Methods for preparing antibodies

Abstract

The present invention particularly provides a method for improving the pairing of the heavy chain and light chain of an antibody (e.g., a bispecific antibody). Antibodies (e.g., bispecific antibodies), libraries, and methods for screening such libraries produced using such methods are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 845,594, filed on May 9, 2019, the contents of which are incorporated herein by reference in their entirety.

[0003] Submit sequence listing as ASCII text file

[0004] The contents of the following submitted ASCII text file are incorporated herein by reference in their entirety: Computer Readable Format (CRF) of a Sequence Listing (File Name: 146392047740SEQLIST.TXT, Record Date: May 4, 2020, Size: 9KB). Background Art

[0005] The development of bispecific antibodies as therapeutic agents for human diseases has great clinical potential. However, the production of bispecific antibodies in the form of IgG is challenging because the antibody heavy chain has evolved to bind to the antibody light chain in a relatively messy manner. As a result of this messy pairing, the simultaneous expression of two antibody heavy chains and two antibody light chains in a single cell naturally leads to confusion such as heavy chain homodimerization and heavy chain / light chain pairing.

[0006] One approach to circumvent the problem of heavy chain homodimerization, called “knobs-into-holes,” aims to H Mutations were introduced into the C3 domain to modify the contact interface, thus forcing the two different antibody heavy chains to pair. On one heavy chain, the original amino acids were replaced by amino acids with short side chains to form a "hole". Conversely, amino acids with large side chains were introduced into the other C H3 domains to form a "knob". By co-expressing these two heavy chains (and two identical light chains, which must apply to these two heavy chains), a high incidence of heterodimer formation ("knob-hole") compared to homodimer formation ("hole-hole" or "knob-hole") was observed (Ridgway, JB, Protein Eng. 9 (1996) 617-621; Merchant et al. "An efficient route to human bispecific IgG." Nat Biotechnol. 1998; 16: 677 81; Jackman et al. "Development of a two-part strategy to identify a therapeutic human bispecific antibody that inhibits IgE receptor signaling." J Biol Chem. 2010; 285: 20850 9; and WO 96 / 027011).

[0007] Due to the complex multi-domain heterodimer interactions within antibody Fab, minimizing the confusion of heavy chain / light chain becomes more difficult. Bispecific antibody formats designed to resolve heavy chain / light chain confusion include: DVD-Ig (dual variable domain Ig) (Nature Biotechnology 25, 1290-1297 (2007)); Cross-Ig (Schaefer W et al (2011) PNAS 108 (27): 11187-11192); Two-in-One Ig (Science 2009, 323, 1610); Antibodies (PNAS 92(15):7021-7025; 1995) and Lewis et al. (2014) "Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface." Nat Biotechnol 32,191-8; Liu et al. (2015) "A NovelAntibody Engineering Strategy for Making Monovalent Bispecific HeterodimericIgG Antibodies by Electrostatic Steering Mechanism." J Biol Chem. Publishedonline January 12, 2015, doi:10.1074 / jbc.M114.620260; Mazor et al. 2015. "Improving target cell specificity using a novel monovalent bispecific IgGdesign." Mabs. Published online January 26, 2015, doi:10.1080 / 19420862.2015.1007816; the strategies described in WO 2014 / 081955, WO 2014 / 082179 and WO 2014 / 150973.

[0008] However, there remains a need in the art for methods to reduce mispaired heavy chain / light chain byproducts and increase the yield of correctly assembled bispecific antibodies. Summary of the invention

[0009] Provided herein is a method for improving the preferential pairing of heavy and light chains of an antibody, the method comprising: modifying the light chain variable domain (V L ) at position 94 or V LIn some embodiments, the method comprises the step of replacing at least one amino acid at position 96 of the heavy chain variable domain from an uncharged residue to a charged residue, the charged residue being selected from the group consisting of aspartic acid (D), arginine (R), glutamic acid (E) and lysine (K), wherein the amino acid numbering is according to Kabat. In some embodiments, the method comprises the step of replacing each of the amino acids at position 94 and at position 96 from an uncharged residue to a charged residue. In some embodiments, the amino acid at position 94 is replaced by D. In some embodiments, the amino acid at position 96 is replaced by R. In some embodiments, the amino acid at position 94 is replaced by D and the amino acid at position 96 is replaced by R. In some embodiments, the heavy chain variable domain (V H ) is substituted from an uncharged residue to a charged residue selected from the group consisting of aspartic acid (D), arginine (R), glutamic acid (E) and lysine (K), wherein the amino acid numbering is according to Kabat. In some embodiments, V L The amino acid at position 94 of L The amino acid at position 96 of H The amino acid at position 95 of is substituted by D.

[0010] In some embodiments, the methods provided herein further comprise subjecting the antibody (e.g., an antibody that has been modified to improve preferential pairing of heavy and light chains) to at least one affinity maturation step, wherein V L The substituted amino acid at position 94 of is not random. Additionally or alternatively, in some embodiments, V L The substituted amino acid at position 96 of is not random. Additionally or alternatively, in some embodiments, V H The substituted amino acid at position 95 of is not random.

[0011] In some embodiments, the antibody is an antibody fragment selected from the group consisting of: Fab, Fab', F(ab')2, one-armed antibody, and scFv or Fv. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody comprises a human IgG Fc region. In some embodiments, the human IgG Fc region is a human IgG1 Fc region, a human IgG2 Fc region, a human IgG3 Fc region, or a human IgG4 Fc region. In some embodiments, the antibody is a monospecific antibody. In some embodiments, the antibody is a multispecific antibody.

