 0.25, 1, 4, 8, 12, 24, 48, 72, 168, 240, 336, 408, 504, 576, 672, 744, 840, 1008, 1176 and 1344 hours post-dose. The antibodies were detected in blood samples and the pharmacokinetic analysis was performed essentially as described above in Example 9.

    [0138] As shown in Figure 9, variant versions of Antibody X containing all variant Fc-fragments tested demonstrated higher mAb concentrations in cynomolgus monkeys at most sampling time points, including all time points beyond 400 hours, compared to unmodified Antibody X. Further, some variants were comparable to X-5-112, which had previously been tested, including X-5y-8, X-5y-127, and X-5y-91.

    [0139] Table 11 below shows the half life (T½), exposure (area under the curve (AUC)) and clearance rate(CI) of Antibody X and variants thereof in each of the cynomolgus monkeys in the study.
    Table 11: Individual values for half life (T½), exposure (AUC), and clearance (CI).
    Antibody (monkey identification number)T½ (hours)AUC (µg*hr/mL)Cl (mL/kg/hr)
    Antibody X (#201) 269 3356 0.29
    Antibody X (#202) 351 3745 0.253
    X-5-112 (#203) 469 5843 0.148
    X-5-112 (#204) 677 7752 0.099
    X-5y-8 (#205) 389 5484 0.168
    X-5y-8 (#206) 520 6611 0.13
    X-5y-132 (#207) 139 5962 0.167
    X-5y-132 (#208) 531 6770 0.127
    X-5y-38 (#209) 258 5454 0.178
    X-5y-38 (#210) 428 4563 0.199
    X-5y-91 (#211) 626 3742 0.213
    X-5y-91 (#212) 684 5162 0.151
    X-5y-119 (#213) 39 4713 0.212
    X-5y-119 (#214) 465 7272 0.12
    X-5y-127 (#215) 529 4825 0.176
    X-5y-127 (#216) 679 5946 0.132
    All variants showed increased half lives and AUC compared to that of Antibody X in at least one of the two monkeys injected with each. In monkeys #207 and #213, X-5y132 and X-5y-119, respectively, had shorter half lives than Antibody X. This could be interpreted to mean that these particular monkeys developed anti-drug antibodies, which led to more rapid clearance of the antibodies. AUC values were also greater than those of Antibody X for most of the variants tested.

    [0140] Averaged data for these pharmacokinetic parameters, which omits monkeys #207 and #213, is shown in Table 12 below. Values from a previous study reported in Table 6 above are included for Antibodies X and X-5-112, which show that the values are relatively constant from one study to the next.
    Table 12: Mean values for half life (T½), exposure (AUC), and clearance (CI).
    Antibody (n)T½ (hours)AUC (µg*hr/mL)Cl (mL/kg/hr)
    Antibody X (n=2)* 335 3890 0.247
    Antibody X (n=2) 310 3550 0.272
    X-5-112 (n=2)* 529 7810 0.108
    X-5-112 (n=2) 573 6795 0.123
    X-5y-8 (n=2) 455 6044 0.149
    X-5y-132 (n=1)# 531 6770 0.127
    X-5y-38 (n=2) 343 5008 0.185
    X-5y-91 (n=2) 655 4452 0.182
    X-5y-119 (n=1)# 465 7272 0.12
    X=5y-127 (n=2) 604 5386 0.154
    *These are values from a previous study reported in Example 9 and Table 6 and included here for comparison.
    #Only one of the two monkeys tested in this study was included because it is suspected that anti-drug antibodies are the cause of rapid clearance observed in the other monkey.


    [0141] These data show that pharmacokinetic parameters for Antibody X and for X-5-112 were relatively constant from one study to the next. They also indicate that half life and exposure are increased in all variants tested. Thus, these data generally indicate that increased binding of a variant Fc-fragment to FcRn at pH 5.5-6.0 (relative to a control Fc-fragment) and rapid dissociation from FcRn at pH 7.4 correlate with a longer in vivo half-life of an antibody containing the variant Fc-fragment in cynomolgus monkeys.

    Example 13: In vivo characterization of a variant Fc-containing antibody having a non-linear PK profile



    [0142] To determine whether the PK properties of an antibody having a non-linear PK profile could be improved by the insertion of one of peptides described herein, the insertion in variant Fc fragment Fc-5-112 was transferred into Antibody Y, a human IgG2 antibody having a non-linear PK profile. This variant form of Antibody Y, a variant IgG2 antibody called Y-5-112, was made essentially as described above, except using different PCR primers appropriate for making the required alteration in Antibody Y.

    [0143] Antibodies were prepared as described above in Example 9. Cynomolgus monkeys (n=2/group) received a single intravenous dose of Antibody Y or Antibody Y-5-112 at a dose of 10 mg/kg and were followed during an 8-week in-life phase. Blood samples were collected at pre-dose, and 0.083 (5 min), 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 384, 432, 480, 528, 576, 624, 672, 720, 768, 816, 864, 912, 960, 1008, 1056, 1104, 1152, 1200, 1248, 1296, and 1344 hours post dose.

    [0144] Concentrations in cynomolgus monkey serum were determined using a sandwich ELISA. A mouse anti-human antibody was diluted in PBS and added to the wells of a 96-well microtiter plate. After a nominal 5°C incubation lasting overnight or up to three days, the well contents were discarded and a blocking buffer comprised of blocker BLOTTO (Thermo® Scientific) was dispensed into plate wells. After a minimum one-hour incubation at ambient room temperature (ART), plate wells were emptied and washed six times with 1X Wash Solution (2 mM imidazole, 0.02% Tween 20, 0.5 mM EDTA, 160 mM NaCl, which is sold by KPL, Inc. as a 20X solution). Study specimens, assay standards and quality control samples prepared in 100% monkey serum, were diluted 50-fold in blocker BLOTTO prior to adding to plate wells. The contents of the plate wells were mixed while incubating for 60 minutes on a plate shaker. Next, plate wells were washed and horseradish peroxidase-labeled mouse anti-human antibody was added to the plate wells. After a final one-hour incubation, plate wells were washed and developed using one component 3,3' ,5,5' - tetramethylbenzidine (TMB) substrate solution. The resulting colorimetric reaction was quenched by adding sulfuric acid to plate wells. Optical densities were determined at dual wavelengths of 450 nm and 650 nm, and the value obtained at 650 nm was subtracted from the value obtained at 450 nm. The conversion of OD values into concentrations for the diluted study specimens was achieved through data regression using a logistic model with weighting set to 1/Y2 in Watson LIMS version 7.0.0.01.

    [0145] As shown in Figure10, Antibody Y has a biphasic PK profile with an approximately linear phase extending from 0 hours to about 216 hours. Thereafter, Antibody Y concentration drops off precipitously. Antibody Y-5-112 has a similar profile except that the linear, gradually-descending portion of the profile extends from about 0 hours to about 432 hours. Thus, the insertion in AntibodyY-5-112 apparently decreases the slope of the linear portion of the PK profile, thus increasing overall exposure as compared to Antibody Y.

    SEQUENCE LISTING



    [0146] 

    <110> AMGEN INC.

    <120> VARIANT FC-POLYPEPTIDES WITH ENHANCED BINDING TO THE NEONATAL FC RECEPTOR

    <130> A-1688-WO-PCT

    <140> TO BE ASSIGNED
    <141> 2012-12-17

    <150> 61/729,050
    <151> 2012-11-21

    <150> 61/585,993
    <151> 2012-01-12

    <150> 61/578,780
    <151> 2011-12-21

    <160> 575

    <170> PatentIn version 3.5

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    <221> VARIANT
    <222> (3)..(8)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (3)..(8)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 16

    <210> 17
    <211> 11
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (3)..(9)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (3)..(9)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 17

    <210> 18
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (3)..(10)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (3)..(10)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 18

    <210> 19
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (4)..(9)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (4)..(9)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 19

    <210> 20
    <211> 13
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (4)..(10)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (4)..(10)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 20

    <210> 21
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (4)..(11)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (4)..(11)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 21

    <210> 22
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (5)..(10)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (5)..(10)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 22

    <210> 23
    <211> 15
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (5)..(11)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (5)..(11)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 23

    <210> 24
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (5)..(12)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (5)..(12)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 24

    <210> 25
    <211> 705
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic polynucleotide"

    <220>
    <221> Variation
    <222> (109)..(111)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (112)..(114)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (115)..(117)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (118)..(120)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (121)..(123)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (124)..(126)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> misc_feature
    <222> (109)..(126)
    <223> /note="nucleotides given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> source
    <223> /note="see specification as filed for detailed description of substitutions and preferred embodiments"

    <400> 25



    <210> 26
    <211> 234
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic polypeptide"

    <220>
    <221> VARIANT
    <222> (37)..(42)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (37)..(42)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 26



    <210> 27
    <211> 711
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic polynucleotide"

    <220>
    <221> Variation
    <222> (277)..(279)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (280)..(282)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (283)..(285)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (286)..(288)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (289)..(291)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (292)..(294)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> misc_feature
    <222> (277)..(294)
    <223> /note="nucleotides given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> source
    <223> /note="see specification as filed for detailed description of substitutions and preferred embodiments"

    <400> 27



    <210> 28
    <211> 236
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic polypeptide"

    <220>
    <221> VARIANT
    <222> (93)..(98)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (93)..(98)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 28



    <210> 29
    <211> 729
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic polynucleotide"

    <220>
    <221> Variation
    <222> (286)..(288)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (289)..(291)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (292)..(294)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (295)..(297)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (298)..(300)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (301)..(303)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> misc_feature
    <222> (286)..(303)
    <223> /note="nucleotides given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> source
    <223> /note="see specification as filed for detailed description of substitutions and preferred embodiments"

    <400> 29

    <210> 30
    <211> 242
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic polypeptide"

    <220>
    <221> VARIANT
    <222> (96)..(101)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (96)..(101)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 30



    <210> 31
    <211> 705
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic polynucleotide"

    <220>
    <221> Variation
    <222> (646)..(648)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (649)..(651)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (652)..(654)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (655)..(657)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (658)..(660)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> misc_feature
    <222> (646)..(660)
    <223> /note="nucleotides given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> Variation
    <222> (664)..(666)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> misc_feature
    <222> (664)..(666)
    <223> /note="nucleotides given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> source
    <223> /note="see specification as filed for detailed description of substitutions and preferred embodiments"

    <400> 31

    <210> 32
    <211> 234
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic polypeptide"

    <220>
    <221> VARIANT
    <222> (215)..(219)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (215)..(219)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> VARIANT
    <222> (221)..(221)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (221)..(221)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 32



    <210> 33
    <211> 735
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic polynucleotide"

    <220>
    <221> Variation
    <222> (223)..(225)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (226)..(228)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (229)..(231)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (232)..(234)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (235)..(237)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (238)..(240)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> misc_feature
    <222> (223)..(240)
    <223> /note="nucleotides given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> source
    <223> /note="see specification as filed for detailed description of substitutions and preferred embodiments"

