(19)
(11)EP 3 647 419 A1

(12)EUROPEAN PATENT APPLICATION

(43)Date of publication:
06.05.2020 Bulletin 2020/19

(21)Application number: 18204106.1

(22)Date of filing:  02.11.2018
(51)Int. Cl.: 
C12N 9/52  (2006.01)
C07K 16/28  (2006.01)
A61K 38/16  (2006.01)
A61K 47/68  (2017.01)
(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
Designated Extension States:
BA ME
Designated Validation States:
KH MA MD TN

(71)Applicant: NBE Therapeutics AG
4057 Basel (CH)

(72)Inventors:
  • GRAWUNDER, Ulf
    4423 Hersberg (CH)
  • BEERLI, Roger
    8106 Adlikon bei Regensdorf (CH)
  • GEBLEUX, Remy
    4600 Olten (CH)
  • PUORGER, Chasper
    4057 Basel (CH)
  • DI GIROLAMO, Salvatore
    4056 Basel (CH)
  • LIPPS, Georg
    4125 Riehen (CH)

(74)Representative: Michalski Hüttermann & Partner Patentanwälte mbB 
Speditionstraße 21
40221 Düsseldorf
40221 Düsseldorf (DE)

  


(54)SORTASE F AND ITS USE IN METHODS FOR CONJUGATION


(57) The present invention relates to a method for producing a conjugate of two substrates, the method comprising the steps of (a) providing two substrates, each substrate being independently selected from the one or more of the groups consisting of small molecules, and proteins, and (b) enzymatically conjugating the two substrates using a sortase F enzyme, or a catalytic domain thereof. One substrate comprises a sortase F recognition motif, while the other substrate comprises at least one motif selected from a Glyn motif, an Alan motif, or a motif consisting of a mixture of Ala and Gly residues the motif totaling n residues, where n is an integer from ≥ 1 to ≤ 21. The reaction takes place in an aqueous reaction medium comprising a salt concentration of from about ≥ 0,01 to ≤ 3 M, thereby producing a conjugated product of the two substrates




Description

FIELD OF THE INVENTION



[0001] The present invention relates to sortase F and its use in methods for conjugation.

BACKGROUND OF THE INVENTION



[0002] Sortases are bacterial transpeptidase enzymes that anchor proteins to the cell wall of gram-positive bacteria or participate in pilus subunit polymerization. sortases sequence-specifically cleave a peptide bond within a cell wall sorting sequence (Ton-That H. et al., J. Biol. Chem., 275 (2000) 9876-9881), forming a thioester bond between the thiol group of an active site cysteine side chain and the carboxylic acid of the cleaved peptide bond (Frankel B.A. et al., Biochemistry, 44 (2005) 11188-11200). Subsequent nucleophilic attack by the amino group of a second peptide leads to formation of a new peptide bond.

[0003] Due to the high sequence specificity of this transpeptidation reaction, sortases are used to ligate or label proteins site-specifically with peptides or peptide-modified small molecules ("sortagging") (Popp M.W. et al., Nat. Chem. Biol., 3 (2007) 707-708). WO 2014/140317, the contents of which are incorporated herein by reference, refers to the use of a sortase enzyme to conjugate antibodies with low molecular weight toxins, and exemplifies the use of sortase A in this context. sortases can be divided into classes based on their amino acid sequence and their structure. Spirig et al., describes sortase classes A-F (Table 1) (Spirig T. et al., Mol. Microbiol., 82 (2011) 1044-1059), a system reflected in the conserved domain database of the NCBI, which however splits the sortase D class into two separate classes: sortase D_1 and sortase D_2.
Table 1: Overview of sortase enzyme family as disclosed in Spirig: Sortases are grouped according to sequence homology. The CD and PDB codes of representative structures are indicated, along with the most common recognition motifs and function.
ClassConserved domain (CD)Representative structure (PDB)sortase motifMain function
A cd06165 3FN6 LPxTG general
B cd05826 1QWZ NPxTG pilus polymerization, heme uptake
C cd05827 2WIJ LPxTG pilus polymerization
D cd05828, 2LN7, LPxTA spore formation
  cd06166 4D70    
E cd05830 5GO5 LAxTG pilus attachment, general
F Cd05829 None available Unknown Unknown


[0004] As can be seen, little is known about sortase F and its characteristics.

[0005] Many sortases have low reaction rates, limiting their widespread biotechnological use. Indeed, most applications rely on the well-studied sortase A from S. aureus, which has a relatively high reaction rate. Further, sortase A variants with improved catalytic efficiency and modified sequence specificity have been reported (Chen I. et al., PNAS USA, 108 (2011) 11399-11404; Dorr B.M. et al., PNAS USA, 111 (2014) 13343-13348).

[0006] Sortases of class B are also well characterized and considerable knowledge has been gathered on sortases of classes C and D (reviewed in Bradshaw W.J. et al., FEBS J., 282 (2015) 2097-2114). In contrast, much less is known on sortases of class F. This applies, inter alia, to the respective recognition motifs, and to conditions that allow successful conjugation.

[0007] There is considerable interest in developing sortases and their methods of use for efficient sortase-mediated conjugation/sortagging.

[0008] In this context, the present invention refers to a method for producing a conjugate of two substrates, the method comprising the steps of:
  1. a. providing two substrates, each substrate being independently selected from the one or more of the groups consisting of:
    1. i. small molecules, and
    2. ii. proteins,
    and
  2. b. enzymatically conjugating the two substrates using a sortase F enzyme, or a catalytic domain thereof,
    wherein
    • one substrate comprises a sortase F recognition motif, while
    • the other substrate comprises at least one motif selected from
      • a Glyn motif,
      • an Alan motif, or
      • a motif consisting of a mixture of Ala and Gly residues
    the motif totaling n residues, where n is an integer from ≥ 1 to ≤ 21, wherein further the reaction takes place in an aqueous reaction medium comprising a salt concentration of from about ≥ 0,01 to ≤ 3 M, thereby producing a conjugated product of the two substrates.


[0009] The inventors have surprisingly found that a particular, relatively high salt concentration is necessary in order to achieve an efficient conjugation reaction in the context of the inventive method.

[0010] As used herein, the terms "Glyn motif" and "Alan motif" refer to peptides comprising Ala or Gly, with a length of N residues

[0011] Preferably, the lower limit of the salt concentration is ≥ 0,05 M; more preferably ≥ 0,1 M; ≥ 0,15 M; ≥ 0,2 M; ≥ 0,25 M; ≥ 0,3 M; ≥ 0,35 M; ≥ 0,4 M; ≥ 0,45 M; ≥ 0,5 M; ≥ 0,55 M; ≥ 0,6 M; ≥ 0,65 M; ≥ 0,7 M; ≥ 0,75 M; ≥ 0,8 M; ≥ 0,85 M; ≥ 0,9 M; ≥ 0,95 M; ≥ 1 M; ≥ 1,05 M; ≥ 1,1 M; ≥ 1,15 M; ≥ 1,2 M; ≥ 1,25 M; ≥ 1,3 M; ≥ 1,35 M; ≥ 1,4 M; ≥ 1,45 M; ≥ 1,5 M; ≥ 1,55 M; ≥ 1,6 M; ≥ 1,65 M; ≥ 1,7 M; ≥ 1,75 M; ≥ 1,8 M; ≥ 1,85 M; ≥ 1,9 M; ≥ 1,95 M; most preferably ≥ 2 M.

[0012] Preferably, the upper limit of the salt concentration is ≤ 3 M; more preferably ≤ 2,95 M; ≤ 2,9 M; ≤ 2,85 M; ≤ 2,8 M; ≤ 2,75 M; ≤ 2,7 M; ≤ 2,65 M; ≤ 2,6 M; ≤ 2,55 M; ≤ 2,5 M; ≤ 2,45 M; ≤ 2,4 M; ≤ 2,35 M; ≤ 2,3 M; ≤ 2,25 M; ≤ 2,2 M; ≤ 2,15 M; ≤ 2,1 M; ≤ 2,05 M; ≤ 2 M; ≤ 1,95 M; ≤ 1,9 M; ≤ 1,85 M; ≤ 1,8 M; ≤ 1,75 M; ≤ 1,7 M; ≤ 1,65 M; ≤ 1,6 M; ≤ 1,55 M; ≤ 1,5 M; ≤ 1,45 M; ≤ 1,4 M; ≤ 1,35 M; ≤ 1,3 M; ≤ 1,25 M; ≤ 1,2 M; ≤ 1,15 M; ≤ 1,1 M; ≤ 1,05 M; and most preferably ≤ 1 M.