[0012] In some embodiments, the multispecific antibody is a bispecific antibody. In some embodiments, the bispecific antibody comprises a first C H 2 domain (CH 21) First C H 3 domains (C H 31) Second C H 2 domain (C H 22) and the second C H 3 domains; among them, C H 32 is changed so that in C H 31 / C H 32 interface, one or more amino acid residues are replaced by one or more amino acid residues with larger side chain volumes, thereby H The surface of 32 is generated with C H 31 interacting protrusions, and among them C H 31 is changed so that in C H 31 / C H 32 interface, one or more amino acid residues are replaced by amino acid residues with smaller side chain volumes, thereby H 31 on the surface produced with C H In some embodiments, the bispecific antibody comprises a first C H 2 domain (C H 21) First C H 3 domains (C H 31) Second C H 2 domain (C H 22) and the second C H 3 domains; among them, C H 31 is changed so that in C H 31 / C H 32 interface, one or more amino acid residues are replaced by one or more amino acid residues with larger side chain volumes, thereby H 31 on the surface produced with C H 32 interacting protrusions, and among them C H 32 is changed so that in C H 31 / C H 32 interface, one or more amino acid residues are replaced by amino acid residues with smaller side chain volumes, thereby H The surface of 32 is generated with C H In some embodiments, the protrusion is a knob mutation. In some embodiments, the knob mutation comprises T366W, wherein the amino acid numbering is according to the EU index. In some embodiments, the cavity is a hole mutation. In some embodiments, the hole mutation comprises at least one, at least two, or all three of T366S, L368A, and Y407V, wherein the amino acid numbering is according to the EU index.

[0013] Also provided are antibodies produced by any one (or combination) of the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A and 1B High-resolution liquid chromatography mass spectrometry (LCMS) data of an anti-LGR5 / anti-IL4 bispecific antibody, a representative example of a low-yield BsIgG, are provided. Figure 1A The mass envelopes for charge states 38+ and 39+ are shown. Figure 1B The corresponding deconvoluted data are shown.

[0015] Figure 1C and 1D High-resolution LCMS data of an anti-SIRPα / anti-IL4 bispecific antibody, a representative example of an intermediate-yield BsIgG, is provided. Figure 1C The mass envelope plots for charge states 38+ and 39+ are shown. Figure 1D The corresponding deconvoluted data are shown.

[0016] Figure 1E and 1F High-resolution LCMS data of an anti-Met / anti-DR5 bispecific antibody, a representative example of a high-yield BsIgG, is provided. Figure 1E The mass envelope plots for charge states 38+ and 39+ are shown. Figure 1F The corresponding deconvoluted data are shown.

[0017] Figure 2 Results of experiments designed to determine the incorporation of C H 1 / C L Whether charge pair substitution mutations can increase the yield of BsIgG that displays a strong intrinsic HC / LC pairing preference.

[0018] Figure 3 The experimental design performed to investigate the mechanistic basis for preferential HC / LC pairing in anti-EGFR / anti-MET BsIgG and anti-IL-4 / anti-IL-13 BsIgG is described. The results of this experiment are provided in Table C.

[0019] Figure 4AProvided are light chain variable domains (V L ... L ). Amino acid residues are numbered according to Kabat. CDRs from the sequence definition of Kabat et al. Sequences of Proteins of Immunological Interest. Bethesda, MD: NIH, 1991 and the structure definition of Chothia and Lesk (1987) J Mol Biol 196: 901-917 are shaded.

[0020] Figure 4B Provided are heavy chain variable domains (V H ). Amino acid residues are numbered according to Kabat. CDRs from the sequence definition of Kabat et al. Sequences of Proteins of Immunological Interest. Bethesda, MD: NIH, 1991 and the structure definition of Chothia and Lesk (1987) J Mol Biol 196: 901-917 are shaded.

[0021] Figure 5A Provided are the results of experiments conducted to evaluate the contribution of complementarity determining regions (CDR) L3 and CDR H3 of the anti-EGFR arm of anti-EGFR / anti-MET bispecific antibodies to BsIgG yield. Also provided are the results of experiments conducted to evaluate the contribution of CDR L3 and CDR H3 of the anti-MET arm of anti-EGFR / anti-MET bispecific antibodies to BsIgG yield.

[0022] Figure 5B The results of experiments performed to evaluate the contribution of CDR L3 and CDR H3 of the anti-IL-4 arm of an anti-IL-4 / anti-IL-13 bispecific antibody to BsIgG yield are provided. The results of experiments performed to evaluate the contribution of CDR L3 and CDR H3 of the anti-IL-13 arm of an anti-IL-4 / anti-IL-13 bispecific antibody to BsIgG yield are also provided.

[0023] Figure 6 Results of experiments performed to evaluate the contribution of CDR-L1+CDR-H1, CDR-L2+CDR-H2, and CDR-L3+CDR-H3 to BsIgG yield of anti-EGFR / anti-MET bispecific antibodies are provided.

[0024] Figure 7 The X-ray structure of anti-MET Fab (PDB 4K3J) is provided, highlighting the CDR L3 and CDR H3 contact residues.