    <400> 33

    <210> 34
    <211> 244
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic polypeptide"

    <220>
    <221> VARIANT
    <222> (75)..(80)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (75)..(80)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 34



    <210> 35
    <211> 735
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic polynucleotide"

    <220>
    <221> Variation
    <222> (517)..(519)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (520)..(522)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (523)..(525)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (526)..(528)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (529)..(531)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (532)..(534)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> misc_feature
    <222> (517)..(534)
    <223> /note="nucleotides given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> source
    <223> /note="see specification as filed for detailed description of substitutions and preferred embodiments"

    <400> 35

    <210> 36
    <211> 244
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic polypeptide"

    <220>
    <221> VARIANT
    <222> (173)..(178)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (173)..(178)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 36



    <210> 37
    <211> 735
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic polynucleotide"

    <220>
    <221> Variation
    <222> (439)..(441)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (442)..(444)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (445)..(447)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (448)..(450)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (451)..(453)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (454)..(456)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> misc_feature
    <222> (439)..(456)
    <223> /note="nucleotides given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> source
    <223> /note="see specification as filed for detailed description of substitutions and preferred embodiments"

    <400> 37

    <210> 38
    <211> 244
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic polypeptide"

    <220>
    <221> VARIANT
    <222> (147)..(152)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (147)..(152)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 38



    <210> 39
    <211> 735
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic polynucleotide"

    <220>
    <221> modified_base
    <222> (439)..(440)
    <223> a, c, t, g, unknown or other

    <220>
    <221> modified_base
    <222> (442)..(443)
    <223> a, c, t, g, unknown or other

    <220>
    <221> modified_base
    <222> (445)..(446)
    <223> a, c, t, g, unknown or other

    <220>
    <221> modified_base
    <222> (448)..(449)
    <223> a, c, t, g, unknown or other

    <220>
    <221> modified_base
    <222> (451)..(452)
    <223> a, c, t, g, unknown or other

    <220>
    <221> modified_base
    <222> (454)..(455)
    <223> a, c, t, g, unknown or other

    <400> 39



    <210> 40
    <211> 244
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic polypeptide"

    <220>
    <221> MOD_RES
    <222> (147)..(152)
    <223> Any amino acid

    <400> 40



    <210> 41
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 41

    <210> 42
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 42

    <210> 43
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 43

    <210> 44
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 44

    <210> 45
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 45

    <210> 46
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 46

    <210> 47
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 47

    <210> 48
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 48

    <210> 49
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 49

    <210> 50
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 50

    <210> 51
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 51

    <210> 52
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 52

    <210> 53
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 53

    <210> 54
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 54

    <210> 55
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 55

    <210> 56
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 56

    <210> 57
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 57

    <210> 58
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 58

    <210> 59
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 59



    <210> 60
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 60

    <210> 61
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 61

    <210> 62
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 62

    <210> 63
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 63

    <210> 64
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 64

    <210> 65
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 65

    <210> 66
    <211> 6
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 66

    <210> 67
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 67

    <210> 68
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 68

    <210> 69
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 69

    <210> 70
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 70

    <210> 71
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 71

    <210> 72
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 72

    <210> 73
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 73

    <210> 74
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 74

    <210> 75
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 75

    <210> 76
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 76

    <210> 77
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 77

    <210> 78
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 78

    <210> 79
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 79

    <210> 80
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 80

    <210> 81
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 81

    <210> 82
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 82

    <210> 83
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 83

    <210> 84
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 84

    <210> 85
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 85

    <210> 86
    <211> 72
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 86

    <210> 87
    <211> 72
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 87

    <210> 88
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (4)..(11)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (4)..(11)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 88

    <210> 89
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (4)..(13)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (4)..(13)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 89

    <210> 90
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 90

    <210> 91
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 91

    <210> 92
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 92

    <210> 93
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 93

    <210> 94
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 94

    <210> 95
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 95

    <210> 96
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 96

    <210> 97
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 97

    <210> 98
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 98

    <210> 99
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 99

    <210> 100
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 100

    <210> 101
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 101

    <210> 102
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 102

    <210> 103
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 103

    <210> 104
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 104

    <210> 105
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 105

    <210> 106
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 106

    <210> 107
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 107

    <210> 108
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 108

    <210> 109
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 109

    <210> 110
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 110

    <210> 111
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 111

    <210> 112
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 112

    <210> 113
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 113

    <210> 114
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 114

    <210> 115
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 115

    <210> 116
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 116

    <210> 117
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 117

    <210> 118
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 118

    <210> 119
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 119

    <210> 120
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 120

    <210> 121
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 121

    <210> 122
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 122

    <210> 123
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 123

    <210> 124
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 124

    <210> 125
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 125

    <210> 126
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 126

    <210> 127
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 127

    <210> 128
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 128

    <210> 129
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 129

    <210> 130
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 130

    <210> 131
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 131

    <210> 132
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 132

    <210> 133
    <211> 11
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 133

    <210> 134
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 134

    <210> 135
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 135

    <210> 136
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 136

    <210> 137
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 137

    <210> 138
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 138

    <210> 139
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 139

    <210> 140
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 140

    <210> 141
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 141

    <210> 142
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 142

    <210> 143
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 143

    <210> 144
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 144

    <210> 145
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 145

    <210> 146
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 146

    <210> 147
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 147

    <210> 148
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 148

    <210> 149
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 149

    <210> 150
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 150

    <210> 151
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 151

    <210> 152
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 152

    <210> 153
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 153

    <210> 154
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 154

    <210> 155
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 155

    <210> 156
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 156

    <210> 157
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 157

    <210> 158
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 158

    <210> 159
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 159

    <210> 160
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 160

    <210> 161
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 161

    <210> 162
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 162

    <210> 163
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 163

    <210> 164
    <211> 13
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 164

    <210> 165
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 165

    <210> 166
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 166

    <210> 167
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 167

    <210> 168
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 168

    <210> 169
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 169

    <210> 170
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 170

    <210> 171
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 171

    <210> 172
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 172

    <210> 173
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 173

    <210> 174
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 174

    <210> 175
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 175

    <210> 176
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 176

    <210> 177
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 177

    <210> 178
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 178

    <210> 179
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 179

    <210> 180
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 180

    <210> 181
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 181

    <210> 182
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 182

    <210> 183
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 183

    <210> 184
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 184

    <210> 185
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 185

    <210> 186
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 186

    <210> 187
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 187

    <210> 188
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 188

    <210> 189
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 189

    <210> 190
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 190

    <210> 191
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 191

    <210> 192
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 192

    <210> 193
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 193

    <210> 194
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 194

    <210> 195
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 195

    <210> 196
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 196

    <210> 197
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 197

    <210> 198
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 198

    <210> 199
    <211> 11
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 199

    <210> 200
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 200

    <210> 201
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 201

    <210> 202
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 202

    <210> 203
    <211> 11
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 203

    <210> 204
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 204

    <210> 205
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 205

    <210> 206
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 206

    <210> 207
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 207

    <210> 208
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 208

    <210> 209
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 209

    <210> 210
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 210

    <210> 211
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 211

    <210> 212
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 212

    <210> 213
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 213

    <210> 214
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 214

    <210> 215
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 215

    <210> 216
    <211> 11
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 216

    <210> 217
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 217

    <210> 218
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 218

    <210> 219
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 219

    <210> 220
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 220

    <210> 221
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 221

    <210> 222
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 222

    <210> 223
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 223

    <210> 224
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 224

    <210> 225
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 225

    <210> 226
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 226

    <210> 227
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 227

    <210> 228
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 228

    <210> 229
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 229

    <210> 230
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 230

    <210> 231
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 231

    <210> 232
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 232

    <210> 233
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 233

    <210> 234
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 234

    <210> 235
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 235

    <210> 236
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 236

    <210> 237
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 237

    <210> 238
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 238

    <210> 239
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 239

    <210> 240
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 240

    <210> 241
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 241

    <210> 242
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 242

    <210> 243
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 243

    <210> 244
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 244

    <210> 245
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 245

    <210> 246
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 246

    <210> 247
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 247

    <210> 248
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 248

    <210> 249
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 249

    <210> 250
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 250

    <210> 251
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 251

    <210> 252
    <211> 13
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 252

    <210> 253
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 253

    <210> 254
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 254

    <210> 255
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 255

    <210> 256
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 256

    <210> 257
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 257

    <210> 258
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 258

    <210> 259
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 259

    <210> 260
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 260

    <210> 261
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 261

    <210> 262
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 262

    <210> 263
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 263

    <210> 264
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 264

    <210> 265
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 265

    <210> 266
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 266

    <210> 267
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 267

    <210> 268
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 268

    <210> 269
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 269

    <210> 270
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 270

    <210> 271
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 271

    <210> 272
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 272

    <210> 273
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 273

    <210> 274
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 274

    <210> 275
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 275

    <210> 276
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 276

    <210> 277
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 277

    <210> 278
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 278

    <210> 279
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 279

    <210> 280
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 280

    <210> 281
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 281

    <210> 282
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 282

    <210> 283
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 283

    <210> 284
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 284

    <210> 285
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 285

    <210> 286
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 286

    <210> 287
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 287

    <210> 288
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 288

    <210> 289
    <211> 15
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 289

    <210> 290
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 290

    <210> 291
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 291

    <210> 292
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 292

    <210> 293
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 293

    <210> 294
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 294

    <210> 295
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 295

    <210> 296
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 296

    <210> 297
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 297

    <210> 298
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 298

    <210> 299
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 299

    <210> 300
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 300

    <210> 301
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 301

    <210> 302
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 302

    <210> 303
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 303

    <210> 304
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 304

    <210> 305
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 305

    <210> 306
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 306

    <210> 307
    <211> 15
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 307

    <210> 308
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 308

    <210> 309
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 309

    <210> 310
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 310

    <210> 311
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 311

    <210> 312
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 312

    <210> 313
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 313

    <210> 314
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 314

    <210> 315
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 315

    <210> 316
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 316

    <210> 317
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 317

    <210> 318
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 318

    <210> 319
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 319

    <210> 320
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 320

    <210> 321
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 321

    <210> 322
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 322

    <210> 323
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 323

    <210> 324
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 324

    <210> 325
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 325

    <210> 326
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 326

    <210> 327
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 327

    <210> 328
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 328

    <210> 329
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 329

    <210> 330
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 330

    <210> 331
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 331

    <210> 332
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 332

    <210> 333
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 333

    <210> 334
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 334

    <210> 335
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 335

    <210> 336
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 336

    <210> 337
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 337

    <210> 338
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 338

    <210> 339
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 339

    <210> 340
    <211> 17
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 340

    <210> 341
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 341



    <210> 342
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 342

    <210> 343
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 343

    <210> 344
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 344

    <210> 345
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 345

    <210> 346
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 346

    <210> 347
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 347

    <210> 348
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 348

    <210> 349
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 349

    <210> 350
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 350

    <210> 351
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 351

    <210> 352
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 352

    <210> 353
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 353

    <210> 354
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 354

    <210> 355
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 355

    <210> 356
    <211> 16
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 356

    <210> 357
    <211> 26
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 357
    ggaaaagtcg actagaccac catgga   26