[0013] All possible combinations of the above upper and lower limits shall be deemed as disclosed herewith.

[0014] The concept of the sortase F recognition motif is shown in Fig. 1. In a preferred embodiment, the sortase F recognition motif is a motif as shown in Fig. 1.

[0015] In another preferred embodiment, the sortase F recognition motif is a motif as shown in Table 2.

DESCRIPTION OF THE FIGURES



[0016] 

Figure 1. Amino acid sequence of the sortase F recognition motif according to relative frequency of reaction of pentapeptide sequences varied over the naturally-occurring amino acids at individual positions. The letter J stands for the amino acid residue Leucine or Isoleucine.

Figure 2. A preferred embodiment of the small molecule toxin of formula (i), comprising a linker with an alkyldiamino group of the form NH2-(CH2)m-NH2, where m ≥ 1 and ≤ 11, preferably m=2, linked to an oligoglycine motif (n is from 1 to 21, preferably from 1 to 5).

Figure 3. Multiple sequence alignment of sortase F representatives having Conserved Domain cd05829 in the NCBI Conserved Domain Database. The species name is given in the first column of the first block (e.g., Propionibacterium acnes), and the database accession number in the first column of the subsequent blocks. The catalytic triad formed of histidine (H), cysteine (C) and arginine (R) residues are in bold. Signal peptides are underlined in the first block. Variable length spacer residues are given in lower case.

Figure 4. Phylogenetic tree representing distances between example sortases, with sortase classes indicated, built with a Neighborhood Joining algorithm using the programs VAST (Gilbrat, J.F., Current Opinion in Structural Biology, 6 (1996), 377-385) and MAFFT (Kuraku, Zmasek, Nishimura, Katoh 2013 (Nucleic Acids Research 41:W22-W28).

Figure 5. sortase activity assay: sortase F activity was evaluated relative to its conjugation of a 60 kDa protein (comprising an N-terminal Gly6-motif) to the following FITC-labeled peptide substrates (from left to right): (1) LPxTGE (known to be a sortase A/C substrate), (2) DNPNTGDE (sortase B substrate), (3) ENPxTNAGT (sortase B substrate), (4) KLPxTASN (sortase D substrate), and (5) ALAxTGSE (sortase E substrate). Conjugation reaction products were loaded on an SDS-PAGE gel and visualized by FITC emission using the FLA-9000 Imager from Fujifilm. sortase F is most active relative to the LPxTGE peptide. (M) PageRuler Plus Prestained Protein Ladder.

Figure 6. (A) sortase F activity as a function of pH after 6 hours conjugation, with detection of conjugated product (products were loaded on an SDS-PAGE gel and visualized by FITC emission): pH was varied by 1 unit increments between 4 and 12, while maintaining other parameters (sortase F concentration, temperature, salt concentration, etc.) constant. pH was preferentially between 8 and 9; (B) sortase F activity as a function of ionic strength after 6 hours conjugation, with detection of conjugated product (FITC emission): NaCl concentration was varied between 0 and 3 M, while maintaining other parameters (sortase F concentration, temperature, pH, etc.) constant. Salt concentration was preferentially between 0.5 and 1.5 M.

Figure 7. (A) Initial sortase F activity rate as a function of pH, with detection of conjugated product (RP-HPLC): pH was varied between 4.5 to 10.5, while maintaining other parameters (sortase F concentration, temperature, salt concentration, etc.) constant. pH was preferentially between 8 and 9; (B) initial sortase F activity rate as a function of ionic strength, with detection of conjugated product (RP-HPLC): NaCl concentration was varied between 0 and 3 M, while maintaining other parameters (sortase F concentration, temperature, pH, etc.) constant. The salt concentration optimum was found to be above 250 mM of NaCl.

Figure 8. (A) Resulting product amount from sortase F activity as a function of pH after 24 hours conjugation, with detection of conjugated product (RP-HPLC): pH was varied between 4.5 to 10.5, while maintaining other parameters (sortase F concentration, temperature, salt concentration, etc.) constant. pH was preferentially around 8; (B) Resulting product amount from sortase F activity as a function of ionic strength after 24 hours conjugation, with detection of conjugated product (RP-HPLC): NaCl concentration was varied between 0 and 3 M, while maintaining other parameters (sortase F concentration, temperature, pH, etc.) constant. Salt concentration was preferentially above 250 mM for maximal sortase F activity, and more preferential above 750 mM.

Figure 9. Temperature dependence of sortase F activity rate in conjugating a quencher-fluorophore peptide (representing a sortase F recognition motif) and a triglycine, as measured with a fluorescence spectrometer. The temperature optimum was found to be about 25°C, and the reaction rate decreased at temperatures above 40°C.

Figure 10. Dose-response curve of the in vitro cell killing assays on the CD30-high Karpas-299 and CD30-negative EMT6 cell lines. The sortase F conjugated CD30-targeting ADCs provide effective in vitro cell killing in an antigen-dependent manner. Viable cells were quantified using a luminescent cell viability assay.


DETAILED DESCRIPTION OF THE INVENTION



[0017] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0018] All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.

[0019] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains.

Preferred embodiments relative to the substrates



[0020] The method of the invention comprises providing two substrates, each substrate being independently selected from the one or more of the groups consisting of:
  1. i. small molecules, and
  2. ii. proteins.


[0021] In one embodiment, one substrate is an antibody, an antibody-based binding protein or an antibody fragment, and the second substrate is a small molecule.

[0022] "Small molecules" as used herein refer to small molecules having a molecular weight not exceeding 2500 Daltons. This small molecule may particularly be a toxin or a label.

[0023] "Small molecule toxins", also referred to as toxins, as used herein refer to cytotoxic or cytostatic small molecules, preferably having a molecular weight not exceeding 2500 Daltons. Such small molecule toxins include, but are not limited to, a maytansinoid (e.g., maytansinol or DM1 maytansinoid), a taxane, a calicheamicin, a cemadotin, a monomethylauristatin (e.g., monomethylauristatin E or monomethylauristatin F), a pyrrolobenzodiazepine (PBD), a campothecin, an amanitin, a cryptophycin or an anthracycline. Small molecule toxins also include vincristine and prednisone. In various embodiments, the small molecule toxin can be an antimetabolite (e.g., an antifolate such as methotrexate, a fluoropyrimidine such as 5-fluorouracil, cytosine arabinoside, or an analogue of purine or adenosine); an intercalating agent (for example, an anthracycline such as doxorubicin, PNU-159682, daunomycin, epirabicin, idarubicin, mitomycin-C, dactinomycin, or mithramycin, or other intercalating agents such as pyrrolobenzodiazepine); a DNA-reactive agent such as calicheamicins, tiancimycins, and other enediynes; a platinum derivative (e.g., cisplatin or carboplatin); an alkylating agent (e.g., nitrogen mustard, melphalan, chlorambucil, busulphan, cyclophosphamide, ifosfamide nitrosoureas or thiotepa); an RNA polymerase inhibitor such as α-amanitin; an antimitotic agent (e.g., a vinca alkaloid such as vincristine, or a taxoid such as paclitaxel or docetaxel); a topoisomerase inhibitor (for example, etoposide, teniposide, amsacrine, topotecan); a cell cycle inhibitor (for example, a flavopyridol); or a microbtubule agent (e.g., an epothilone, a tubulysine, a pre-tubulysine, discodermolide analog, or eleutherobin analog). A small molecule toxin can be a proteosome inhibitor or a topoisomerase inhibitor such as bortezomib, amsacrine, etoposide, etoposide phosphate, teniposide, or doxorubicin. A small molecule toxin can include therapeutic radioisotopes such as iodine (131I), yttrium (90Y), lutetium (177Lu), actinium (225Ac), praseodymium, astatine (At), rhenium (Re), bismuth (Bi or Bi), and rhodium (Rh). Antiangiogenic agents that may be small molecule toxins include linomide, bevacuzimab, angiostatin, and razoxane.