[0025] Fig. 8A Provided are light chain variable domains (V L domains) of the anti-IL-13 antibody lebrikizumab (see Ultsch et al. (2013) J Mol Biol 425:1330-1339) (SEQ ID NO:5) and the anti-IL-4 antibody 19C11 (see Spiess et al. (2013) J Biol Chem 288:265:83-93) (SEQ ID No:6). L ). The CDRs from the sequence definition of Kabat and the structure definition of Chothia and Lesk are shaded.

[0026] Figure 8B Provided are heavy chain variable domains (V H ). Amino acid residues are numbered according to Kabat. CDRs from the sequence definition of Kabat and the structure definition of Chothia and Lesk are shaded.

[0027] Fig. 9 The X-ray structure of anti-IL-13 Fab (PDB 4177) is provided, highlighting the CDR L3 and CDR H3 contact residues.

[0028] Fig. 10AExperimental results are provided, which were performed to evaluate the effects of the following on BsIgG yield: (a) replacing CDR L3 and CDR H3 of the anti-CD3 arm of an anti-CD3 / anti-HER2 bispecific antibody with CDR L3 and CDR H3 of anti-MET; (b) replacing CDR L3 and CDR H3 of the anti-HER2 arm of an anti-CD3 / anti-HER2 bispecific antibody with CDR L3 and CDR H3 of anti-MET; (c) replacing CDR L3 and CDR H3 of the anti-CD3 arm of an anti-CD3 / anti-HER2 bispecific antibody with CDR L3 and CDR H3 of anti-IL-13; and (d) replacing CDR L3 and CDR H3 of the anti-HER2 arm of an anti-CD3 / anti-HER2 bispecific antibody with CDR L3 and CDR H3 of anti-IL-13.

[0029] Fig. 10B Results of experiments are provided to evaluate the effects of: (a) replacing CDR L3 and CDR H3 of the anti-VEGFA arm of an anti-VEGFA / anti-ANG2 bispecific antibody with CDR L3 and CDR H3 of anti-MET; (b) replacing CDR L3 and CDR H3 of the anti-ANG2 arm of an anti-VEGFA / anti-ANG2 bispecific antibody with CDR L3 and CDR H3 of anti-MET; (c) replacing CDR L3 and CDR H3 of the anti-VEGFA arm of an anti-VEGFA / anti-ANG2 bispecific antibody with CDR L3 and CDR H3 of anti-IL-13; and (d) replacing CDR L3 and CDR H3 of the anti-ANG2 arm of an anti-VEGFA / anti-ANG2 bispecific antibody with CDR L3 and CDR H3 of anti-IL-13 on BsIgG yield.

[0030] Fig.11 Results are provided for experiments performed to assess the contribution of interchain disulfide bonds to BsIgG yields of the following bispecific antibodies: (1) anti-HER2 / anti-CD3; (2) anti-VEGFA / anti-VEGFC; (3) anti-EGFR / anti-MET; and (4) anti-IL13 / anti-IL-4. DETAILED DESCRIPTION

[0031] Bispecific antibodies are promising therapeutic agents because they have dual specificity, for example, payload is delivered to target, two signaling pathways are blocked simultaneously, immune cells are delivered to tumor cells, etc. However, the production of bispecific antibodies (e.g., bispecific IgG, or "BsIgG") remains a technical challenge because the co-expression of two antibody heavy chains and two antibody light chains in a cell may naturally lead to, for example, confusion of heavy chain homologation and heavy chain / light chain pairing. The method described herein is based on the applicant's discovery that preferred antibody heavy chain / antibody light chain will be strongly affected by specific amino acid position residues in CDR-H3 and CDR-L3. In addition, the applicant finds that in many cases, transferring these residues to the corresponding amino acid positions in other unrelated antibodies increases the yield of correctly assembled BsIgG.

[0032] Unless otherwise defined herein, the meaning of all scientific and technical terms used herein is the same as that generally understood by ordinary technicians in the field to which the present invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D Ed., John Wiley and Sons, New York (1994), and Hale & Margham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide a general dictionary of many terms used in the present invention. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. Numerical ranges include the numbers defining the range. Unless otherwise stated, nucleic acids are written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the direction of amino to carboxyl, respectively. For definitions and terms in the art, practitioners are particularly directed to Sambrook et al., 1989, and Ausubel FM et al., 1993. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

[0033] Numeric ranges are inclusive of the numbers defining the range.

[0034] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0035] The headings provided herein are not limitations of the various aspects or embodiments, which can be obtained by reference to the specification as a whole. Therefore, the terms defined below are more fully defined by reference to the specification as a whole.

[0036] definition

[0037] The term "antibody" is used in the broadest sense herein and refers to any immunoglobulin (Ig) molecule comprising two heavy chains and two light chains, as well as any fragment, mutant, variant or derivative thereof, as long as they exhibit the desired biological activity (e.g., epitope binding activity). Examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments described herein. The antibody may be a mouse antibody, a chimeric antibody, a human antibody, a humanized antibody and / or an affinity matured antibody.

[0038] As a frame of reference, immunoglobulin as used herein refers to the structure of immunoglobulin G (IgG). However, those skilled in the art will understand / recognize that antibodies of any immunoglobulin class can be used in the methods of the invention described herein. For the sake of clarity, an IgG molecule comprises a pair of heavy chains (HC) and a pair of light chains (LC). Each LC has a variable domain (V L ) and a constant domain (C L ), and each HC has a variable domain (V H ) and three constant domains (C H 1. C H 2 and C H 3) C H 1 and C H The two domains are connected by a hinge region. This structure is well known in the art.