    <210> 358
    <211> 21
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 358
    ctttgcggcc gctcattatt t   21

    <210> 359
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 359

    <210> 360
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 360

    <210> 361
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 361

    <210> 362
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 362

    <210> 363
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 363

    <210> 364
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 364

    <210> 365
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 365

    <210> 366
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 366

    <210> 367
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 367

    <210> 368
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 368

    <210> 369
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 369

    <210> 370
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 370

    <210> 371
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 371

    <210> 372
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 372

    <210> 373
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 373

    <210> 374
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 374

    <210> 375
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 375

    <210> 376
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 376

    <210> 377
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 377

    <210> 378
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 378

    <210> 379
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 379

    <210> 380
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 380

    <210> 381
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 381

    <210> 382
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 382

    <210> 383
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 383

    <210> 384
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 384

    <210> 385
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 385

    <210> 386
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 386

    <210> 387
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 387

    <210> 388
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 388

    <210> 389
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 389

    <210> 390
    <211> 63
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 390

    <210> 391
    <211> 63
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 391

    <210> 392
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 392

    <210> 393
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 393

    <210> 394
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 394

    <210> 395
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 395

    <210> 396
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 396

    <210> 397
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 397

    <210> 398
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 398

    <210> 399
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 399

    <210> 400
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 400

    <210> 401
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 401

    <210> 402
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 402

    <210> 403
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 403

    <210> 404
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 404

    <210> 405
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 405

    <210> 406
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 406

    <210> 407
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 407

    <210> 408
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 408

    <210> 409
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 409

    <210> 410
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 410

    <210> 411
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 411

    <210> 412
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 412

    <210> 413
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 413

    <210> 414
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 414

    <210> 415
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 415

    <210> 416
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 416

    <210> 417
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 417

    <210> 418
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 418

    <210> 419
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 419

    <210> 420
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 420

    <210> 421
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 421

    <210> 422
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 422

    <210> 423
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 423

    <210> 424
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 424

    <210> 425
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 425

    <210> 426
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 426

    <210> 427
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 427

    <210> 428
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 428

    <210> 429
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 429

    <210> 430
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 430

    <210> 431
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 431

    <210> 432
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 432

    <210> 433
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 433

    <210> 434
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 434

    <210> 435
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 435

    <210> 436
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 436

    <210> 437
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 437

    <210> 438
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 438

    <210> 439
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 439

    <210> 440
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 440

    <210> 441
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 441

    <210> 442
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 442

    <210> 443
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 44

    <210> 444
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 444

    <210> 445
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 445

    <210> 446
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 446

    <210> 447
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 447

    <210> 448
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 448

    <210> 449
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 449

    <210> 450
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 450

    <210> 451
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 451

    <210> 452
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 452

    <210> 453
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 453

    <210> 454
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 454

    <210> 455
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 455

    <210> 456
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 456

    <210> 457
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 457

    <210> 458
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 458

    <210> 459
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 459

    <210> 460
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 460

    <210> 461
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 461

    <210> 462
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 462

    <210> 463
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 463

    <210> 464
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 464

    <210> 465
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 465

    <210> 466
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 466

    <210> 467
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 467

    <210> 468
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 468

    <210> 469
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 469

    <210> 470
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 470

    <210> 471
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 471

    <210> 472
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 472

    <210> 473
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 473

    <210> 474
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 474

    <210> 475
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 475

    <210> 476
    <211> 14
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 476

    <210> 477
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 477

    <210> 478
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 478

    <210> 479
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 479

    <210> 480
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 480

    <210> 481
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 481

    <210> 482
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 482

    <210> 483
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 483

    <210> 484
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 484

    <210> 485
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 485

    <210> 486
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 486

    <210> 487
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 487

    <210> 488
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 488

    <210> 489
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 489

    <210> 490
    <211> 13
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 490

    <210> 491
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 491

    <210> 492
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 492

    <210> 493
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 493

    <210> 494
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 494

    <210> 495
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 495

    <210> 496
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 496

    <210> 497
    <211> 36
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 497
    gagagcaatg gtggttgttt cccgctgcag gactac   36

    <210> 498
    <211> 36
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 498
    gtagtcctgc agcgggaaac aaccaccatt gctctc   36

    <210> 499
    <211> 36
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 499
    gagagcaatg gtggttgtta cccgctgcag gactac   36

    <210> 500
    <211> 36
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 500
    gtagtcctgc agcgggtaac aaccaccatt gctctc   36

    <210> 501
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 501
    ggtggttgtg ttttcaacgc gttcaactgt ggtggtggg   39

    <210> 502
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 502
    cccaccacca cagttgaacg cgttgaaaac acaaccacc   39

    <210> 503
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 503
    ggtggttgtg ttttcaacgg gttcaactgt ggtggtggg   39

    <210> 504
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 504
    cccaccacca cagttgaacc cgttgaaaac acaaccacc   39

    <210> 505
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 505
    ggtggttgtg ttttcaacca tttcaactgt ggtggtggg   39

    <210> 506
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 506
    cccaccacca cagttgaaat ggttgaaaac acaaccacc   39

    <210> 507
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 507
    ggtggttgtg ttttcaacat cttcaactgt ggtggtggg   39

    <210> 508
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 508
    cccaccacca cagttgaaga tgttgaaaac acaaccacc   39

    <210> 509
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 509
    ggtggttgtg ttttcaactt gttcaactgt ggtggtggg   39

    <210> 510
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 510
    cccaccacca cagttgaaca agttgaaaac acaaccacc   39

    <210> 511
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 511
    ggtggttgtg ttttcaacaa cttcaactgt ggtggtggg   39

    <210> 512
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 512
    cccaccacca cagttgaagt tgttgaaaac acaaccacc   39

    <210> 513
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 513
    ggtggttgtg ttttcaacca gttcaactgt ggtggtggg   39

    <210> 514
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 514
    cccaccacca cagttgaact ggttgaaaac acaaccacc   39

    <210> 515
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 515
    ggtggttgtg ttttcaactc gttcaactgt ggtggtggg   39

    <210> 516
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 516
    cccaccacca cagttgaacg agttgaaaac acaaccacc   39

    <210> 517
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 517
    ggtggttgtg ttttcaacac gttcaactgt ggtggtggg   39

    <210> 518
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 518
    cccaccacca cagttgaacg tgttgaaaac acaaccacc   39

    <210> 519
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 519
    ggtggttgtg ttttcaacgt gttcaactgt ggtggtggg   39

    <210> 520
    <211> 39
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 520
    cccaccacca cagttgaaca cgttgaaaac acaaccacc   39

    <210> 521
    <211> 18
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 521
    gttcctttct attctcac   18

    <210> 522
    <211> 24
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 522
    gagggtgtcc ttgggttttg gggg   24

    <210> 523
    <211> 24
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 523
    ggtgaggacg ctgaccacac ggta   24

    <210> 524
    <211> 24
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 524
    atgcatcacg gagcatgaga agac   24

    <210> 525
    <211> 24
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 525
    attatgcacc tccacgccgt ccac   24

    <210> 526
    <211> 24
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 526
    attgctctcc cactccacgg cgat   24

    <210> 527
    <211> 18
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 527
    cccattcaga tcctcttc   18

    <210> 528
    <211> 54
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <220>
    <221> Variation
    <222> (19) .. (21)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (22) .. (24)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (25) .. (27)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (28)..(30)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (31)..(33)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (34)..(36)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> misc_feature
    <222> (19) .. (36)
    <223> /note="nucleotides given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> source
    <223> /note="see specification as filed for detailed description of substitutions and preferred embodiments"

    <400> 528
    aaacccaagg acaccctcaa aaaaaaaaaa aaaaaaaccc ctgaggtcac atgc   54

    <210> 529
    <211> 54
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <220>
    <221> Variation
    <222> (19) .. (21)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (22) .. (24)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (25) .. (27)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (28)..(30)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (31)..(33)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (34)..(36)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> misc_feature
    <222> (19) .. (36)
    <223> /note="nucleotides given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> source
    <223> /note="see specification as filed for detailed description of substitutions and preferred embodiments"

    <400> 529
    gtggtcagcg tcctcaccaa aaaaaaaaaa aaaaaacacc aggactggct gaat   54

    <210> 530
    <211> 72
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <220>
    <221> Variation
    <222> (28)..(30)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (31)..(33)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (34)..(36)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (37)..(39)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (40)..(42)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (43)..(45)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> misc_feature
    <222> (28)..(45)
    <223> /note="nucleotides given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> source
    <223> /note="see specification as filed for detailed description of substitutions and preferred embodiments"

    <400> 530

    <210> 531
    <211> 57
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <220>
    <221> Variation
    <222> (19) .. (21)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (22) .. (24)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (25) .. (27)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (28)..(30)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (31)..(33)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> misc_feature
    <222> (19)..(33)
    <223> /note="nucleotides given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> Variation
    <222> (37)..(39)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> misc_feature
    <222> (37)..(39)
    <223> /note="nucleotides given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> source
    <223> /note="see specification as filed for detailed description of substitutions and preferred embodiments"

    <400> 531
    tcatgctccg tgatgcataa aaaaaaaaaa aaacacaaac actacacgca gaagagc   57

    <210> 532
    <211> 72
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <220>
    <221> Variation
    <222> (28)..(30)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (31)..(33)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (34)..(36)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (37)..(39)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (40)..(42)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (43)..(45)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> misc_feature
    <222> (28)..(45)
    <223> /note="nucleotides given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> source
    <223> /note="see specification as filed for detailed description of substitutions and preferred embodiments"

    <400> 532

    <210> 533
    <211> 72
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <220>
    <221> Variation
    <222> (28)..(30)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (31)..(33)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (34)..(36)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (37)..(39)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (40)..(42)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (43)..(45)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> misc_feature
    <222> (28)..(45)
    <223> /note="nucleotides given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> source
    <223> /note="see specification as filed for detailed description of substitutions and preferred embodiments"

    <400> 533

    <210> 534
    <211> 18
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 534
    gttcctttct attctcac 18

    <210> 535
    <211> 18
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 535
    cccattcaga tcctcttc   18

    <210> 536
    <211> 84
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <220>
    <221> Variation
    <222> (31)..(33)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (34)..(36)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (37)..(39)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (40)..(42)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (43)..(45)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (46)..(48)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (49)..(51)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> Variation
    <222> (52)..(54)
    <223> /replace="AAC" or "ACT" or "ATC" or "ATG" or "CAG" or "CAT" or "CCG" or "CGT" or "CTG" or "GAA" or "GAC" or "GCT" or "GGT" or "GTT" or "TAC" or "TCT" or "TGG" or "TTC"

    <220>
    <221> misc_feature
    <222> (31)..(54)
    <223> /note="nucleotides given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> source
    <223> /note="see specification as filed for detailed description of substitutions and preferred embodiments"