[0024] In another embodiment, the small molecule is a toxin selected from the group consisting of: maytansinoids, auristatins, anthracyclins, calcheamicins, tubulysins, duocarmycins, taxanes, and pyrrolbenzodiazepines.

[0025] In another embodiment, the small molecule toxin is selected from PNU-159682 as described in Quintieri et al. (Clin. Cancer Res., 11 (2005) 1608-1617) and derivatives thereof, maytansine, monomethyl auristatin MMAE, and monomethyl auristatin MMAF.

[0026] In another embodiment, the toxin has the following formula (i),

conjugated, directly or indirectly, to the other substrate via its wavy line.

[0027] "Small molecule labels", also referred to as labels, as used herein refer to a fluorescent label (including a fluorescent dye or a fluorescent protein), a near infrared dye, a chromophore label, a radioisotope label containing iodine (e.g., 125I), gallium (67Ga), indium (111I), technetium (99mTc), phosphorus (32P), carbon (14C), tritium (3H), and/or other radioisotope (e.g., a radioactive ion).

[0028] "Proteins" refer to molecules made up of stretches of amino acid monomers linked by peptide bonds. In a preferred embodiment, proteins are selected from one or more of the following:
  1. i. oligopeptides,
  2. ii. antibodies,
  3. iii. antibody-based binding proteins containing at least one antibody-derived VH, VL, or CH immunoglobulin domain,
  4. iv. antibody fragments binding to a receptor, antigen, growth factor, cytokine and/or hormone,
  5. v. antibody mimetics, and
  6. vi. modified antibody formats.


[0029] In one embodiment, the protein is useful in a biochemical assay, such as avidin or streptavidin.

[0030] "Oligopeptides" as used herein refer to short, generally 2 to 20, stretches of amino acid monomers linked by peptide bonds. Some peptides may present inhibitory or toxic properties that make them useful as therapeutics.

[0031] "Antibodies", also synonymously called "immunoglobulins" (Ig), generally comprise four polypeptide chains, two heavy (H) chains and two light (L) chains, and are therefore multimeric proteins, or an equivalent Ig homologue thereof (e.g., a camelid nanobody, which comprises only a heavy chain, single domain antibodies (dAbs) which can be either derived from a heavy or light chain); including full length functional mutants, variants, or derivatives thereof (including, but not limited to, murine, chimeric, humanized and fully human antibodies, which retain the essential epitope binding features of an Ig molecule, and including dual specific, bispecific, multispecific, and dual variable domain immunoglobulins; Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) and allotype.

[0032] An "antibody-based binding protein", as used herein, may represent any protein that contains at least one antibody-derived VH, VL, or CH immunoglobulin domain in the context of other non-immunoglobulin, or non-antibody derived components. Such antibody-based proteins include, but are not limited to (i) Fc-fusion proteins of binding proteins, including receptors or receptor components with all or parts of the immunoglobulin CH domains, (ii) binding proteins, in which VH and or VL domains are coupled to alternative molecular scaffolds, or (iii) molecules, in which immunoglobulin VH, and/or VL, and/or CH domains are combined and/or assembled in a fashion not normally found in naturally occurring antibodies or antibody fragments.

[0033] An "antibody fragment", as used herein, relates to a molecule comprising at least one polypeptide chain derived from an antibody that is not full length, including, but not limited to (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CH1) domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a heavy chain portion of a Fab (Fd) fragment, which consists of the VH and CH1 domains; (iv) a variable fragment (Fv) fragment, which consists of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) fragment, which comprises a single variable domain; (vi) an isolated complementarity determining region (CDR); (vii) a single chain Fv Fragment (scFv); (viii) a diabody, which is a bivalent, bispecific antibody in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites; and (ix) a linear antibody, which comprises a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementarity light chain polypeptides, form a pair of antigen binding regions; and (x) other non-full length portions of immunoglobulin heavy and/or light chains, or mutants, variants, or derivatives thereof, alone or in any combination.

[0034] The term "antibody mimetic", as used herein, refers to proteins not belonging to the immunoglobulin family, and even non-proteins such as aptamers, or synthetic polymers. Some types have an antibody-like beta-sheet structure. Potential advantages of "antibody mimetics" or "alternative scaffolds" over antibodies are better solubility, higher tissue penetration, higher stability towards heat and enzymes, and comparatively low production costs.

[0035] The term "modified antibody format", as used herein, encompasses antibody-drug-conjugates, polyalkylene oxide-modified scFv, Monobodies, Diabodies, Camelid Antibodies, Domain Antibodies, bi- or trispecific antibodies, IgA, or two IgG structures joined by a J chain and a secretory component, shark antibodies, new world primate framework + non-new world primate CDR, IgG4 antibodies with hinge region removed, IgG with two additional binding sites engineered into the CH3 domains, antibodies with altered Fc region to enhance affinity for Fc gamma receptors, dimerised constructs comprising CH3+VL+VH, and the like.

[0036] In one embodiment, the antibody, antibody-based binding protein or antibody fragment binds to at least one of the mouse and/or cynomolgus and/or protein targets selected from the group consisting of: CD30, HER2, ROR1, ROR2, mesothelin, and/or or CS1.

[0037] In one embodiment, the antibody, antibody-based binding protein or antibody fragment comprises all, substantially all, or part of the sequence of anti-CD30 antibody Ac10 as disclosed in US2008213289A1.

[0038] Preferably, the antibody comprises at least the 6 CDR sequences or the VH/VL sequences of Ac10.

Preferred embodiments relative to the sortase F recognition motif



[0039] The method of the invention comprises providing two substrates, wherein one substrate comprises a sortase F recognition motif.

[0040] In a preferred embodiment, the sortase F recognition motif corresponds to a recognition motif according to Figure 1, preferably where the residue at each position is a residue having a relative frequency of at least 4%, and preferably of at least 8%, according to Figure 1.

[0041] The data of Figure 1, i.e., the approximate percentage preference of individual amino acids at different positions along the pentameric sortase F recognition motif, rounded to one decimal point, is given below in tabular form (Positions 1-5 of the pentamer are listed in N to C-terminal direction). The amino acid residue at each position having a relative frequency of at least 4% are given in bold:
Table 2: Amino acid composition of sortase F recognition motifs
Residue (single letter code)Position 1Position 2Position 3Position 4Position 5
Alanine (A) 0 0 8,1 17,3 0
Arginine (R) 0 0 12,6 0 0
Asparagine (N) 0,3 0 7,8 0 0
Aspartate (D) 0,1 0 12,7 0,3 0
Cysteine (C) 0 0 0,8 1,4 0
Glutamine (Q) 0,5 0 13,4 0 0
Glutamate (E) 0,4 0 3,1 0 0
Glycine (G) 0 0 2,3 1,8 100
Histidine (H) 0 0 5,2 0 0
Leucine/Isoleucine (J) 66,5 0 7,5 0 0
Lysine (K) 0 0 8,5 0 0
Methionine (M) 12,1 0 3,0 1,4 0
Phenylalanine (F) 2,4 0 1,3 0 0
Proline (P) 4,7 100 0 0 0
Serine (S) 0,3 0 3,5 6,2 0
Threonine (T) 0 0 3,8 71,1 0
Tryptophan (W) 9,5 0 3,2 0 0
Tyrosine (Y) 1,3 0 1,9 0,4 0
Valine (V) 2,0 0 1,4 0 0


[0042] In one embodiment, the sortase F recognition motif is LPXTG, where X is any amino acid with the exception of proline.

[0043] In a preferred embodiment, X is selected from amino acid residues having the following single letter codes: A, R, N, D, Q, I, L and K. In a preferred embodiment, the sortase F recognition motif is LPQTG (in N- to C-terminal orientation).