[0039] In short, the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two light (L) chains and two heavy (H) chains (IgM antibodies are composed of 5 basic heterotetrameric units plus an additional polypeptide called the J chain and therefore contain 10 antigen binding sites, while secretory IgA antibodies can polymerize to form multivalent assemblies containing 2-5 basic 4-chain units plus the J chain). In the case of IgG, the 4-chain unit is usually about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the isotype of the H chain. Each H chain and L chain also has regularly spaced intrachain disulfide bonds. Each H chain has a variable domain (V H ), followed by three constant domains (C H ) (for each α and γ chain) and four C HEach L chain has a variable domain (V L ), followed by a constant domain (C L ). L With V H Align, and C L The first constant domain of the heavy chain (C H 1) Alignment. Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. H and V L The pairing together forms a single antigen binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6.

[0040] The L chains from any vertebrate can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Immunoglobulins can be assigned to different classes, or isotypes, based on the amino acid sequence of their heavy chain constant domains (CH). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains called α, δ, γ, ε, and μ, respectively. The γ and α classes are based on the amino acid sequence of the C constant domain. H Relatively minor differences in sequence and function further divide into subclasses, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2.

[0041] The term "C L The "domain" comprises the constant region domain of an immunoglobulin light chain, which, for example, extends from about Kabat positions 107A-216 (EU positions 108-214 (κ)). Eu / Kabat conversion tables for Kappa C domains are available online at www(dot)imgt(dot)org / IMGTScientificChart / Numbering / Hu_IGKCnber.html, and Eu / Kabat conversion tables for Lambda C domains are available online at www(dot)imgt(dot)org / IMGTScientificChart / Numbering / Hu_IGLCnber.html. C L Domain and V LThe domains are adjacent to and include the carboxyl terminus of the immunoglobulin light chain.

[0042] As used herein, the term "C H The "1 domain" comprises the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain, extending from about positions 114-223 in the Kabat numbering system (EU positions 118-215), for example. H 1 domain and the V H The domain is adjacent to the amino terminus of the hinge region, does not form part of the Fc region of the immunoglobulin heavy chain, and is capable of dimerizing with the immunoglobulin light chain constant domain (i.e., "CL"). The EU / Kabat conversion table for IgG1 heavy chain is available online at www(dot)imgt(dot)org / IMGTScientificChart / Numbering / Hu_IGHGnber.html.

[0043] According to the EU numbering system, the term “C H The "2 domain" generally comprises residues from about 231 to about 340 of IgG. H The 2 domain is unique in that it is not tightly paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two C H It is hypothesized that carbohydrates may provide substitution for domain-domain pairing and help stabilize C H 2 domain. Burton, Mol. Immunol. 22: 161-206 (1985).

[0044] The term "C H 3 domains" including the Fc region C H 2 domain (ie, from about amino acid residue 341 to about amino acid residue 447 of IgG according to the EU numbering system).

[0045] As used herein, the term "Fc region" generally refers to a dimeric complex comprising the C-terminal polypeptide sequence of an immunoglobulin heavy chain, wherein the C-terminal polypeptide sequence is obtainable by papain digestion of an intact antibody. The Fc region may comprise a native or variant Fc sequence. Although the boundaries of the Fc sequence of an immunoglobulin heavy chain may be different, the human IgG heavy chain Fc sequence comprises approximately Cys226, or from approximately Pro230 to the carboxyl terminus of the Fc sequence. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The Fc sequence of an immunoglobulin generally comprises two constant domains, C H 2 domains and C H 3 domains, and optionally includes C H 4 domains. "Fc polypeptide" herein refers to one of the polypeptides that make up the Fc region, such as a monomeric Fc. Fc polypeptides can be obtained from any suitable immunoglobulin, such as human IgG1, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD or IgM. Fc polypeptides can be obtained from mice, such as mouse IgG2a. The Fc region contains the carboxyl terminal portion of two H chains linked together by a disulfide. The effector function of the antibody is determined by the sequence in the Fc region, which is also the part recognized by the Fc receptor (FcR) present on certain types of cells. In some embodiments, the Fc polypeptide contains part or all of the wild-type hinge sequence (usually at its N-terminus). In some embodiments, the Fc polypeptide does not contain a functional or wild-type hinge sequence.

[0046] As used herein, the "Fc component" refers to the hinge region, C H 2 domain or C H 3 domains.

[0047] In certain embodiments, the Fc region comprises an IgG Fc region, preferably derived from a wild-type human IgG Fc region. In certain embodiments, the Fc region is derived from a "wild-type" mouse IgG, such as mouse IgG2a. "Wild-type" human IgG Fc or "wild-type" mouse IgG Fc refers to an amino acid sequence that occurs naturally in a human population or a mouse population, respectively. Of course, just as the Fc sequence may vary slightly between individuals, one or more changes may be made to the wild-type sequence and still be within the scope of the present invention. For example, the Fc region may comprise changes, such as mutations in glycosylation sites or the inclusion of non-natural amino acids.