    <400> 536

    <210> 537
    <211> 84
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <220>
    <221> modified_base
    <222> (31) .. (32)
    <223> a, c, t, g, unknown or other

    <220>
    <221> modified_base
    <222> (34)..(35)
    <223> a, c, t, g, unknown or other

    <220>
    <221> modified_base
    <222> (37) .. (38)
    <223> a, c, t, g, unknown or other

    <220>
    <221> modified_base
    <222> (40)..(41)
    <223> a, c, t, g, unknown or other

    <220>
    <221> modified_base
    <222> (43)..(44)
    <223> a, c, t, g, unknown or other

    <220>
    <221> modified_base
    <222> (46) .. (47)
    <223> a, c, t, g, unknown or other

    <220>
    <221> modified_base
    <222> (49) .. (50)
    <223> a, c, t, g, unknown or other

    <220>
    <221> modified_base
    <222> (52) .. (53)
    <223> a, c, t, g, unknown or other

    <400> 537

    <210> 538
    <211> 21
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 538
    ggccccgtga tggtgatgat g   21

    <210> 539
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 539

    <210> 540
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 540

    <210> 541
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 541

    <210> 542
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 542

    <210> 543
    <211> 63
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 543

    <210> 544
    <211> 63
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 544

    <210> 545
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 545

    <210> 546
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 546

    <210> 547
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 547

    <210> 548
    <211> 66
    <212> DNA
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic primer"

    <400> 548

    <210> 549
    <211> 6
    <212> PRT
    <213> Homo sapiens

    <400> 549

    <210> 550
    <211> 8
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (2)..(7)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (2) .. (7)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 550

    <210> 551
    <211> 8
    <212> PRT
    <213> Homo sapiens

    <400> 551

    <210> 552
    <211> 12
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (2)..(7)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (2) .. (7)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 552

    <210> 553
    <211> 18
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (5)..(10)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (5)..(10)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 553

    <210> 554
    <211> 9
    <212> PRT
    <213> Homo sapiens

    <400> 554

    <210> 555
    <211> 11
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (3)..(7)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (3)..(7)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <220>
    <221> VARIANT
    <222> (9) .. (9)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (9) .. (9)
    <223> /note="Residue given in the sequence has no preference with respect to the annotations for said position"

    <400> 555

    <210> 556
    <211> 5
    <212> PRT
    <213> Homo sapiens

    <400> 556

    <210> 557
    <211> 17
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (8)..(13)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (8)..(13)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 557

    <210> 558
    <211> 6
    <212> PRT
    <213> Homo sapiens

    <400> 558

    <210> 559
    <211> 18
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (5)..(10)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (5)..(10)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 559

    <210> 560
    <211> 7
    <212> PRT
    <213> Homo sapiens

    <400> 560

    <210> 561
    <211> 21
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> MOD_RES
    <222> (7)..(14)
    <223> Any amino acid

    <400> 561

    <210> 562
    <211> 21
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <220>
    <221> VARIANT
    <222> (7)..(14)
    <223> /replace="Phe" or "Asn" or "Arg" or "Ser" or "Thr" or "Met" or "Ile" or "Glu" or "Asp" or "Gly" or "Ala" or "Val" or "Gln" or "His" or "Pro" or "Leu" or "Tyr" or "Trp"

    <220>
    <221> misc_feature
    <222> (7)..(14)
    <223> /note="Residues given in the sequence have no preference with respect to those in the annotations for said positions"

    <400> 562

    <210> 563
    <211> 13
    <212> PRT
    <213> Homo sapiens

    <400> 563

    <210> 564
    <211> 25
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 564

    <210> 565
    <211> 25
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 565

    <210> 566
    <211> 25
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 566

    <210> 567
    <211> 25
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 567

    <210> 568
    <211> 25
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 568

    <210> 569
    <211> 25
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 569

    <210> 570
    <211> 25
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 570

    <210> 571
    <211> 25
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 571

    <210> 572
    <211> 25
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 572

    <210> 573
    <211> 22
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 573

    <210> 574
    <211> 24
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 574

    <210> 575
    <211> 9
    <212> PRT
    <213> Artificial Sequence

    <220>
    <221> source
    <223> /note="Description of Artificial Sequence: Synthetic peptide"

    <400> 575




    Claims

    1. A variant Fc-polypeptide comprising a human IgG 1, IgG2, IgG3, or IgG4 variant Fc-fragment;
    wherein the variant Fc-fragment comprises an insertion of 3 to 20 amino acids in comparison to a control Fc-fragment, wherein the insertion into the variant Fc-fragment is an insertion into an amino acid sequence corresponding to a portion of the control Fc-fragment selected from:

    (a) amino acids 383-387 using the EU numbering system shown in Table 1,

    (b) amino acids 383-387 using the EU numbering system shown in Table 1, wherein amino acids 384-386 are deleted, and

    (c) amino acids 382-391 using the EU numbering system shown in Table 1, wherein the insertion is between amino acids 382 and 383, 383 and 384, 384 and 385, 385 and 386, 386 and 387, 387 and 388, 388 and 389, 389 and 390, or 390 and 391;
    wherein the control Fc-fragment comprises an amino acid sequence selected from:

    amino acids 169-175 of SEQ ID NO:1 (IgG1 Fc-fragment),

    amino acids 165-171 of SEQ ID NO:3 (IgG2 Fc-fragment),

    amino acids 216-222 of SEQ ID NO:5 (IgG3 Fc-fragment), and

    amino acids 166-172 of SEQ ID NO:7 (IgG4 Fc-fragment);

    wherein the control Fc-polypeptide comprises the control Fc-fragment and has the same amino acid sequence as the variant Fc-polypeptide except that the control Fc-polypeptide does not contain the insertion of 3 to 20 amino acids;
    wherein the variant Fc-polypeptide binds to a human neonatal Fc receptor (hFcRn) with higher binding activity at pH 6.0 than the control Fc-polypeptide;
    wherein the variant Fc-polypeptide has little or no binding activity for binding to hFcRn at pH 7.4 and the residual binding response detected at pH 7.4 is no more than 0,2 nanometer more than that detected using the control Fc-polypeptide; and
    wherein the comparative values for binding activity and residual binding response are determined by bio-layer interferometry.
     
    2. The variant Fc-polypeptide of claim 1, wherein the residual binding response detected at pH 7.4 is no more than 0.1 nanometer more than that detected using the control Fc-polypeptide.
     
    3. The variant Fc-polypeptide of claim 1 or claim 2, wherein the control Fc-fragment comprises an amino acid sequence selected from:

    SEQ ID NO:1 (IgG1 Fc-fragment),

    SEQ ID NO:3 (IgG2 Fc-fragment),

    SEQ ID NO:5 (IgG3 Fc-fragment), and

    SEQ ID NO:7 (IgG4 Fc-fragment).


     
    4. The variant Fc-polypeptide of any one of claims 1 to 3, wherein the insertion of the variant Fc-fragment is into an amino acid sequence corresponding to the portion of the control Fc-fragment which is amino acids 383-387 using the EU numbering system shown in Table 1.
     
    5. The variant Fc-polypeptide of any one of claims 1 to 3, wherein the insertion of the variant Fc-fragment is into an amino acid sequence corresponding to the portion of the control Fc-fragment which is amino acids 383-387 using the EU numbering system shown in Table 1, wherein amino acids 384-386 are deleted.
     
    6. The variant Fc-polypeptide of any one of claims 1 to 3, wherein the insertion of the variant Fc fragment is into an amino acid sequence corresponding to a portion of the control Fc-fragment selected from: amino acids 382-391 using the EU numbering system shown in Table 1, wherein the insertion is between amino acids 382 and 383, amino acids 383 and 384, amino acids 384 and 385, amino acids 385 and 386, amino acids 386 and 387, amino acids 387 and 388, amino acids 388 and 389, amino acids 389 and 390, or amino acids 390 and 391.
     
    7. The variant Fc-polypeptide of any one of claims 1 to 6, wherein the control Fc-fragment comprises the amino acid sequence of amino acids 165-171 of SEQ ID NO:3 (IgG2 Fc-fragment).
     
    8. The variant Fc-polypeptide of any one of claims 1 to 7, wherein the insertion of 3 to 20 amino acids is at least six amino acids long.
     
    9. The variant Fc-polypeptide of any one of claims 1 to 8, wherein the insertion of 3 to 20 amino acids is 10 to 20 amino acids long.
     
    10. The variant Fc-polypeptide of any one of claims 1 to 9, wherein the insertion of 3 to 20 amino acids is 6 to 16 amino acids long.
     
    11. The variant Fc-polypeptide of any one of claims 1-4 and 6-10, wherein the insertion is between amino acids 382 and 383, amino acids 383 and 384, amino acids 384 and 385, amino acids 385 and 386, amino acids 386 and 387, or amino acids 387 and 388 of the variant Fc-fragment using the EU numbering system shown in Table 1.
     
    12. The variant Fc-polypeptide of claim 9, wherein the insertion is between amino acids 384 and 385, using the EU numbering system; or wherein amino acids 384-386 are deleted and the insertion is between amino acids 383 and 387, using the EU numbering system shown in Table 1.
     
    13. The variant Fc-polypeptide of any one of claims 1 to 12, wherein the insertion is from position 382 to 386, 387 or 388, using the EU numbering system shown in Table 1.
     
    14. The variant Fc-polypeptide of claim 13, wherein the control Fc-fragment is a human IgG2 Fc-fragment.
     
    15. The variant Fc-polypeptide of any one of claims 1 to 14, wherein the insertion comprises at least one cysteine among the first four inserted amino acids and at least one cysteine among the last four inserted amino acids.
     
    16. The variant Fc-polypeptide of claim 15, wherein the first three amino acids of the insertion are Gly-Gly-Cys and the last three amino acids of the insertion are Cys-Gly-Gly.
     
    17. The variant Fc-polypeptide of any one of claims 1 to 16, wherein the insertion comprises the amino acid sequence of any one of SEQ ID NO:13-24, 41-67, 90-356, 359-367, 369, 372, 373, 375-379, amino acids 4-8 of SEQ ID NO:216; amino acids 4-9 of any one of SEQ ID NO:41-53, 67, 90-163, 165-215, 217-247, and 359-378, amino acids 4-10 of SEQ ID NO: 164, amino acids 4-11 of any one of SEQ ID NO:248-288, 290-306, and 342, amino acids 4-12 of SEQ ID NO:289 or 307, and amino acids 4-13 of any one of SEQ ID NO:308-341 and 343-356.
     
    18. The variant Fc-polypeptide of claim 17, wherein the insertion has the amino acid sequence of any one of SEQ ID NO:41-45, 54-58, 97, 127, 180, 208, 216, and 221.
     
    19. The variant Fc-polypeptide of claim 17, wherein the insertion is between amino acids 384 and 385, using the EU numbering system shown in Table 1.
     