[0044] In a preferred embodiment, prior to sortase F-mediated conjugation, the sortase F recognition motif is linked, directly or indirectly, to one or more affinity purification and/or detection tags. Affinity purification tags, for example, may be such as to be removed as part of the transpeptidation reaction. Affinity resins can then be used to remove non-conjugated substrates and to enrich fully conjugated substrates. Affinity purification or detection tags include, but are not limited to, small peptide tags (e.g. histidine tags, strep-tag, twinstrep-tag, MYC-tag or HA-tag) or larger protein affinity purification tags (e.g. maltose-binding protein (MBP) tag, Glutathione-S-transferase (GST) tag, or Chitin-binding tag).

[0045] In the case where the substrate comprising the sortase F recognition motif is an oligopeptide, an antibody, an antibody-based binding protein containing at least one antibody-derived VH, VL, or CH immunoglobulin domain, an antibody fragment binding to a receptor, antigen, growth factor, cytokine and/or hormone, an antibody mimetic, or a modified antibody format (jointly referred to as "protein substrates"), the sortase F recognition motif is preferably located at one or more C-termini.

[0046] In the case of one substrate being an antibody, wherein the sortase F recognition motif is at the C-terminus, and the other substrate being a small molecule toxin, the average toxin ("drug") to antibody ratio can be determined by methods known to the skilled person, and is generally referred to as the DAR (Drug to Antibody Ratio). In a preferred embodiment, the DAR is any number between 1 and 4.

[0047] In case the other substrate is a small molecule toxin, the average toxin ("drug") to antibody ratio can be determined by methods known to the skilled person, and is generally referred to as the DAR (Drug to Antibody Ratio). In a preferred embodiment, the DAR is any number between 1 and 4.

[0048] In some cases (e.g. at the C-terminus of the Ig kappa light chains, see: Beerli et al., PloS One 10, (2015) e131177) it may be beneficial to add additional amino acids (hereafter "spacer amino acids") between the C-terminus of protein substrates and the sortase recognition motif. In the case of sortase A, this has been shown to improve sortase enzyme conjugation efficiencies of payloads to a binding protein. In the case of Ig kappa light chains, it was observed that by adding 5 amino acids between the last C-terminal cysteine amino acid of the Ig kappa light chain and the sortase recognition motif improved the kinetic of conjugation, so that the C-termini of Ig kappa light chains and Ig heavy chains could be conjugated with similar kinetics (see: Beerli et al., PloS One 10, (2015) e131177). Therefore, it is another preferred embodiment that optionally ≥ 1 and ≤ 11 amino acids are added in between the last C-terminal amino acid of a protein substrate and the sortase F recognition motif. In a preferred embodiment, a GnS peptide (wherein n is an integer is from 1 to 10, preferably from 1 to 5) is added between the last C-terminal amino acid of a protein substrate and the sortase F recognition motif. Finally, in another preferred embodiment, additional amino acids between the C-terminus of a protein substrate and the sortase F recognition motif may beneficially be included that comprise a sequence and/or linker that is cleavable by hydrolysis, by a pH change or by a change in redox potential, or that is cleavable by a non-sortase enzyme, e.g., by proteases.

[0049] In the case where the substrate comprising the sortase F recognition motif is a small molecule, this motif may be joined directly thereto, or indirectly via a chemical or peptidic linker. Such chemical or peptidic linkers are further discussed herebelow.

Preferred embodiments relative to the Glyn or Alan motif



[0050] The method of the invention comprises providing two substrates, wherein one substrate comprises a Glyn motif, an Alan motif or a motif consisting of a mixture of Ala and Gly residues totaling n residues, where n is any integer from 1 to 21.

[0051] Preferably, the substrate being a small molecule comprises the Glyn motif, the Alan motif, or the motif consisting of a mixture of Ala and Gly residues.

[0052] Preferably, the substrate being a small molecule comprisies the Glyn motif. Preferably, n is an integer from ≥ 1 to ≤ 5

[0053] In the case where the substrate comprising a Glyn motif, an Alan motif or a motif consisting of a mixture of Ala and Gly residues (jointly referred to as "Gly/Ala motif") is a protein substrate, the Gly/Ala motif is preferably located at one or more N-termini. Preferential embodiments described hereabove with regards to spacer amino acids apply in this context as well.

[0054] In the case where the substrate comprising the Gly/Ala motif is a small molecule, this motif may be joined directly thereto, or indirectly via a chemical or peptidic linker. Such linkers include, but are not limited to, one or more of: an oligopeptide linker (including cleavable and non-cleavable oligopeptide linkers), a hydrazine linker, a thiourea linker, a self-immolative linker, a succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC) linker, a disulfide linker, a thioether linker and a maleimide linker. The skilled person understands that further linkers may be suitable. Such linkers may be non-cleavable or may be cleaved by changes in pH, redox potential or specific intracellular or extracellular enzymes. Cleavable oligopeptide linkers include protease-cleavable linkers. It is understood that the linker may comprise combinations of the above. For example, the linker may be a valine-citrulline PAB linker.

[0055] In the embodiment where the small molecule toxin is of formula (i), it is preferred that the linker comprise an alkyldiamino group of the form NH2-(CH2)m-NH2, where m ≥ 1 and ≤ 11, preferably m=2, such that one amino group is directly linked at the wavy line of formula (i) to form an amide bond. It is moreover preferred that the second amino group is linked to the Gly/Ala motif. A preferred embodiment of the small molecule toxin comprising a Gly/Ala motif is depicted in Figure 2.

Preferred embodiments relative to sortase F or a catalytic domain thereof



[0056] The present invention refers to a method for producing a conjugate of two substrates, the method comprising enzymatically-conjugating the two substrates using sortase F, or a catalytic domain thereof.

[0057] In a one embodiment, sortase F, or a catalytic domain thereof, is capable of binding and acting upon a sortase F recognition motif as defined herein.

[0058] In one embodiment, sortase F, or a catalytic domain thereof, comprises conserved domain cd05829 according to the NCBI Conserved Domain Database (CDD). The CDD is a protein annotation resource that consists of a collection of well-annotated multiple sequence alignment models for ancient domains and full-length proteins. Sequences can be searched for conserved domains at the CDD database (ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). See, Marchler-Bauer et al. (2015) Nuclic Acids Res. 43(D) 222-226.

[0059] Figure 3 shows a multiple sequence alignment of sortase F representatives having Conserved Domain cd05829 in the CDD. The species name is given in the first column of the first block (e.g., Propionibacterium acnes), and the database accession number in the first column of the subsequent blocks. The catalytic triad formed of histidine (H), cysteine (C) and arginine (R) residues are outlined. Signal peptides are underlined in the first block. Variable length spacer residues are given in lower case.

[0060] In one embodiment, sortase F, or a catalytic domain thereof, originates from the class of Actinomycetales. In a preferred embodiment, sortase F, or a catalytic domain thereof, originates from the order of Propionibacterinaea. In a preferred embodiment, sortase F, or a catalytic domain thereof, originates from the family of Propionibacteriaceae. In a preferred embodiment, sortase F, or a catalytic domain thereof, originates from the genus of Propionibacterium. In particular, it is preferred that sortase F, or a catalytic domain thereof, originates from Propionibacterium acnes (P. acnes).

[0061] Figure 4 shows a phylogenetic tree representing distances between example sortases, with sortase classes indicated, built with a Neighborhood Joining algorithm using the programs VAST (Gilbrat, J.F., Current Opinion in Structural Biology, 6 (1996), 377-385) and MAFFT (Kuraku, Zmasek, Nishimura, Katoh 2013 (Nucleic Acids Research 41:W22-W28). In a preferred embodiment, the class of sortase F is defined, as depicted here, as comprising sortases having a distance of up to and including 3, preferably up to an including 2.5 from sortase F of P. acnes.

[0062] In one embodiment, sortase F, or a catalytic domain thereof, comprises or consists of the sequence of sortase F of P. acnes (SEQ ID NO. 1). In one embodiment, one or more predicted non-essential amino acid residues in SEQ ID NO. 1 are removed or replaced with another amino acid residue. A "non-essential" amino acid residue is a residue that can be altered from SEQ ID NO. 1 without abolishing or substantially altering its sortase activity (also referred to as "biological activity"). Suitably, its sortase activity is at least 20%, 40%, 60%, 70% or 80% 100%, 500%, 1000% or more of the activity of the sortase F enzyme of SEQ ID NO. 1.