[0048] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ) generally have a similar structure, wherein each domain comprises four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al. Kuby Immunology, 61st ed., WH Freeman and Co., page 91 (2007).) A single V H or V L The domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen may use V domains from antibodies that bind to that antigen, respectively. H or V L domains to separate and screen for complementary V L or V H Libraries of domains. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0049] As used herein, the term "hypervariable region" or "HVR" refers to individual regions of an antibody variable domain that are hypervariable in sequence and determine antigen binding specificity, such as the "complementarity determining region" ("CDR").

[0050] Typically, antibodies contain six CDRs; three in V H (CDR-H1, CDR-H2, CDR-H3), and three in V L (CDR-L1, CDR-L2, CDR-L3) in. Exemplary CDRs herein include:

[0051] (a) hypervariable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0052] (b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

[0053] (c) Antigenic contact points present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)).

[0054] Unless otherwise indicated, CDRs are determined according to the method described in Kabat et al. (supra). Those skilled in the art will appreciate that CDR names may also be determined according to the method described in Chothia (supra), McCallum (supra), or any other scientifically accepted nomenclature system.

[0055] "Framework" or "FR" refers to the variable domain residues other than the complementarity determining regions (CDRs). The FR of a variable domain is usually composed of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, CDR and FR sequences are usually in the V H (or V L ) in the following sequence: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0056] As used herein, the phrases "antigen binding arm", "target molecule binding arm", "target binding arm" and variants thereof refer to a component of an antibody (e.g., a bispecific antibody) that has the ability to specifically bind to a target of interest. Typically and preferably, the antigen binding arm is a complex of immunoglobulin polypeptide sequences, such as CDRs and / or variable domain sequences of immunoglobulin light and heavy chains.

[0057] "Target" or "target molecule" refers to the part recognized by the binding arm of an antibody (e.g., a bispecific antibody). For example, if the antibody is a multispecific antibody (e.g., a bispecific antibody), the target can be a single molecule or different molecules, or an epitope on a pathogen or tumor cell, depending on the background. It will be understood by those skilled in the art that the target is determined by the binding specificity of the target binding arm, and different target binding arms can recognize different targets. The target preferably binds to the antibody (e.g., a bispecific antibody) with an affinity greater than 1 μM Kd (according to methods known in the art, including methods described herein). Examples of target molecules include, but are not limited to, serum soluble proteins and / or their receptors, such as cytokines and / or cytokine receptors, adhesins, growth factors and / or their receptors, hormones, viral particles (e.g., RSV F protein, CMV, Staph A, influenza, hepatitis C virus), microorganisms (e.g., bacterial cell proteins, fungal cells), adhesins, CD proteins and their receptors.

[0058] As used herein, the term "interface" refers to the associative surface created by the interaction of one or more amino acids in a first antibody domain with one or more amino acids in a second antibody domain. Exemplary interfaces include, for example, C H 1 / C L 、V H / V L and C H 3 / C H 3. In some embodiments, the interface includes, for example, hydrogen bonds, electrostatic interactions, or salt bridges between the amino acids that form the interface.

[0059] An example of a "complete" or "full-length" antibody is one comprising an antigen-binding arm and a C L and at least the heavy chain constant domain C H 1. C H 2 and C H 3. The constant domain may be a native sequence constant domain (eg, a human native sequence constant domain) or an amino acid sequence variant thereof.

[0060] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody preparations, such variants are usually present in small amounts), each antibody comprising the population is identical and / or binds to the same epitope. Contrary to polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" represents that the feature of an antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibody to be produced by any ad hoc method. For example, monoclonal antibodies according to the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0061] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical compositions.

[0062] "Native antibodies" refer to natural immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (V H ), also called the variable heavy chain domain or heavy chain variable region, followed by three constant heavy chain domains (C H 1. C H 2 and C H 3). Similarly, from N-terminus to C-terminus, each light chain has a variable domain (V L ), also called the variable light chain domain or light chain variable region, followed by the constant light chain (C L ) domain.

[0063] "Monospecificity" refers to the ability of an antibody to bind only one epitope. "Bispecificity" refers to the ability of an antibody to bind to two different epitopes. "Multispecificity" refers to the ability of an antibody to bind to multiple epitopes. In certain embodiments, multispecific antibodies include bispecific antibodies. For bispecific and multispecific antibodies, the epitopes can be on the same antigen, or each epitope can be on a different antigen. In certain embodiments, bispecific antibodies bind to two different antigens. In certain embodiments, bispecific antibodies bind to two different epitopes on one antigen. In certain embodiments, the dissociation constant (Kd) for binding of a multispecific antibody (e.g., a bispecific antibody) to each epitope is about ≤1 μM, about ≤100 nM, about ≤10 nM, about ≤1 nM, about ≤0.1 nM, about ≤0.01 nM, or about ≤0.001 nM (e.g., about 10 -8 M or less, for example, from about 10 -8 M to about 10 -13 M, for example, from about 10 -9 M to about 10 -13 M)

[0064] The term "multispecific antibody" herein refers in the broadest sense to an antibody capable of binding to two or more antigens. In certain aspects, the multispecific antibody refers to a bispecific antibody, such as a human bispecific antibody, a humanized bispecific antibody, a chimeric bispecific antibody or a mouse bispecific antibody.

[0065] "Antibody fragments" comprise a portion of an intact antibody, preferably the V H and V L Examples of antibody fragments include Fab, Fab', F(ab')2, ScFv and Fv fragments; one-armed antibodies and multispecific antibodies formed from antibody fragments.