    20. The variant Fc-polypeptide of any one of claims 1 to 19,
    which is a variant Fc fusion protein comprising a non-antibody polypeptide,
    which is an antibody comprising a VH region, a VL region, a CH2 region, and a CH3 region,
    which is monovalent, or divalent, and/or
    which is a dimer or a tetramer.
     
    21. A nucleic acid encoding the variant Fc-polypeptide of any one of claims 1 to 20.
     
    22. A host cell containing the nucleic acid of claim 21.
     
    23. A method for making a variant Fc-polypeptide comprising culturing the host cell of claim 22 under conditions such that the nucleic acid is expressed, and recovering the expressed variant Fc-polypeptide from the culture medium or the cell mass.
     
    24. A method for extending the half-life of an Fc-polypeptide comprising a human IgG Fc-fragment, wherein the method comprises:

    (1) selecting a site for insertion of a peptide into an amino acid sequence of the human IgG Fc-fragment, wherein said site for insertion is selected from:

    (a) between amino acids 383-387 using the EU numbering system shown in Table 1,

    (b) between amino acids 383-387 using the EU numbering system shown in Table 1, wherein amino acids 384-386 are deleted, and

    (c) between amino acids 382-391 using the EU numbering system shown in Table 1, wherein the insertion is between amino acids 382 and 383, amino acids 383 and 384, amino acids 384 and 385, amino acids 385 and 386, amino acids 386 and 387, amino acids 387 and 388, amino acids 388 and 389, amino acids 389 and 390, or amino acids 390 and 391, using the EU numbering system shown in Table 1;
    wherein the control Fc-fragment comprises an amino acid sequence of the human IgG Fc-fragment selected from:

    amino acids 169-175 of SEQ ID NO:1 (IgG1 Fc-fragment),

    amino acids 165-171 of SEQ ID NO:3 (IgG2 Fc-fragment),

    amino acids 216-222 of SEQ ID NO:5 (IgG3 Fc-fragment), and

    amino acids 166-172 of SEQ ID NO:7 (IgG4 Fc-fragment);

    and

    (2) inserting a peptide into the selected site, wherein the peptide comprises an amino acid sequence selected from the group consisting of:

    (a) SEQ ID NO:41-67, 90-356, 359-367, 369, 372, 373, and 375-379,

    (b) amino acids 4-8 of SEQ ID NO:216,

    (c) amino acids 4-9 of any one of SEQ ID NOs: 41-53, 67, 90-163, 165-215, 217-247, and 359-378,

    (d) amino acids 4-10 of SEQ ID NO:164,

    (e) amino acids 4-11 of any one of SEQ ID NO:248-288, 290-306, and 342,

    (f) amino acids 4-12 of SEQ ID NO:289 or 307, and

    (g) amino acids 4-13 of any one of SEQ ID NO:308-341 and 343-356.


     
    25. The method of claim 24, wherein the insertion is between amino acids 382 and 383, amino acids 383 and 384, amino acids 384 and 385, or amino acids 385 and 386 of the variant Fc-fragment, using the EU numbering system shown in Table 1.
     
    26. A method for identifying a human IgG variant Fc-fragment that confers a longer in vivo half-life on a variant Fc-polypeptide that comprises the variant Fc-fragment, as compared to a control Fc-polypeptide, wherein the control Fc-polypeptide comprises a control Fc-fragment comprising an amino acid sequence selected from:

    amino acids 169-175 of SEQ ID NO:1 (IgG1 Fc-fragment),

    amino acids 165-171 of SEQ ID NO:3 (IgG2 Fc-fragment),

    amino acids 216-222 of SEQ ID NO:5 (IgG3 Fc-fragment),

    amino acids 166-172 of SEQ ID NO:7 (IgG4 Fc-fragment);

    said method comprising:

    (1) creating a library of nucleic acids encoding Fc-fragments containing an insertion comprising 4-20 randomized amino acids, wherein the insertion is into an amino acid sequence of the control Fc polypeptide selected from:

    (a) amino acids 383-387 using the EU numbering system shown in Table 1,

    (b) amino acids 383-387 using the EU numbering system shown in Table 1, wherein amino acids 384-386 are deleted, and

    (c) amino acids 382-391 using the EU numbering system shown in Table 1, wherein the insertion is between amino acids 382 and 383, amino acids 383 and 384, amino acids 384 and 385, amino acids 385 and 386, amino acids 386 and 387, amino acids 387 and 388, amino acids 388 and 389, amino acids 389 and 390, or amino acids 390 and 391;

    (2) screening Fc-fragments encoded by the library to identify the variant Fc-fragments that (i) bind to hFcRn with higher binding activity at pH 6 than a control Fc-fragment and (ii) have little or no binding activity for binding to hFcRn at pH 7.4, as determined by bio-layer interferometry;

    (3) constructing a nucleic acid encoding a variant Fc-polypeptide comprising a variant Fc-fragment identified in (2), wherein the concentration of a control Fc-polypeptide, which comprises a control Fc-fragment rather than the variant Fc-fragment, is known to decrease linearly over time when administered to an animal in vivo;

    (4) introducing the nucleic acid of (3) into a host cell and culturing the host cell under conditions such that the variant Fc-polypeptide encoded by the nucleic acid can be expressed;

    (5) recovering the variant Fc-polypeptide from the cell mass or cell culture medium;

    (6) administering the variant Fc-polypeptide to a first non-human animal and administering the control Fc-polypeptide to a second non-human animal; and

    (7) monitoring the concentrations of the variant and control Fc-polypeptides in peripheral blood over time subsequent to administration, thereby identifying a variant Fc-fragment that confers a longer in vivo half life and greater exposure on a variant Fc-polypeptide;

    wherein the control Fc-polypeptide has the same amino acid sequence as the variant Fc-polypeptide except that the control Fc-polypeptide does not contain the insertion comprising 4-20 randomized amino acids.
     
    27. The method of any one of claims 24 to 26, wherein the control Fc-fragment comprises an amino acid sequence selected from:

    SEQ ID NO:1 (IgG1 Fc-fragment),

    SEQ ID NO:3 (IgG2 Fc-fragment),

    SEQ ID NO:5 (IgG3 Fc-fragment), and

    SEQ ID NO:7 (IgG4 Fc-fragment).


     
    28. The method of any one of claims 24 to 27, wherein the control Fc-fragment comprises the amino acid sequence of amino acids 165-171 of SEQ ID NO:3.
     
    29. A pharmaceutical composition comprising the variant Fc-polypeptide according to any one of claims 1-20 and a physiologically acceptable carrier, excipient or diluent.
     
    30. The variant Fc-polypeptide according to any one of claims 1 to 20, or the pharmaceutical composition according to claim 28, for use in treating a chronic disease.
     
    31. The variant Fc-polypeptide for use, or the pharmaceutical composition for use, according to claim 30 wherein the use is for treating a human patient, and the chronic disease is selected from the group of chronic diseases consisting of:

    (a) an oncologic indication,

    (b) an inflammatory condition,

    (c) a bone-related condition,

    (d) a metabolic condition,

    (e) a neurologic condition, and

    (f) chronic pain.


     


    Ansprüche

    1. Fc-Polypeptidvariante, die eine menschliche IgG1, IgG2, IgG3 oder IgG4 Fc-Fragmentvariante umfasst; wobei die Fc-Fragmentvariante eine Insertion von 3 bis 20 Aminosäuren im Vergleich zu einem Kontroll-Fc-Fragment umfasst, wobei die Insertion in die Fc-Fragmentvariante eine Insertion in eine Aminosäuresequenz ist, die einem Teil des Kontroll-Fc-Fragments entspricht, der ausgewählt ist aus:

    (a) Aminosäuren 383-387, unter Verwendung des in Tabelle 1 gezeigten EU-Nummerierungssystems,

    (b) Aminosäuren 383-387, unter Verwendung des in Tabelle 1 gezeigten EU-Nummerierungssystems, wobei die Aminosäuren 384-386 deletiert sind, und

    (c) Aminosäuren 382-391, unter Verwendung des in Tabelle 1 gezeigten EU-Nummerierungssystems, wobei die Insertion zwischen den Aminosäuren 382 und 383, 383 und 384, 384 und 385, 385 und 386, 386 und 387, 387 und 388, 388 und 389, 389 und 390 oder 390 und 391 liegt;

    wobei das Kontroll-Fc-Fragment eine Aminosäuresequenz umfasst, die ausgewählt ist aus:

    Aminosäuren 169-175 von SEQ ID NO: 1 (IgG1 Fc-Fragment),

    Aminosäuren 165-171 von SEQ ID NO: 3 (IgG2 Fc-Fragment),

    Aminosäuren 216-222 von SEQ ID NO: 5 (IgG3 Fc-Fragment) und

    Aminosäuren 166-172 von SEQ ID NO: 7 (IgG4 Fc-Fragment);

    wobei das Kontroll-Fc-Polypeptid das Kontroll-Fc-Fragment umfasst und dieselbe Aminosäuresequenz aufweist wie die Fc-Polypeptidvariante, mit der Ausnahme, dass das Kontroll-Fc-Polypeptid keine Insertion von 3 bis 20 Aminosäuren enthält;

    wobei die Fc-Polypeptidvariante an einen menschlichen neonatalen Fc-Rezeptor (hFcRn) mit höherer Bindungsaktivität bei pH 6,0 bindet als das Kontroll-Fc-Polypeptid;

    wobei die Fc-Polypeptidvariante eine geringe oder keine Bindungsaktivität für die Bindung an hFcRn bei pH 7,4 aufweist und die bei pH 7,4 nachgewiesene Restbindungsantwort nicht mehr als 0,2 Nanometer höher ist als diejenige, die unter Verwendung des Kontroll-Fc-Polypeptids nachgewiesen wird; und

    wobei die Vergleichswerte für die Bindungsaktivität und die restliche Bindungsantwort durch Bioschicht-Interferometrie bestimmt werden.


     
    2. Fc-Polypeptidvariante nach Anspruch 1, wobei die bei pH 7,4 nachgewiesene Restbindungsantwort nicht mehr als 0,1 Nanometer größer ist als diejenige, die unter Verwendung des Kontroll-Fc-Polypeptids nachgewiesen wird.
     
    3. Fc-Polypeptidvariante nach Anspruch 1 oder Anspruch 2, wobei das Kontroll-Fc-Fragment eine Aminosäuresequenz umfasst, die ausgewählt ist aus:

    SEQ ID NO: 1 (IgG1 Fc-Fragment)

    SEQ ID NO: 3 (IgG2 Fc-Fragment)

    SEQ ID NO: 5 (IgG3 Fc-Fragment) und

    SEQ ID NO: 7 (IgG4 Fc-Fragment).


     
    4. Fc-Polypeptidvariante nach einem der Ansprüche 1 bis 3, wobei die Insertion der Fc-Fragmentvariante in einer Aminosäuresequenz liegt, die dem Teil des Kontroll-Fc-Fragments entspricht, das die Aminosäuren 383-387 darstellt, unter Verwendung des in Tabelle 1 gezeigten EU-Nummerierungssystems.
     