[0063] An "essential" amino acid residue is a residue that, when altered from the wild-type sequence of a reference polypeptide, results in abolition of an activity of the parent molecule such that less than 20% of the wild-type activity is present. For example, such essential amino acid residues may include those that are conserved in reference polypeptides across different species, including those sequences that are conserved in the enzymatic sites of reference polypeptides from various sources. In one embodiment, sortase F, or a catalytic domain thereof, comprises a catalytic triad of residues essential for enzymatic activity. In a preferred embodiment, the catalytic triad consists of a histidine residue, a cysteine residue and an arginine residue.

[0064] In one embodiment, sortase F, or a catalytic domain thereof, is a biologically active variant (i.e., having a sortase activity of at least 20%, 40%, 60%, 70% or 80% 100%, 500%, 1000% or more of the activity of the sortase F enzyme of SEQ ID NO. 1) of the sortase F enzyme of SEQ ID NO. 1 that has a sequence comprising at least 30%, 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, usually about 90% to 95% or more, and typically about 97% or 98% or more sequence identity to SEQ ID NO. 1, as determined by sequence alignment programs described elsewhere herein, particularly using default parameters.

[0065] A biologically active variant of a SEQ ID NO. 1 may differ from SEQ ID NO. 1 generally by as much 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10 amino acid residues, including about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or even 1 amino acid residues. In some embodiments, a biologically active variant differs from SEQ ID NO. 1 by at least one but by less than 15, 10 or 5 amino acid residues. In other embodiments, it differs from SEQ ID NO. 1 by at least one residue but less than 20%, 15%, 10% or 5% of the residues.

[0066] The percent identity between two sequences is a function of the number of identical positions shared by two aligned sequences, considering the number of gaps, and the length of each gap, which need to be introduced for optimal alignment. To determine the percent identity of two amino acid sequences, sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid sequence for optimal alignment). The comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms known to the skilled person in the art. In comparing two sequences, it may be advantageous to first remove any signal peptide sequences known to the skilled person.

[0067] In one embodiment, polypeptides can be aligned to all or part of SEQ ID NO. 1 using a BLAST alignment tool. A local alignment consists of a pair of sequence segments, one from each of the sequences being compared. A modification of Smith-Waterman or Sellers algorithms will find all segment pairs whose scores cannot be improved by extension or trimming, called high-scoring segment pairs (HSPs). The results of the BLAST alignments include statistical measures to indicate the likelihood that the BLAST score can be expected from chance alone. In a preferred embodiment, a BLAST alignment is used with the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1. The BLOSUM62 matrix assigns a probability score for each position in an alignment that is based on the frequency with which that substitution is known to occur among consensus blocks within related proteins. In a preferred embodiment, the algorithm of Needleman and Wunsch (J Mol Biol. 48:443-453, 1970) is used to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.

[0068] The sequence similarity score, S, may be calculated from the number of gaps and substitutions associated with the aligned sequence; higher similarity scores indicate a more significant alignment. Substitution scores are given by a look-up table. Gap scores are typically calculated as the sum of G, the gap opening penalty and L, the gap extension penalty. For a gap of length n, the gap cost would be G+Ln. The choice of gap costs, G and L is empirical, but it is customary to choose a high value for G (10-15), e.g., 11, and a low value for L (1-2) e.g., 1.

[0069] The program GAP uses either a BLOSUM62 or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters are a BLOSUM62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Cabios. 4:11-17, 1989) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In one embodiment, GAP Version 10 (GCG, Accelrys, San Diego, Calif.) is used with the following parameters: GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, PNAS USA. 89:10915-10919, 1992).

[0070] In one embodiment, sortase F, or a catalytic domain thereof, comprises an amino acid sequence that can be optimally aligned with all or part of SEQ ID NO. 1 to generate a sequence similarity (or bit) score of at least about 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440,450,460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, or more, including all integers and ranges in between.

[0071] In a further aspect, the case by case analysis may be performed using PSI-BLAST (Position-Specific iterative Basic Local Alignment Search Tool). PSI-BLAST derives a position-specific scoring matrix (PSSM) or profile from the multiple sequence alignment of sequences detected above a given score threshold using protein-protein BLAST. This PSSM is used to further search the database for new matches, and is updated for subsequent iterations with these newly detected sequences. Thus, PSI-BLAST provides a means of detecting distant relationships between proteins.

[0072] In another aspect, the case by case analysis is performed using HHpred, a method for sequence database searching and structure prediction that is more sensitive than BLAST or PSI-BLAST in finding remote homologs. HHpred is based on the pairwise comparison of profile hidden Markov models (HMMs). HHpred accepts a single query sequence or a multiple alignment as input. Search options include local or global alignment and scoring secondary structure similarity. HHpred can produce pairwise query-template sequence alignments, merged query-template multiple alignments (e.g. for transitive searches), as well as 3D structural models calculated by the MODELLER software from HHpred alignments.

[0073] All or part of the sequence of SEQ ID NO. 1 can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences that may be sortase F. Such databases include, but are not limited to, UniProtKB, SwissProt, PIR, PRF and PDB. BLAST protein searches can be performed with, e.g., the BLASTX program, score=50, wordlength=3 to obtain amino acid sequences homologous to SEQ ID NO. 1. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTX) can be used.

[0074] In certain embodiments, the length of SEQ ID NO. 1 aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100%. The amino acid residues at corresponding positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position.

[0075] sortase F may be a biologically active variant which has, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 or more contiguous amino acids, including all integers in between, relative to SEQ ID NO. 1.

[0076] The expectation value (E) threshold is a statistical measure of the number of expected matches in a random database. The lower the e-value, the more likely the match is to be significant. The smaller the E-Value, the more significant the alignment. For example, an alignment having an E value of e<-117 >means that a sequence with a similar score is very unlikely to occur simply by chance. In one embodiment, sortase F, or a catalytic domain thereof, is a biologically active variant having an alignment to all or part of SEQ ID NO. 1 with an E value of less than -0.1, -0.01, -0.001, -0.0001, or a lower value.

[0077] In one embodiment, sortase F, or a catalytic domain thereof, may correspond to SEQ ID NO. 1 that is altered by various ways including by amino acid substitutions, deletions, truncations, and/or insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (PNAS USA. 82: 488-492, 1985); Kunkel et al., (Methods in Enzymol. 154: 367-382, 1987), U.S. Pat. No. 4,873,192, Watson, J. D. et al., ("Molecular Biology of the Gene," Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).

[0078] Such variants may result from, for example, genetic polymorphism and/or from human manipulation. In one embodiment, mutations are introduced randomly along all or part of the nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1, such as by saturation mutagenesis, and the resultant mutants are screened for sortase activity to identify mutants which retain that activity. Following mutagenesis of the coding sequences, the encoded peptide can be expressed recombinantly and the activity of the peptide can be determined. In one embodiment, sortase F, or the catalytic domain thereof, is the result of mutation of a wild type sortase F, and preferably is the result of mutation of SEQ ID NO. 1, to maintain or improve its conjugation efficiency.

[0079] Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of reference polypeptides. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify polypeptide variants (Arkin and Yourvan, PNAS USA 89: 7811-7815, 1992; Delgrave et al., Protein Engineering. 6: 327-331, 1993). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be desirable as discussed in more detail below.

[0080] sortase F may be a polypeptide variant that contains conservative amino acid substitutions at various locations along the sequence of all or part of SEQ ID NO. 1. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:

[0081] Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.

[0082] Basic: The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having a basic side chain include arginine, lysine and histidine.

[0083] Charged: The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine).

[0084] Hydrophobic: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.

[0085] Neutral/polar: The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.