[0066] The antibody may be a "chimeric" antibody in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to the corresponding sequence of an antibody from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)). The chimeric antibodies of interest herein include primate antibodies comprising variable domain antigen binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0067] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies comprising minimal sequences derived from non-human antibodies. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies), wherein the residues in the hypervariable regions of the receptor are replaced by residues from the hypervariable regions of non-human species (donor antibodies), such as mice, rats, rabbits or non-human primates, which have the desired antibody specificity, affinity and function. In some cases, the framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues not found in receptor antibodies or donor antibodies. These modifications are intended to further improve antibody performance. In general, humanized antibodies will substantially include at least one variable domain in all, typically two variable domains, wherein all or substantially all hypervariable loops correspond to hypervariable loops of non-human immunoglobulins, and all or substantially all FRs are FRs of human immunoglobulin sequences. Humanized antibodies also optionally include at least a portion of an immunoglobulin constant region (Fc), which is typically a human immunoglobulin. For more details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0068] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0069] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or preparation other than the active ingredient, which is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0070] As used herein, "complex" or "complexed" refers to the association of two or more molecules that interact with each other through bonds and / or forces other than peptide bonds (e.g., van der Waals forces, hydrophobic forces, hydrophilic forces). In one embodiment, the complex is a heteromultimer. It should be understood that the term "protein complex" or "polypeptide complex" as used herein includes complexes of non-protein entities (e.g., including but not limited to chemical molecules such as toxins or detection agents) conjugated to proteins in the protein complex.

[0071] An antibody that "binds to an antigen of interest" (such as a monospecific or multispecific antibody) is an antibody that binds to an antigen (e.g., a protein) with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent targeting cells or tissues expressing the protein, and does not significantly cross-react with other proteins. In such embodiments, the degree of binding of the antibody to a "non-target" protein is less than about 10% of the degree of binding of the antibody to its specific target protein, as measured by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA) or ELISA. With respect to the binding of an antibody to a target molecule, the term "specific binding" or "specific binding to" or "specific for" an epitope on a specific polypeptide or specific polypeptide target means that a different binding can be measured from a nonspecific interaction (e.g., a nonspecific interaction may bind to bovine serum albumin or casein). For example, specific binding can be measured by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by competing with a control molecule similar to the target (an excess of unlabeled target). In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target, specific binding is indicated. As used herein, the term "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or an epitope on a particular polypeptide target can be expressed, for example, as having an affinity for the target of at least about 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM, or greater. In one embodiment, the term "specific binding" refers to the binding of a multispecific antibody to a specific polypeptide or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0072] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody, such as a bispecific or multispecific antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X to its partner Y can generally be represented by a dissociation constant (Kd). For example, Kd may be about 200 nM or less, about 150 nM or less, about 100 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 8 nM or less, about 6 nM or less, about 4 nM or less, about 2 nM or less, or about 1 nM or less. Affinity can be measured by conventional methods known in the art, including those described herein. Low affinity antibodies generally bind antigen slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigen quickly and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.

[0073] In one embodiment, "Kd" or "Kd value" is measured using surface plasmon resonance assay. For example, -2000 or -3000 (BIAcore, Inc., Piscataway, NJ) at 25 ° C. The response unit of the fixed target (e.g., antigen) CM5 chip is -10 (RU). Briefly, in one example, a carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5 μg / ml (about 0.2 μM) with 10 mM sodium acetate pH 4.8 and then injected at a flow rate of 5 μL / min to obtain about 10 response units (RU) of coupled protein. After the injection of the antigen, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (e.g., 0.78 nM to 500 nM) in PBS containing 0.05% Tween 20 (PBST) were injected at 25°C at a flow rate of approximately 25 μL / min. The association rates (k) were calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2). on ) and dissociation rate (k offThe equilibrium dissociation constant (KD) was calculated as the ratio k off / k on See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate measured by the surface plasmon resonance above exceeds 10 6 M -1 s -1 , the association rate can be determined by using a fluorescence quenching technique, i.e., measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25°C in the presence of increasing concentrations of antigen as measured in a spectrometer such as a spectrophotometer equipped with a stop-flow device (Aviv Instruments) or a 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) using a stirred cuvette.

[0074] "Biological activity" and "biological activity" and "biological properties" with respect to an antibody (e.g., a modified antibody, e.g., a modified bispecific antibody), such as an antibody (e.g., a bispecific antibody), a fragment or derivative thereof, prepared according to the methods provided herein means the ability to bind to a biological molecule, unless otherwise indicated.

[0075] When used to describe various heteromultimeric polypeptides, "isolated" means a heteromultimer that has been separated and / or recovered from the cell or cell culture in which it is expressed. Contaminant components of its natural environment are materials that would interfere with the diagnostic or therapeutic use of the heteromultimer, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In certain embodiments, the heteromultimer will be purified to (1) greater than 95% by weight protein (as determined by the Lowry method), and most preferably greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence (by use of a spinning cup sequencer), or (3) to homogeneity (by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain). Generally, however, an isolated polypeptide will be prepared by at least one purification step.