    5. Fc-Polypeptidvariante nach einem der Ansprüche 1 bis 3, wobei die Insertion der Fc-Fragmentvariante in einer Aminosäuresequenz liegt, die dem Teil des Kontroll-Fc-Fragments entspricht, das die Aminosäuren 383-387 darstellt, unter Verwendung des in Tabelle 1 gezeigten EU-Nummerierungssystems, wobei die Aminosäuren 384-386 deletiert sind.
     
    6. Fc-Polypeptidvariante nach einem der Ansprüche 1 bis 3, wobei die Insertion der Fc-Fragmentvariante in einer Aminosäuresequenz liegt, die einem Teil des Kontroll-Fc-Fragments entspricht, der ausgewählt ist aus: Aminosäuren 382-391, unter Verwendung des in Tabelle 1 gezeigten EU-Nummerierungssystems, wobei die Insertion zwischen Aminosäuren 382 und 383, Aminosäuren 383 und 384, Aminosäuren 384 und 385, Aminosäuren 385 und 386, Aminosäuren 386 und 387, Aminosäuren 387 und 388, Aminosäuren 388 und 389, Aminosäuren 389 und 390 oder Aminosäuren 390 und 391 liegt.
     
    7. Fc-Polypeptidvariante nach einem der Ansprüche 1 bis 6, wobei das Kontroll-Fc-Fragment die Aminosäuresequenz der Aminosäuren 165-171 von SEQ ID NO: 3 (IgG2 Fc-Fragment) umfasst.
     
    8. Fc-Polypeptidvariante nach einem der Ansprüche 1 bis 7, wobei die Insertion von 3 bis 20 Aminosäuren mindestens sechs Aminosäuren lang ist.
     
    9. Fc-Polypeptidvariante nach einem der Ansprüche 1 bis 8, wobei die Insertion von 3 bis 20 Aminosäuren 10 bis 20 Aminosäuren lang ist.
     
    10. Fc-Polypeptidvariante nach einem der Ansprüche 1 bis 9, wobei die Insertion von 3 bis 20 Aminosäuren 6 bis 16 Aminosäuren lang ist.
     
    11. Fc-Polypeptidvariante nach einem der Ansprüche 1-4 und 6-10,
    wobei die Insertion zwischen Aminosäuren 382 und 383, Aminosäuren 383 und 384, Aminosäuren 384 und 385, Aminosäuren 385 und 386, Aminosäuren 386 und 387 oder Aminosäuren 387 und 388 der Fc-Fragmentvariante liegt, unter Verwendung des in Tabelle 1 gezeigten EU Nummerierungssystems.
     
    12. Fc-Polypeptidvariante nach Anspruch 9, wobei die Insertion zwischen den Aminosäuren 384 und 385 liegt, unter Verwendung des EU-Nummerierungssystems; oder wobei die Aminosäuren 384 bis 386 deletiert sind und die Insertion zwischen den Aminosäuren 383 und 387 liegt, unter Verwendung des in Tabelle 1 gezeigten EU-Nummerierungssystems.
     
    13. Fc-Polypeptidvariante nach einem der Ansprüche 1 bis 12, wobei die Insertion von Position 382 bis 386, 387 oder 388 liegt, unter Verwendung des in Tabelle 1 gezeigten EU-Nummerierungssystems.
     
    14. Fc-Polypeptidvariante nach Anspruch 13, wobei das Kontroll-Fc-Fragment ein menschliches IgG2 Fc-Fragment ist.
     
    15. Fc-Polypeptidvariante nach einem der Ansprüche 1 bis 14, wobei die Insertion mindestens ein Cystein unter den ersten vier inserierten Aminosäuren und mindestens ein Cystein unter den letzten vier inserierten Aminosäuren umfasst.
     
    16. Fc-Polypeptidvariante nach Anspruch 15, wobei die ersten drei Aminosäuren der Insertion Gly-Gly-Cys sind und die letzten drei Aminosäuren der Insertion Cys-Gly-Gly sind.
     
    17. Fc-Polypeptidvariante nach einem der Ansprüche 1 bis 16, wobei die Insertion die Aminosäuresequenz einer der SEQ ID NO: 13-24, 41-67, 90-356, 359-367, 369, 372, 373, 375-379 umfasst, Aminosäuren 4-8 von SEQ ID NO: 216; Aminosäuren 4-9 einer der SEQ ID NO: 41-53, 67, 90-163, 165-215, 217-247 und 359-378, Aminosäuren 4-10 von SEQ ID NO: 164, Aminosäuren 4-11 einer der SEQ ID NO: 248-288, 290-306 und 342, Aminosäuren 4-12 der SEQ ID NO: 289 oder 307 und Aminosäuren 4-13 einer der SEQ ID NO: 308-341 und 343-356.
     
    18. Fc-Polypeptidvariante nach Anspruch 17, wobei die Insertion die Aminosäuresequenz einer der SEQ ID NO: 41-45, 54-58, 97, 127, 180, 208, 216 und 221 aufweist.
     
    19. Fc-Polypeptidvariante nach Anspruch 17, wobei die Insertion zwischen den Aminosäuren 384 und 385 liegt, unter Verwendung des in Tabelle 1 gezeigten EU-Nummerierungssystems.
     
    20. Fc-Polypeptidvariante nach einem der Ansprüche 1 bis 19,
    die eine Fc-Fusionsproteinvariante ist, die ein Nicht-Antikörper-Polypeptid umfasst,
    die ein Antikörper ist, der eine VH-Region, eine VL-Region, eine CH2-Region und eine CH3-Region umfasst, die einwertig oder zweiwertig ist und/oder die ein Dimer oder ein Tetramer ist.
     
    21. Nukleinsäure, welche für die Fc-Polypeptidvariante nach einem der Ansprüche 1 bis 20 kodiert.
     
    22. Wirtszelle, welche die Nukleinsäure nach Anspruch 21 enthält.
     
    23. Verfahren zur Herstellung einer Fc-Polypeptidvariante, welches das Kultivieren der Wirtszelle nach Anspruch 22 unter solchen Bedingungen umfasst, dass die Nukleinsäure exprimiert wird, und das Gewinnen der Fc-Polypeptidvariante aus dem Kulturmedium oder der Zellmasse.
     
    24. Verfahren zur Verlängerung der Halbwertszeit eines Fc-Polypeptids, das ein humanes IgG Fc-Fragment umfasst, wobei das Verfahren Folgendes umfasst:

    (1) Auswählen einer Stelle für die Insertion eines Peptids in eine Aminosäuresequenz des menschlichen IgG Fc-Fragments, wobei die Insertionsstelle ausgewählt wird aus:

    (a) zwischen den Aminosäuren 383-387, unter Verwendung des in Tabelle 1 gezeigten EU-Nummerierungssystems,

    (b) zwischen den Aminosäuren 383-387, unter Verwendung des in Tabelle 1 gezeigten EU-Nummerierungssystems, wobei die Aminosäuren 384-386 deletiert sind, und

    (c) zwischen den Aminosäuren 382-391, unter Verwendung des In Tabelle 1 gezeigten EU-Nummerierungssystems, wobei die Insertion zwischen Aminosäuren 382 und 383, Aminosäuren 383 und 384, Aminosäuren 384 und 385, Aminosäuren 385 und 386, Aminosäuren 386 und 387, Aminosäuren 387 und 388, Aminosäuren 388 und 389, Aminosäuren 389 und 390 oder Aminosäuren 390 und 391 erfolgt, unter Verwendung des in Tabelle 1 gezeigten EU-Nummerierungssystems;

    wobei das Kontroll-Fc-Fragment eine Aminosäuresequenz des menschlichen IgG Fc-Fragments umfasst, die ausgewählt wird aus:

    Aminosäuren 169-175 von SEQ ID NO: 1 (IgG1 Fc-Fragment),

    Aminosäuren 165-171 von SEQ ID NO: 3 (IgG2 Fc-Fragment),

    Aminosäuren 216-222 von SEQ ID NO: 5 (IgG3 Fc-Fragment) und

    Aminosäuren 166-172 von SEQ ID NO: 7 (IgG4 Fc-Fragment);

    und

    (2) Inserieren eines Peptids in die ausgewählte Stelle, wobei das Peptid eine Aminosäuresequenz umfasst, die ausgewählt wird aus der Gruppe, bestehend aus:

    (a) SEQ ID NO: 41-67, 90-356, 359-367, 369, 372, 373 und 375-379,

    (b) Aminosäuren 4-8 von SEQ ID NO: 216,

    (c) Aminosäuren 4-9 einer der SEQ ID NOs: 41-53, 67, 90-163, 165-215, 217-247 und 359-378,

    (d) Aminosäuren 4-10 von SEQ ID NO: 164,

    (e) Aminosäuren 4-11 einer der SEQ ID NO: 248-288, 290-306 und 342,

    (f) Aminosäuren 4-12 von SEQ ID NO: 289 oder 307 und

    (g) Aminosäuren 4-13 einer der SEQ ID NO: 308-341 und 343-356.


     
    25. Verfahren nach Anspruch 24, wobei die Insertion zwischen Aminosäuren 382 und 383, Aminosäuren 383 und 384, Aminosäuren 384 und 385 oder Aminosäuren 385 und 386 der Fc-Fragmentvariante erfolgt, unter Verwendung des in der Tabelle 1 gezeigten EU-Nummerierungssystems.
     