[0086] This description also characterizes certain amino acids as "small" since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, "small" amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains. The structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the α-amino group, as well as the α-carbon. Several amino acid similarity matrices (e.g., PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff et al., (1978), A model of evolutionary change in proteins. Matrices for determining distance relationships In M. O. Dayhoff, (ed.), Atlas of protein sequence and structure, Vol. 5, pp. 345-358, National Biomedical Research Foundation, Washington D.C.; and by Gonnet et al., (Science. 256: 14430-1445, 1992), however, include proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a "small" amino acid.

[0087] Amino acid residues can be further sub-classified as cyclic or non-cyclic, and aromatic or non-aromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always non-aromatic. Dependent on their structural properties, amino acid residues may fall in two or more classes.

Preferred embodiments relative to the salt concentration



[0088] The present invention refers to a method for producing a conjugate of two substrates wherein the reaction takes place in an aqueous environment comprising a salt concentration of from ≥ 0.5 to ≤ 3 M.

[0089] In one embodiment, the salt concentration is adjusted to a range from ≥ 0.5 to ≤ 1.5 M.

[0090] This embodiment is particularly preferred in case the antibody, antibody-based binding protein or antibody fragment comprises at least
  • one antibody-derived VH, VL, or CH immunoglobulin domain,
  • an antibody fragment binding to a receptor, antigen, growth factor, cytokine and/or hormone, an antibody mimetic, or
  • a modified antibody format,
and wherein the second substrate is a small molecule,

[0091] In one embodiment, the salt is a chloride salt. In another embodiment, the salt is a sodium, lithium or potassium salt. In a preferred embodiment, the salt is sodium chloride.

Further preferred embodiments



[0092] In one embodiment, the reaction medium is essentially free of Zn, Rb, Mg, Ni, Ca and/or Mn cations.

[0093] Indeed, it is surprising that the reaction does not require any of these cations, as such cations are required by some sortases of classes outside of sortase F.

[0094] As used herein, the term "essentially free of" means that the aqueous solution comprises ≤ 10 mM, preferably ≤ 5 mM, ≤ 1 mM, ≤ 0,5 mM, ≤ 0,1 mM, ≤ 0,05 mM, ≤ 10 µM, ≤ 5 µM, ≤ 1 µM, ≤ 0,5 µM, ≤ 0,1 µM, ≤ 0,05 µM, ≤ 10 nM, ≤ 5 nM, ≤ 1 nM, ≤ 0,5 nM, ≤ 0,1 nM, ≤ 0,05 nM, most preferably 0,01 nM.

[0095] In one embodiment, the temperature in the aqueous reaction medium is adjusted, at least for a given time period, to a range from ≥ 3 to ≤ 45°C, preferably from ≥ 15 to ≤ 40°C, more preferably from ≥ 20 to ≤ 30°C, and most preferably at around 25°C.

[0096] In one embodiment, the pH in the aqueous reaction medium is adjusted, at least for a given time period, to a range from ≥ 4.5 to ≤ 10.5, more preferably from ≥ 6.5 to ≤ 8.5, and even more preferably of from ≥ 7.0 to ≤ 8.0.

[0097] In one embodiment, the reaction takes place over a time period of 1h to 10 days, and preferably 3 days.

[0098] In one embodiment, one or more reducing agents are added to the aqueous environment before, during and/or after the conjugation reaction.

[0099] Preferred reducing agents include, but are not limited to, 1,4-dithiothreitol (DTT) and tris-2-carboxyethylphosphine hydrochloride (TCEP-HCl). Such reducing agents may be added during purification of the conjugated product.

[0100] In one embodiment, one or more of the following are added to the aqueous environment of the conjugation reaction:
  • glycerol
  • organic solvents such as, but not limited to, dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO).


[0101] Such organic solvents are selected by the skilled person to be compatible with the substrates.

[0102] In one embodiment wherein:
  • one substrate is a protein substrate comprising at least one sortase recognition motif,
  • the second substrate is a small molecule comprising a Gly/Ala motif, it is preferred that the small molecule be added to the reaction in an amount corresponding to:
    1. a. 2 to 50, preferably 5 to 20, and more preferably around 10 molar equivalents per molar equivalent of sortase F recognition motif; and/or
    2. b. 10 molar equivalents per molar equivalent of sortase F.

EXAMPLES



[0103] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0104] All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.

[0105] Example 1: Cloning and expression of recombinant sortase F enzyme from P. acnes. The ORF of sortase F from P. acnes KPA171202 is found in GenBank or Uniprot under: AAT82533.1 or Q6A9N3, respectively, and has SEQ ID No 1 herein
Table 3: Sequence of sortase F enzyme from P. acnes
SEQ ID NO. 1

 


[0106] The coding region for sortase F fragment of P. acnes, as provided in SEQ ID NO. 1 (but with an N-terminal MS peptide and a C-terminal TwinStrep-comprising tag: AGSWSHPQFEKGGGSGGGSGGSAWSHPQFEK), was custom synthesized in a proprietary bacterial expression vector at GenScript, and introduced into the p7X expression vector backbone (see: Geertsma E., James C. Samuelson (ed.), Enzyme Engineering: Methods and Protocols, Methods in Molecular Biology, vol. 978, DOI 10.1007/978-1-62703-293-3_10, © Springer Science+Business Media New York 2013). E. coli BL21(DE3), transformed with the p7X vector comprising the sortase F sequence, was cultured overnight at 37 °C in lysogeny broth (LB) medium with 50 µg/mL kanamycin until an OD600 = 0.6 was reached. IPTG was then added to a final concentration of 0.5 mM and protein expression was induced for 24 hours at 16°C. Cells were then harvested by centrifugation and washed (100 mM Tris/HCl, pH 8.0, 300 mM NaCl, 1 mM EDTA), prior to a second centrifugation and then resuspension in lysis buffer (100 mM Tris/HCl, pH 8.0, 300 mM NaCl, 1 mM EDTA, 0.1% Triton X 100, 1 mg/mL lysozyme). Cells were incubated on ice 30 min., and then lysed by sonication. Clarified supernatant was purified by StrepTactin affinity chromatography (IBA GmbH) following the manufacturer's instructions, adjusting the buffer to 500 mM NaCl and 5 mM DTT.

[0107] Fractions of >90% purity, as judged by SDS-PAGE, were then consolidated and diluted 10-fold with 10 mM sodium phosphate, pH 7.0, and purified on a column using HiTrap SP XL with a linear gradient in 10 mM sodium phosphate, pH 7.0, 1 M NaCl. sortase F-containing fractions were pooled and dialyzed against 50 mM sodium phosphate, pH 8.5, 500 mM NaCl, 1 mM EDTA and 10% glycerol. Optionally, cooling was undertaken and/or protease inhibitors were added to limit degradation.

Example 2: sortase activity assay



[0108] sortase F, in a concentration range from 2.5 to 20 µM, was incubated with FITC-labelled LPXTGE, DNPNTGDE, ENPXTNAGT, KLPXTASN and ALAXTGSE peptides (where X was varied over the natural amino acids), and 10 µM of a 60 kDa protein comprising an N-terminal Gly6-motif. The reaction was performed at 25°C for 6 to 24 hours, and stopped by addition of reducing SDS-PAGE loading buffer, followed by 5 min. incubation at 95°C. Reaction products were loaded on a 12% SDS- PAGE gel and run according to standard protocols. Labelled reaction products were visualized by FITC emission using the FLA-9000 Imager from Fujifilm, and quantified using ImageJ software. As per Figure 5, sortase F is principally active on the LPXTGE peptide.

Example 3: Determination of preferential sortase F recognition motif



[0109] sortase F sequence specificity analysis was conducted in accordance with Puorger C., et al. (Biochemistry, 56 (2017), 2641-2650). Briefly, 250 µM of fluorescein-labelled peptide mixture (with each position being independently varied over the 20 naturally-occurring amino acids), and 250 µM biotin-labelled nucleophile (GGGK-biotin) were incubated with 5 µM of sortase F in 50 mM of sodium phosphate, pH 8.5, and 500 mM NaCl at 25°C. Conjugated products were isolated using magnetic streptavidin beads, separated by reverse phase HPLC and identified by MS. Ion intensities of the products were normalized based on initial abundance of the corresponding fluorescein-labelled peptide. Figure 1 presents the relative normalized abundance of the different amino acids within the sortase recognition motifs of the conjugated products.