[0076] Antibodies (e.g., bispecific antibodies) are typically purified to substantial homogeneity. The phrases "substantially homogeneous," "substantially homogeneous form," and "substantially homogeneous" are used to indicate that the product is substantially free of byproducts (e.g., heavy chain homodimers and / or missense heavy chain / light chain pairs) derived from undesirable polypeptide combinations.

[0077] Expressed in terms of purity, substantially homogeneous means that the amount of by-products does not exceed 10 wt%, 9 wt%, 8 wt%, 7 wt%, 6 wt%, 4 wt%, 3 wt%, 2 wt% or 1 wt%, or less than 1 wt%. In one embodiment, the by-products are less than 5%.

[0078] "Biomolecule" refers to nucleic acids, proteins, carbohydrates, lipids, and combinations thereof. In one embodiment, the biomolecule exists in nature.

[0079] Unless the context indicates otherwise, the terms "first" polypeptide (such as heavy chain (HC1 or HC1) or light chain (LC1 or LC1)) and "second" polypeptide (such as heavy chain (HC2 or HC2) or light chain (LC2 or LC2)) and variants thereof are merely general identifiers and are not to be construed as identifying specific or particular polypeptides or components of the antibodies (e.g., bispecific antibodies) produced using the methods provided herein.

[0080] Unless otherwise noted, commercially available reagents mentioned in the examples were used according to the manufacturer's instructions. The source of those cells identified by ATCC accession numbers in the following examples and throughout the specification is the American Type Culture Collection, Manassas, VA. Unless otherwise indicated, the present invention uses standard procedures of recombinant DNA technology, such as those described above and in the following textbooks: Sambrook et al. (supra), Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, NY, 1989); Innis et al., PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc., NY, 1990); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press, Oxford, 1984); Freshney, Animal Cell Culture, 1987; Coligan et al., Current Protocols in Immunology, 1991.

[0081] References herein to "about" values ​​or parameters refer to the common error ranges of the corresponding values ​​that are readily known to those skilled in the art. References herein to "about" values ​​or parameters include (and describe) aspects related to the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0082] It should be understood that aspects and embodiments of the invention described herein include aspects and embodiments referred to as "comprising," "consisting of," and "consisting essentially of."

[0083] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0084] Methods for improving heavy chain / light chain pairing selectivity

[0085] The present application is based on the V L and (e.g., an antibody heavy chain or fragment thereof) V H Identification of residues at amino acid positions in the molecule that play a role in preferential heavy chain / light chain pairing

[0086] As described in further detail below, the methods provided herein include introducing one or more substitutions at specific residues within the variable domains of heavy and / or light chain polypeptides (e.g., particularly within CDR sequences). It will be appreciated by those of ordinary skill in the art that various numbering conventions may be used to specify specific amino acid residues within antibody variable region sequences. Commonly used numbering conventions include Kabat and EU index numbers (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Other conventions that include corrected or alternative numbering systems for variable domains include Chothia (Chothia C, Lesk AM (1987), J Mal Biol 196:901-917; Chothia et al. (1989), Nature 342:877-883), IMGT (Lefranc et al. (2003), Dev Comp Immunol 27:55-77) and AHo (Honegger A, Plückthun A (2001) J Mol Biol 309:657-670). These references provide amino acid sequence numbering schemes for immunoglobulin variable regions that define the positions of variable region amino acid residues in the antibody sequence.

[0087] Unless otherwise expressly stated herein, all references to immunoglobulin heavy chain variable regions (i.e., V H ) amino acid residues (i.e., numbers) are based on the Kabat numbering system, and unless otherwise specifically stated, all references to V L Unless otherwise specifically stated, all references to the immunoglobulin heavy chain constant region C residues appearing in the examples and claims are H 1. C H 2 and C H References to residues (i.e., numbering) are based on the EU system, with all references to C L The same applies to references to residues. Knowing that residue numbers are numbered according to the Kabat or EU index, one of ordinary skill can identify amino acid sequence modifications described herein according to any commonly used numbering convention.

[0088] Although items, components, or elements provided herein (eg, "antibody," "substitution," or "substitution mutation") may be described or claimed in the singular, the plural is contemplated to be within the scope thereof unless limitation to the singular is explicitly stated.

[0089] As described in more detail below, provided herein are methods for improving correct heavy chain / light chain pairing in antibodies (including bispecific antibodies), the methods comprising introducing one or more substitutions into V H and / or V L Also provided is a method for improving the yield of an antibody (e.g., a correctly assembled bispecific antibody), the method comprising introducing one or more substitutions into the V H and / or V L , wherein the yield of an antibody (e.g., a bispecific antibody) comprising substitutions produced using a particular method (e.g., a method known in the art) is higher than the yield of an unsubstituted antibody (e.g., a bispecific antibody) produced using the same method. Previous efforts have focused on introducing one or more amino acid substitutions into the framework region of the variable domain. See, e.g., Froning et al., Protein Science, 2017, 26:2021-38. Liu et al., J. Biol. Chem. 2015, 290:7535-62. Lewis et al., Nature Biotechnology, 2014, 32:191-202.

[0090] In some embodiments, the methods provided herein further comprise introducing modifications in the Fc region to promote heterodimerization of the two heavy chains of the antibody (eg, bispecific antibody).