    26. Verfahren zum Identifizieren einer menschlichen IgG Fc-Fragmentvariante, die einer Fc-Polypeptidvariante, welche die Fc-Fragmentvariante umfasst, eine längere in-vivo Halbwertszeit verleiht, verglichen mit einem Kontroll-Fc-Polypeptid, wobei das Kontroll-Fc-Polypeptid ein Kontroll-Fc-Fragment umfasst, das eine Aminosäuresequenz umfasst, die ausgewählt wird aus:

    Aminosäuren 169-175 von SEQ ID NO: 1 (IgG1 Fc-Fragment),

    Aminosäuren 165-171 von SEQ ID NO: 3 (IgG2 Fc-Fragment),

    Aminosäuren 216-222 von SEQ ID NO: 5 (IgG3 Fc-Fragment),

    Aminosäuren 166-172 von SEQ ID NO: 7 (IgG4 Fc-Fragment);

    wobei das Verfahren Folgendes umfasst:

    (1) Erstellen einer Bibliothek von Nukleinsäuren, die für Fc-Fragmente kodieren, die eine Insertion enthalten, welche 4-20 randomisierte Aminosäuren umfasst, wobei die Insertion in eine Aminosäuresequenz des Kontroll-Fc-Polypeptids erfolgt, das ausgewählt wird aus:

    (a) Aminosäuren 383-387, unter Verwendung des in Tabelle 1 gezeigten EU-Nummerierungssystems,

    (b) Aminosäuren 383-387, unter Verwendung des in Tabelle 1 gezeigten EU-Nummerierungssystems, wobei die Aminosäuren 384-386 deletiert sind, und

    (c) Aminosäuren 382-391, unter Verwendung des in Tabelle 1 gezeigten EU-Nummerierungssystems, wobei die Insertion zwischen Aminosäuren 382 und 383, Aminosäuren 383 und 384, Aminosäuren 384 und 385, Aminosäuren 385 und 386, Aminosäuren 386 und 387, Aminosäuren 387 und 388, Aminosäuren 388 und 389, Aminosäuren 389 und 390 oder Aminosäuren 390 und 391 erfolgt;

    (2) Durchforsten von Fc-Fragmenten, die von der Bibliothek kodiert werden, um die Fc-Fragmentvarianten zu identifizieren, die (i) mit höherer Bindungsaktivität bei pH 6 an hFcRn binden als ein Kontroll-Fc-Fragment und (ii) eine geringe oder keine Bindungsaktivität für die Bindung an hFcRn bei pH 7,4 aufweisen, wie durch Bioschicht-Interferometrie bestimmt;

    (3) Konstruieren einer Nukleinsäure, die für eine Fc-Polypeptidvariante kodiert, die eine in (2) identifizierte Fc-Fragmentvariante umfasst, wobei die Konzentration eines Kontroll-Fc-Polypeptids, das ein Kontroll-Fc-Fragment anstelle der Fc-Fragmentvariante umfasst, dafür bekannt ist, mit der Zeit linear abzunehmen, wenn es einem Tier in-vivo verabreicht wird;

    (4) Einführen der Nukleinsäure von (3) in eine Wirtszelle und Kultivieren der Wirtszelle unter solchen Bedingungen, dass die von der Nukleinsäure kodierte Fc-Polypeptidvariante exprimiert werden kann;

    (5) Gewinnen der Fc-Polypeptidvariante aus der Zellmasse oder dem Zellkulturmedium;

    (6) Verabreichen der Fc-Polypeptidvariante an ein erstes nicht-menschliches Tier und Verabreichen des Kontroll-Fc-Polypeptids an ein zweites nicht-menschliches Tier; und

    (7) Überwachen der Konzentrationen der Variante und des Kontroll-Fc-Polypeptids im peripheren Blut über einen längeren Zeitraum nach der Verabreichung, wodurch eine Fc-Fragmentvariante identifiziert wird, die einer Fc-Polypeptidvariante eine längere in vivo Halbwertszeit und eine größere Exposition verleiht;

    wobei das Kontroll-Fc-Polypeptid die gleiche Aminosäuresequenz wie die Fc-Polypeptidvariante aufweist, mit der Ausnahme, dass das Kontroll-Fc-Polypeptid nicht die Insertion enthält, die 4-20 randomisierte Aminosäuren umfasst.
     
    27. Verfahren nach einem der Ansprüche 24 bis 26, wobei das Kontroll-Fc-Fragment eine Aminosäuresequenz umfasst, die ausgewählt wird aus:

    SEQ ID NO: 1 (IgG1 Fc-Fragment)

    SEQ ID NO: 3 (IgG2 Fc-Fragment)

    SEQ ID NO: 5 (IgG3 Fc-Fragment), und

    SEQ ID NO: 7 (IgG4 Fc-Fragment).


     
    28. Verfahren nach einem der Ansprüche 24 bis 27, wobei das Kontroll-Fc-Fragment die Aminosäuresequenz der Aminosäuren 165-171 von SEQ ID NO: 3 umfasst.
     
    29. Pharmazeutische Zusammensetzung, welche die Fc-Polypeptidvariante nach einem der Ansprüche 1 bis 20 und einen physiologisch verträglichen Träger, Hilfsstoff oder Verdünnungsmittel umfasst.
     
    30. Fc-Polypeptidvariante nach einem der Ansprüche 1 bis 20 oder pharmazeutische Zusammensetzung nach Anspruch 28 zur Verwendung bei der Behandlung einer chronischen Erkrankung.
     
    31. Fc-Polypeptidvariante zur Verwendung oder pharmazeutische Zusammensetzung zur Verwendung nach Anspruch 30, wobei die Verwendung zur Behandlung eines menschlichen Patienten dient und die chronische Erkrankung ausgewählt ist aus der Gruppe chronischer Erkrankungen, bestehend aus:

    (a) einer onkologischen Indikation,

    (b) einem entzündlichen Zustand,

    (c) einem knochenbezogenen Zustand,

    (d) einem Stoffwechselzustand,

    (e) einem neurologischen Zustand und

    (f) chronischem Schmerz.


     


    Revendications

    1. Variant polypeptidique Fc comprenant un variant de fragment Fc d'IgG1, d'IgG2, d'IgG3 ou d'IgG4 humaine ; dans lequel le variant de fragment Fc comprend une insertion de 3 à 20 acides aminés comparé à un fragment Fc témoin, dans lequel l'insertion dans le variant de fragment Fc est une insertion dans une séquence d'acides aminés correspondant à une partie du fragment Fc témoin sélectionnée parmi :

    (a) les acides aminés 383 à 387 en utilisant le système de numérotation EU présenté dans le tableau 1,

    (b) les acides aminés 383 à 387 en utilisant le système de numérotation EU présenté dans le tableau 1, dans lequel les acides aminés 384 à 386 sont délétés, et

    (c) les acides aminés 382 à 391 en utilisant le système de numérotation EU présenté dans le tableau 1, dans lequel l'insertion est entre les acides aminés 382 et 383, 383 et 384, 384 et 385, 385 et 386, 386 et 387, 387 et 388, 388 et 389, 389 et 390, ou 390 et 391 ;

    dans lequel le fragment Fc témoin comprend une séquence d'acides aminés sélectionnée parmi :

    les acides aminés 169 à 175 de SEQ ID NO : 1 (fragment Fc d'IgG1),

    les acides aminés 165 à 171 de SEQ ID NO : 3 (fragment Fc d'IgG2),

    les acides aminés 216 à 222 de SEQ ID NO : 5 (fragment Fc d'IgG3), et

    les acides aminés 166 à 172 de SEQ ID NO : 7 (fragment Fc d'IgG4) ;

    dans lequel le polypeptide Fc témoin comprend le fragment Fc témoin et a la même séquence d'acides aminés que le variant polypeptidique Fc à l'exception que le polypeptide Fc témoin ne contient pas l'insertion de 3 à 20 acides aminés ;

    dans lequel le variant polypeptidique Fc se lie à un récepteur Fc néonatal humain (hFcRn) avec une plus grande activité de liaison à pH 6,0 que le polypeptide Fc témoin ;

    dans lequel le variant polypeptidique Fc a peu ou pas d'activité de liaison pour la liaison à hFcRn à pH 7,4 et la réponse de liaison résiduelle détectée à pH 7,4 n'est pas supérieure à 0,2 nanomètre de plus que celle détectée en utilisant le polypeptide Fc témoin ; et

    dans lequel les valeurs comparatives pour l'activité de liaison et la réponse de liaison résiduelle sont déterminées par interférométrie de la bio-couche.


     
    2. Variant polypeptidique Fc selon la revendication 1, dans lequel la réponse de liaison résiduelle détectée à pH 7,4 n'est pas supérieure à 0,1 nanomètre de plus que celle détectée en utilisant le polypeptide Fc témoin.
     
    3. Variant polypeptidique Fc selon la revendication 1 ou la revendication 2, dans lequel le fragment Fc témoin comprend une séquence d'acides aminés sélectionnée parmi :

    SEQ ID NO : 1 (fragment Fc d'IgG1),

    SEQ ID NO : 3 (fragment Fc d'IgG2),

    SEQ ID NO : 5 (fragment Fc d'IgG3), et

    SEQ ID NO : 7 (fragment Fc d'IgG4).


     
    4. Variant polypeptidique Fc selon l'une quelconque des revendications 1 à 3, dans lequel l'insertion du variant de fragment Fc est dans une séquence d'acides aminés correspondant à la partie du fragment Fc témoin qui est les acides aminés 383 à 387 en utilisant le système de numérotation EU présenté dans le tableau 1.
     
    5. Variant polypeptidique Fc selon l'une quelconque des revendications 1 à 3, dans lequel l'insertion du variant de fragment Fc est dans une séquence d'acides aminés correspondant à la partie du fragment Fc témoin qui est les acides aminés 383 à 387 en utilisant le système de numérotation présenté dans le tableau 1, dans lequel les acides aminés 384 à 386 sont délétés.
     
    6. Variant polypeptidique Fc selon l'une quelconque des revendications 1 à 3, dans lequel l'insertion du variant de fragment Fc est dans une séquence d'acides aminés correspondant à une partie du fragment Fc témoin sélectionnée parmi : les acides aminés 382 à 391 en utilisant le système de numérotation EU présenté dans le tableau 1, dans lequel l'insertion est entre les acides aminés 382 et 383, les acides aminés 383 et 384, les acides aminés 384 et 385, les acides aminés 385 et 386, les acides aminés 386 et 387, les acides aminés 387 et 388, les acides aminés 388 et 389, les acides aminés 389 et 390, ou les acides aminés 390 et 391.
     
    7. Variant polypeptidique Fc selon l'une quelconque des revendications 1 à 6, dans lequel le fragment Fc témoin comprend la séquence d'acides aminés des acides aminés 165 à 171 de SEQ ID NO : 3 (fragment Fc d'IgG2).
     
    8. Variant polypeptidique Fc selon l'une quelconque des revendications 1 à 7, dans lequel l'insertion de 3 à 20 acides aminés est d'une longueur d'au moins six acides aminés.
     
    9. Variant polypeptidique Fc selon l'une quelconque des revendications 1 à 8, dans lequel l'insertion de 3 à 20 acides aminés est d'une longueur de 10 à 20 acides aminés.
     
    10. Variant polypeptidique Fc selon l'une quelconque des revendications 1 à 9, dans lequel l'insertion de 3 à 20 acides aminés est d'une longueur de 6 à 16 acides aminés.
     
    11. Variant polypeptidique Fc selon l'une quelconque des revendications 1 à 4 et 6 à 10, dans lequel l'insertion est entre les acides aminés 382 et 383, les acides aminés 383 et 384, les acides aminés 384 et 385, les acides aminés 385 et 386, les acides aminés 386 et 387, ou les acides aminés 387 et 388 du variant de fragment Fc en utilisant le système de numérotation présenté dans le tableau 1.
     
    12. Variant polypeptidique Fc selon la revendication 9, dans lequel l'insertion est entre les acides aminés 384 et 385, en utilisant le système de numérotation EU ; ou dans lequel les acides aminés 384 à 386 sont délétés et l'insertion est entre les acides aminés 383 et 387, en utilisant le système de numérotation EU présenté dans le tableau 1.
     
    13. Variant polypeptidique Fc selon l'une quelconque des revendications 1 à 12, dans lequel l'insertion est de la position 382 à 386, 387 ou 388 en utilisant le système de numérotation présenté dans le tableau 1.
     