Example 4: Optimization of sortase conjugation reaction conditions using a gel-based assay



[0110] sortase F, at concentration of 2.5 µM, was incubated with 10 µM of FITC-labelled LPQTGE peptide, 10 µM of a 60 kDa protein comprising an N-terminal Gly6-motif, and 5 mM DTT. The reaction was performed at 25°C over a period of 6 hours, independently varying the following parameters:
  • pH over a range of 4 to 12 by increments of 1 (using a mixture of boric and citric acid, along with phosphate buffer to achieved the desired pH), while maintaining the concentration of NaCl at 0.5 M;
  • NaCl concentration over a range of 0 to 3 M, while maintaining the pH at 8.5 (using 50 mM of sodium phosphate to establish the pH).


[0111] The reaction was stopped by addition of reducing SDS-PAGE loading buffer followed by 5 min. incubation at 95°C. Reaction products were loaded on a 12% SDS-PAGE gel and run according to standard protocols. Labelled reaction products were visualized by FITC emission using the FLA-9000 Imager from Fujifilm, and quantified using ImageJ software.

[0112] Figure 6 shows (A) sortase F activity as a function of pH after 6 hours conjugation, with detection of conjugated product (products were loaded on an SDS-PAGE gel and visualized by FITC emission): pH was varied by 1 unit increments between 4 and 12, while maintaining other parameters (sortase F concentration, temperature, salt concentration, etc.) constant. pH was preferentially between 8 and 9; (B) sortase F activity as a function of ionic strength after 6 hours conjugation, with detection of conjugated product (FITC emission): NaCl concentration was varied between 0 and 3 M, while maintaining other parameters (sortase F concentration, temperature, pH, etc.) constant. Salt concentration was preferentially between 0.5 and 1.5 M.

Example 5: Optimization of sortase conjugation reaction conditions using HPLC-based assay



[0113] sortase F, at concentration of 5 µM, was incubated with 250 µM of FITC-labelled LPQTGE peptide, 1 mM of GGGK-biotin, and 5 mM DTT. The reaction was performed at 25°C over a period of 24 hours (with 6 samplings in the period up to 3 hours), independently varying the following parameters:
  • pH over a range of 4 to 11 (using a mixture of boric and citric acid, along with phosphate buffer to achieved the desired pH), while the concentration of NaCl at 500 mM;
  • NaCl concentration over a range of 0 to 3 M, while maintaining the pH at 8.5 (using 50 mM of sodium phosphate to establish the desired pH).


[0114] The reaction was stopped by adding 95 µL of 0.05 M HCl per 5 µL of reaction mixture. Reaction products were loaded into XBridge C18 HPLC columns equilibrated in 0.1% NH4OH in 5% methanol. Elution was performed with a linear gradient from 0 to 100% of 0.1% NH4OH in 95% methanol over 10 mL at flow rate of 0.5 mL/min and a temperature of 30°C. Eluted peptides were detected by absorbance at 497 nm and fluorescence at 522 nm (excitation at 497 nm) and peak areas were quantified.

[0115] Initial rate of sortase F-mediated ligation under different pH and salt conditions was evaluated based on regression of the product concentrations over time within samples collected up to 3 hours reaction time. Figure 7 shows (A) initial sortase F activity as a function of pH, with detection of conjugated product (RP-HPLC): pH was varied between 4.5 to 10.5, while maintaining other parameters (sortase F concentration, temperature, salt concentration, etc.) constant. pH was preferentially between 8 and 9; (B) initial sortase F activity as a function of ionic strength, with detection of conjugated product (RP-HPLC): NaCl concentration was varied between 0 and 3 M, while maintaining other parameters (sortase F concentration, temperature, pH, etc.) constant. The salt concentration optimum was found to be above 250 mM of NaCl.

[0116] Product yields after 24 hours of sortase F-mediated ligation under different pH and salt conditions revealed that ligation was most efficient at slightly lower pH, i.e., from 7 to 8. Figure 8 shows (A) Resulting product amount from sortase F activity as a function of pH after 24 hours conjugation, with detection of conjugated product (RP-HPLC): pH was varied between 4.5 to 10.5, while maintaining other parameters (sortase F concentration, temperature, salt concentration, etc.) constant. pH was preferentially around 8; (B) Resulting product amount from sortase F activity as a function of ionic strength after 24 hours conjugation, with detection of conjugated product (RP-HPLC): NaCl concentration was varied between 0 and 3 M, while maintaining other parameters (sortase F concentration, temperature, pH, etc.) constant. Salt concentration was preferentially above 250 mM for maximal sortase F activity, and more preferential above 750 mM.

Example 6: Optimization of sortase conjugation reaction conditions using a fluorescence assay



[0117] sortase F, at concentration of 5 µM, was incubated with 250 µM of self-quenching peptide Dabcyl-QALPQTGET-Edans, 2 mM of triglycine in the following buffer: 50 mM Tris-HCl, 500 mM NaCl, 5mM DTT, pH 8.5. Reactions were performed over a period of 1 hour, varying the reaction temperature between 15 and 45°C and measuring fluorescence over time (using a plate reader or fluorescence spectrometer, with excitation at 350 nm and emission at 495 nm).

[0118] Initial rate of sortase F-mediated ligation at different temperatures was evaluated based on regression of the fluorescence over time. Figure 9 shows the temperature dependence of sortase F activity in conjugating a quencher-fluorophore peptide (representing a sortase F recognition motif) and a triglycine, as measured with a fluorescence spectrometer. The temperature optimum was found to be about 25°C, and the reaction rate decreased at temperatures above 40°C.

Example 7: Antibody expression and purification



[0119] The anti-CD30 antibody Ac10, with its sequence as described in US2008213289A1 but modified to include the following N-terminal sequence on its heavy chain (HC): LPQTGG-TwinStrep, was transiently expressed in HEK293T cells and purified as per Waldmeier et al. 2016.

Example 8: sortase-mediated antibody conjugation.



[0120] The above-mentioned toxin was conjugated to the heavy chain (HC) of AclO by incubating 10 µM of antibody with 200 µM glycine-modified toxin (glycine-modified anthracycline toxin as per Figure 2 with m=2 and n=3, obtained from Levena) and 18 µM sortase F in conjugation buffer (50 mM HEPES, pH 8.5, 1 mM CaCl2, 500 mM NaCl, 10% by vol. glycerol) for 3 days at 16°C. The reaction was stopped by passing it through a Protein A column (rProtein A Gravitrap column, GE Healthcare). Bound conjugate was eluted with 5 column volumes of elution buffer (0.1 M glycine pH 2.7, 50 nM NaCl, 10% by vol. glycerol), with 0.5 column volume fractions collected into tubes containing up to 5% v/v 1M HEPES (pH 8) base to neutralize the acid. Protein containing fractions were pooled and formulated in PBS without calcium or magnesium.

ADC analytics.



[0121] DAR was assessed by Reverse Phase Chromatography performed on a Polymer Labs PLRP 2.1mm x 5cm, 5 µm column run at 1 mL/min/70°C with a 25-min. linear gradient between 0.05% TFA/H2O and 0.05 % TFA/CH3CN. Samples were first treated by FabRICATOR at pH 8.0 at 37°C for 15 min. The DAR determined by Reverse Phase Chromatography was 1.93.

Example 9: ADC Cytotoxicity.