    14. Variant polypeptidique Fc selon la revendication 13, dans lequel le fragment Fc témoin est un fragment Fc d'IgG2 humaine.
     
    15. Variant polypeptidique Fc selon l'une quelconque des revendications 1 à 14, dans lequel l'insertion comprend au moins une cystéine parmi les quatre premiers acides aminés insérés et au moins une cystéine parmi les quatre derniers acides aminés insérés.
     
    16. Variant polypeptidique Fc selon la revendication 15, dans lequel les trois premiers acides aminés de l'insertion sont Gly-Gly-Cys et les trois derniers acides aminés de l'insertion sont Cys-Gly-Gly.
     
    17. Variant polypeptidique Fc selon l'une quelconque des revendications 1 à 16, dans lequel l'insertion comprend la séquence d'acides aminé selon l'une quelconque de SEQ ID NO : 13 à 24, 41 à 67, 90 à 356, 359 à 367, 369, 372, 373, 375 à 379, les acides aminés 4 à 8 de SEQ ID NO : 216 ; les acides aminés 4 à 9 de l'une quelconque de SEQ ID NO : 41 à 53, 67, 90 à 163, 165 à 215, 217 à 247, et 359 à 378, les acides aminés 4 à 10 de SEQ ID NO : 164, les acides aminés 4 à 11 de l'une quelconque SEQ ID NO : 248 à 288, 290 à 306, et 342, les acides aminés 4 à 12 de SEQ ID NO : 289 ou 307, et les acides aminés 4 à 13 de l'une quelconque de SEQ ID NO : 308 à 341 et 343 à 356.
     
    18. Variant polypeptidique Fc selon la revendication 17, dans lequel l'insertion a la séquence d'acides aminés selon l'une quelconque de SEQ ID NO : 41 à 45, 54 à 58, 97, 127, 180, 208, 216, et 221.
     
    19. Variant polypeptidique Fc selon la revendication 17, dans lequel l'insertion est entre les acides aminés 384 et 385, en utilisant le système de numérotation EU présenté dans le tableau 1.
     
    20. Variant polypeptidique Fc selon l'une quelconque des revendications 1 à 19,
    qui est une protéine de fusion Fc variante comprenant un polypeptide non anticorps,
    qui est un anticorps comprenant une région VH, une région VL, une région CH2, et une région CH3,
    qui est monovalent, ou divalent, et/ou qui est un dimère ou un tétramère.
     
    21. Acide nucléique codant pour le variant polypeptidique Fc selon l'une quelconque des revendications 1 à 20.
     
    22. Cellule hôte contenant l'acide nucléique selon la revendication 21.
     
    23. Procédé de préparation d'un variant polypeptidique Fc comprenant la culture de la cellule hôte selon la revendication 22 dans des conditions telles que l'acide nucléique est exprimé, et la récupération du variant polypeptidique Fc exprimé du milieu de culture ou de la masse cellulaire.
     
    24. Procédé d'allongement de la demi-vie d'un polypeptide Fc comprenant un fragment Fc d'IgG humaine, dans lequel le procédé comprend :

    (1) la sélection d'un site pour l'insertion d'un peptide dans une séquence d'acides aminés du fragment Fc d'IgG humaine, dans lequel ledit site d'insertion est sélectionné parmi :

    (a) entre les acides aminés 383 à 387 en utilisant le système de numérotation EU présenté dans le tableau 1,

    (b) entre les acides aminés 383 à 387 en utilisant le système de numérotation EU présenté dans le tableau 1, dans lequel les acides aminés 384 à 386 sont délétés, et

    (c) entre les acides aminés 382 à 391 en utilisant le système de numérotation EU présenté dans le tableau 1, dans lequel l'insertion est entre les acides aminés 382 et 383, les acides aminés 383 et 384, les acides aminés 384 et 385, les acides aminés 385 et 386, les acides aminés 386 et 387, les acides aminés 387 et 388, les acides aminés 388 et 389, les acides aminés 389 et 390, ou les acides aminés 390 et 391, en utilisant la numérotation EU présentée dans le tableau 1 ;

    dans lequel le fragment Fc témoin comprend une séquence d'acides aminés du fragment Fc d'IgG humaine sélectionnée parmi :

    les acides aminés 169 à 175 de SEQ ID NO : 1 (fragment Fc d'IgG1),

    les acides aminés 165 à 171 de SEQ ID NO : 3 (fragment Fc d'IgG2),

    les acides aminés 216 à 222 de SEQ ID NO : 5 (fragment Fc d'IgG3), et

    les acides aminés 166 à 172 de SEQ ID NO : 7 (fragment Fc d'IgG4) ; et

    (2) l'insertion d'un peptide dans le site sélectionné, dans lequel le peptide comprend une séquence d'acides aminés sélectionnée dans le groupe constitué de :

    (a) SEQ ID NO : 41 à 67, 90 à 356, 359 à 367, 369, 372, 373, et 375 à 379,

    (b) les acides aminés 4 à 8 de SEQ ID NO : 216,

    (c) les acides aminés 4 à 9 de l'une quelconque de SEQ ID NO : 41 à 53, 67, 90 à 163, 165 à 215, 217 à 247, et 359 à 378,

    (d) les acides aminés 4 à 10 de SEQ ID NO : 164,

    (e) les acides aminés 4 à 11 de l'une quelconque de SEQ ID NO : 248 à 288, 290 à 306, et 342,

    (f) les acides aminés 4 à 12 de SEQ ID NO : 289 ou 307, et

    (g) les acides aminés 4 à 13 de l'une quelconque de SEQ ID NO : 308 à 341 et 343 à 356.


     
    25. Procédé selon la revendication 24, dans lequel l'insertion est entre les acides aminés 382 et 383, les acides aminés 383 et 384, les acides aminés 384 et 385, ou les acides aminés 385 et 386 du variant de fragment Fc, en utilisant le système de numérotation EU présenté dans le tableau 1.
     
    26. Procédé d'identification d'un variant de fragment Fc d'IgG humaine qui confère une plus longue demi-vie in vivo sur un variant polypeptidique Fc qui comprend le variant de fragment Fc, comparé à un polypeptide Fc témoin, dans lequel le polypeptide Fc témoin comprend un fragment Fc témoin comprenant une séquence d'acides aminés sélectionnée parmi :

    les acides aminés 169 à 175 de SEQ ID NO : 1 (fragment Fc d'IgG1),

    les acides aminés 165 à 171 de SEQ ID NO : 3 (fragment Fc d'IgG2),

    les acides aminés 216 à 222 de SEQ ID NO : 5 (fragment Fc d'IgG3),

    les acides aminés 166 à 172 de SEQ ID NO : 7 (fragment Fc d'IgG4) ;

    ledit procédé comprenant :

    (1) la création d'une banque d'acides nucléiques codant pour des fragments Fc contenant une insertion comprenant 4 à 20 acides aminés randomisés, dans lequel l'insertion est dans une séquence d'acides aminés du polypeptide Fc témoin sélectionnée parmi :

    (a) acides aminés 383 à 387 en utilisant le système de numérotation présenté dans le tableau 1,

    (b) les acides aminés 383 à 387 en utilisant le système de numérotation EU présenté dans le tableau 1, dans lequel les acides aminés 384 à 386 sont délétés, et

    (c) les acides aminés 382 à 391 en utilisant le système de numérotation EU présenté dans le tableau 1, dans lequel l'insertion est entre les acides aminés 382 et 383, les acides aminés 383 et 384, les acides aminés 384 et 385, les acides aminés 385 et 386, les acides aminés 386 et 387, les acides aminés 387 et 388, les acides aminés 388 et 389, les acides aminés 389 et 390, ou les acides aminés 390 et 391 ;

    (2) le criblage de fragments Fc codés par la banque pour identifier les variants de fragment Fc qui (i) se lient à hFcRn avec une plus grande activité de liaison à pH 6 qu'un fragment Fc témoin et (ii) ont peu ou pas d'activité de liaison à hFcRn à pH 7,4, tel que déterminé par interférométrie de bio-couche ;

    (3) construction d'un acide nucléique codant pour un variant polypeptidique Fc comprenant un variant de fragment Fc identifié en (2), dans lequel la concentration d'un polypeptide Fc témoin, qui comprend un fragment Fc témoin plutôt que le variant de fragment Fc, est connu pour diminuer linéairement dans le temps lorsqu'il est administré à un animal in vivo ;

    (4) introduction de l'acide nucléique de (3) dans une cellule hôte et culture de la cellule hôte dans des conditions telles que le variant polypeptidique Fc codé par l'acide nucléique peut être exprimé ;

    (5) récupération du variant polypeptidique Fc de la masse cellulaire ou du milieu de culture cellulaire ;

    (6) administration du variant polypeptidique Fc à un premier animal non humain et administration du polypeptide Fc témoin à un second animal non humain ; et

    (7) surveillance des concentrations du variant polypeptidique Fc et du polypeptide Fc témoin dans le sang périphérique dans le temps après l'administration, identifiant ainsi un variant de fragment Fc qui confère une demi-vie in vivo plus longue et une plus grande exposition sur un variant polypeptidique Fc ;

    dans lequel le polypeptide Fc témoin a la même séquence d'acides aminés que le variant polypeptidique Fc à l'exception que le polypeptide Fc témoin ne contient pas l'insertion comprenant 4 à 20 acides aminés randomisés.


     
    27. Procédé selon l'une quelconque des revendications 24 à 26, dans lequel le fragment Fc témoin comprend une séquence d'acides aminés sélectionnée parmi :

    SEQ ID NO : 1 (fragment Fc d'IgG1),

    SEQ ID NO : 3 (fragment Fc d'IgG2),

    SEQ ID NO : 5 (fragment Fc d'IgG3), et

    SEQ ID NO : 7 (fragment Fc d'IgG4).


     
    28. Procédé selon l'une quelconque des revendications 24 à 27, dans lequel le fragment Fc témoin comprend la séquence d'acides aminés des acides aminés 165 à 171 de SEQ ID NO : 3.
     
    29. Composition pharmaceutique comprenant le variant polypeptidique Fc selon l'une quelconque des revendications 1 à 20 et un vecteur, excipient ou diluant pharmaceutiquement acceptables.
     
    30. Variant polypeptidique Fc selon l'une quelconque des revendications 1 à 20, ou la composition pharmaceutique selon la revendication 28, pour son utilisation dans le traitement d'une maladie chronique.
     
    31. Variant polypeptidique Fc pour son utilisation, ou la composition pharmaceutique pour son utilisation, selon la revendication 30 dans lequel l'utilisation est destinée au traitement d'un patient humain, et la maladie chronique est sélectionnée dans le groupe de maladies chroniques constitué par :

    (a) une indication oncologique,

    (b) une affection inflammatoire,

    (c) une affection liée aux os,

    (d) une affection métabolique,

    (e) une affection neurologique, et

    (f) la douleur chronique.


     




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    REFERENCES CITED IN THE DESCRIPTION



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    Patent documents cited in the description




    Non-patent literature cited in the description