[0122] Cytotoxicity of the CD30-targeting ADC above was investigated using the CD30-expressing human cell line Karpas-299; CD30-negative EMT6 cells were used as control. For this, 5000 Karpas-299 cells and 1000 EMT6 cells, per well, were each plated on 96-well plates (excluding edge wells, which contained water) in 75 µL RPMI or DMEM, respectively, supplemented with 10% by vol. FCS, 100 IU/mL Pen-Strep-Fungizone and 2 mM L-Glutamine at a density of 6.66×104 or 1.33×104 cells per well, respectively. Cells were grown at 37°C in a humidified incubator at 5% CO2 atmosphere. After a 1-day incubation, ADC was added to respective wells in an amount of 25 µL of 3.5-fold serial dilutions in growth medium (starting ADC concentration of 280 µg/mL or 1.9 µg/mL for negative and positive cells, respectively). After 4 additional days, plates were removed from the incubator and equilibrated to room temperature. After approximately 30 min, 50 µL of CellTiter-Glo® 2.0 Luminescent Solution (Promega, G9243) was added to each well. After shaking the plates at 450 rpm for 5 min. followed by 10 min. incubation in the dark without shaking, luminescence was measured on a Spark 10M plate reader with an integration time of 1 second per well. Curves of luminescence versus ADC concentration (ng/mL) were fitted with Graphpad Prism Software.

[0123] Figure 10 shows the dose-response curve of the in vitro cell killing assays on the CD30-high Karpas-299 and CD30-negative EMT6 cell lines. The sortase F conjugated CD30-targeting ADC provides effective in vitro cell killing in an antigen dependent manner.

References



[0124] 

Ton-That H. et al., J. Biol. Chem., 275 (2000) 9876-9881

Frankel B.A. et al., Biochemistry, 44 (2005) 11188-11200

Popp M.W. et al., Nat. Chem. Biol., 3 (2007) 707-708).

Spirig T. et al., Mol. Microbiol., 82 (2011) 1044-1059

Chen I. etal., PNAS USA, 108 (2011) 11399-11404; Dorr B.M. etal., PNAS USA, 111 (2014) 13343-13348

Bradshaw W.J. et al., FEBS J., 282 (2015) 2097-2114

Gilbrat, J.F., Current Opinion in Structural Biology, 6 (1996), 377-385

Katoh, Standley 2013 (Molecular Biology and Evolution 30:772-780)

Kuraku, Zmasek, Nishimura, Katoh 2013 (Nucleic Acids Research 41:W22-W28).

Quintieri et al. (Clin. Cancer Res., 11 (2005) 1608-1617)

Beerli et al., PloS One 10, (2015) e131177

Marchler-Bauer et al. (2015) Nuclic Acids Res. 43(D) 222-226.

Gilbrat, J.F., Current Opinion in Structural Biology, 6 (1996), 377-385)

Katoh, Standley 2013 (Molecular Biology and Evolution 30:772-780).

Needleman and Wunsch (J Mol Biol. 48:443-453, 1970

E. Meyers and W. Miller (Cabios. 4:11-17,1989

Henikoff and Henikoff, PNAS USA. 89:10915-10919,1992

Kunkel (PNAS USA. 82: 488-492, 1985)

Kunkel et al., (Methods in Enzymol. 154: 367-382, 1987), U.S. Pat. No. 4,873,192

Watson, J. D. et al., ("Molecular Biology of the Gene," Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987)

Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.

Arkin and Yourvan, PNAS USA 89: 7811-7815, 1992

Delgrave et al., Protein Engineering. 6: 327-331, 1993

Dayhoff et al., (1978), A model of evolutionary change in proteins. Matrices for determining distance relationships In M. O. Dayhoff, (ed.), Atlas of protein sequence and structure, Vol. 5, pp. 345-358, National Biomedical Research Foundation, Washington D.C.

Gonnet et al., (Science. 256: 14430-1445, 1992



[0125] WO 2014/140317

[0126] WO 2016/102679

[0127] US2008213289A1

Sequences



[0128] The following sequences shown herein form part of the disclosure of the present application. A WIPO ST 25 compatible electronic sequence listing is provided with this application, too. For the avoidance of doubt, if discrepancies exist between the sequences in disclosure herein and the electronic sequence listing, the sequences in the present text shall be deemed to be the correct ones.




































Claims

1. A method for producing a conjugate of two substrates, the method comprising the steps of:

a. providing two substrates, each substrate being independently selected from the one or more of the groups consisting of:

i. small molecules, and

ii. proteins,

and

b. enzymatically conjugating the two substrates using a sortase F enzyme, or a catalytic domain thereof,
wherein

• one substrate comprises a sortase F recognition motif, while

• the other substrate comprises at least one motif selected from

• a Glyn motif,

• an Alan motif, or

• a motif consisting of a mixture of Ala and Gly residues

the motif totaling n residues, where n is an integer from ≥ 1 to ≤ 21,

wherein further the reaction takes place in an aqueous reaction medium comprising a salt concentration of from about ≥ 0,01 to ≤ 3 M,
thereby producing a conjugated product of the two substrates.
 
2. The method according to claim 1, wherein one substrate is an antibody, an antibody-based binding protein or an antibody fragment, and the second substrate is a small molecule.
 
3. The method according to any one of the aforementioned claims, wherein the small molecule is a toxin selected from the group consisting of: maytansinoids, auristatins, anthracyclins, calcheamicins, tubulysins, duocarmycins, taxanes, and pyrrolbenzodiazepines.
 
4. The method according to claim 3, wherein the toxin has the following formula (i),

conjugated, directly or indirectly, to the other substrate via its wavy line.
 
5. The method according to any one of claims 2 - 4, wherein the antibody, antibody-based binding protein or antibody fragment binds to at least one of the mouse and/or cynomolgus and/or protein targets selected from the group consisting of: CD30, HER2, ROR1, ROR2, mesothelin, and/or or CS1.
 
6. The method according to claim 5, wherein the antibody, antibody-based binding protein or antibody fragment comprises all, substantially all, or part of the sequence of anti-CD30 antibody Ac10 as disclosed in US2008213289A1.
 
7. The method according to any one of the aforementioned claims, wherein the sortase F recognition motif is LPXTG, where X is any amino acid with the exception of proline.
 
8. The method according to claim 7, wherein, in the sortase F recognition motif, X is selected from the amino acid residues having the following single letter codes: A, R, N, D, Q, I, L and K.
 
9. The method according to any one of the aforementioned claims, wherein, in a substrate being an antibody, an antibody-based binding protein or an antibody fragment, the sortase F recognition motif is at the N- and/or C-terminus thereof.
 
10. The method according to any one of the aforementioned claims, wherein the substrate being a small molecule comprises the Glyn motif, the Alan motif, or the motif consisting of a mixture of Ala and Gly residues.
 
11. The method according to any one of the aforementioned claims, wherein

a) the salt concentration is adjusted to a range from ≥ 0.5 to ≤ 1.5 M, and/or

b) the salt is a chloride salt, and/or

c) the salt is a sodium, lithium or potassium salt.


 
12. The method according any one of the aforementioned claims, wherein the reaction medium is essentially free of Zn, Rb, Mg, Ni, Ca and/or Mn cations.
 
13. The method according any one of the aforementioned claims, wherein

a) the temperature in the aqueous reaction medium is adjusted, at least for a given time period, to a range from ≥ 3 to ≤ 45°C, preferably from ≥ 15 to ≤ 40°C, more preferably from ≥ 20 to ≤ 30°C, and most preferably at around 25°C, and/or

b) the pH in the aqueous reaction medium is adjusted, at least for a given time period, to a range from ≥ 4.5 to ≤ 10.5, more preferably from ≥ 6.5 to ≤ 8.5, and even more preferably of from ≥ 7.0 to ≤ 8.0, and/pr.

c) one or more reducing agents are added to the aqueous environment before, during and/or after the conjugation reaction.


 
14. The method according any one of the aforementioned claims, wherein one or more of the following agents are added to the aqueous environment:

• glycerol

• organic solvents such as, but not limited to, dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO).


 
15. The method according any one of the aforementioned claims, wherein:

• one substrate is a protein substrate comprising at least one sortase F recognition motif,

• the second substrate is a small molecule comprising a Glyn motif, an Alan motif, or a motif consisting of a mixture of Ala and Gly residues

and wherein further the small molecule is added to the reaction in an amount corresponding to:

a. ≥ 2 to ≤ 50, preferably ≥ 5 to ≤ 20, and more preferably around 10 molar equivalents per molar equivalent of sortase F recognition motif; and/or

b. 10 molar equivalents per molar equivalent of sortase F.


 




Drawing












































REFERENCES CITED IN THE DESCRIPTION



This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description




Non-patent literature cited in the description