[0091] Substitution mutations in the heavy and light chain variable domains

[0092] Provided herein is a method for improving the pairing (e.g., preferential pairing) of heavy and light chains of an antibody, the method comprising: modifying the light chain variable domain (V L ) at position 94 or V L The method comprises the step of replacing at least one amino acid at position 96 (e.g., the "original amino acid") from an uncharged residue to a charged residue selected from aspartic acid (D), arginine (R), glutamic acid (E) and lysine (K), wherein the amino acid numbering is according to Kabat. In some embodiments, the method comprises the step of replacing both the amino acid at position 94 and at position 96 (e.g., the original amino acid) from an uncharged residue to a charged residue (e.g., D, R, E or K). In some embodiments, the method comprises providing an antibody into which the above-mentioned substitutions are introduced. In some embodiments, the method comprises providing an antibody (such as a bispecific or multispecific antibody) that binds to one (or more) exemplary targets described elsewhere herein.

[0093] Preferential pairing describes the pairing pattern of a first polypeptide (e.g., a heavy chain) with a second polypeptide (e.g., a light chain) when pairing occurs between the first polypeptide and the second polypeptide in the presence of one or more additional different polypeptides (e.g., additional heavy chains and / or light chains). In some embodiments, when HC1 is co-expressed with at least LC1 and LC2, if the amount of HC1 / LC1 heavy chain-light chain pairing is greater than the amount of HC1 / LC2 pairing, then preferential pairing occurs between, for example, HC1 and LC1 of an antibody (e.g., a bispecific antibody). Similarly, when HC2 is co-expressed with at least LC1 and LC2, if the amount of HC2 / LC2 heavy chain-light chain pairing is greater than the amount of HC2 / LC1 pairing, then preferential pairing occurs between, for example, HC2 and LC2 of a multispecific antibody (e.g., a bispecific antibody). HC1 / LC1, HC1 / LC2, HC2 / LC1, and HC2 / LC2 pairing can be measured by methods known in the art (e.g., liquid chromatography mass spectrometry (LCMS)), as described in further detail elsewhere herein.

[0094] In some embodiments, the term "original amino acid" refers to the amino acid that was present at a particular position (e.g., V) immediately before substitution, e.g., with a charged amino acid (such as D, R, E, or K). LIn some embodiments, the term "uncharged amino acid" or "uncharged residue" refers to an amino acid that is neither positively charged (e.g., protonated) nor negatively charged (e.g., deprotonated) at physiological pH (e.g., a pH between about 6.8 and about 7.5, between about 6.9 and about 7.355, or between about 6.95 and 7.45). In some embodiments, a "charged amino acid" refers to an amino acid that is positively charged (e.g., protonated) or negatively charged (e.g., deprotonated) at physiological pH (e.g., a pH between about 6.8 and about 7.5, between about 6.9 and about 7.355, or between about 6.95 and 7.45). In some embodiments, an uncharged amino acid residue is an amino acid residue that is not D, R, E, or K. In some embodiments, an amino acid at position 94 (e.g., an original amino acid) is substituted with D. In some embodiments, an amino acid at position 96 (e.g., an original amino acid) is substituted with R. In some embodiments, the amino acid at position 94 (eg, the original amino acid) is substituted with D, and the amino acid at position 96 (eg, the original amino acid) is substituted with R.

[0095] In some embodiments, the method further comprises converting the heavy chain variable domain (V H ) is substituted from an uncharged residue to a charged residue selected from aspartic acid (D), arginine (R), glutamic acid (E) and lysine (K), wherein the amino acid numbering is according to Kabat. In some embodiments, the amino acid at position 95 (e.g., the original amino acid) is substituted with D. In some embodiments, V L The amino acid at position 94 (e.g., the original amino acid) is replaced by D, V L The amino acid at position 96 of (eg, the original amino acid) is substituted with R, and V H The amino acid at position 95 (eg, the original amino acid) is substituted with D.

[0096] Also provided is a method for improving the pairing of heavy and light chains of an antibody (e.g., homologous pairing, i.e., homologous V H and V L , Fab and preferential pairing of HC and LC), the method comprising the step of combining the heavy chain variable domain (V H ) from an uncharged residue to a charged residue selected from aspartic acid (D), arginine (R), glutamic acid (E) and lysine (K), wherein the amino acid numbering is according to Kabat. In some embodiments, the amino acid at position 95 (e.g., the original amino acid) is substituted with D.

[0097] The present invention also provides a method for improving the pairing (e.g., cognate pairing) of the heavy and light chains of an antibody, the method comprising: L ) at position 91, V L 94 or V L In some embodiments, the method comprises a step of replacing at least one amino acid at position 96 (e.g., "original amino acid") from a non-aromatic residue to an aromatic residue selected from tryptophan (W), phenylalanine (F) and tyrosine (Y), wherein the amino acid numbering is according to Kabat. In some embodiments, the method comprises a step of replacing at least two amino acids at position 91, position 94 or position 96 (e.g., original amino acids) from non-aromatic residues to aromatic residues selected from W, F and Y. In some embodiments, the method comprises a step of replacing amino acids at positions 94 and 96 (e.g., original amino acids) from non-aromatic residues to aromatic residues selected from W, F and Y. In some embodiments, the method comprises a step of replacing each of the amino acids at positions 91, 94 and 96 (e.g., original amino acids) from non-aromatic residues to aromatic residues selected from W, F and Y. In some embodiments, the method comprises providing an antibody in which the above-mentioned substitutions are introduced. In some embodiments, the method comprises providing an antibody (such as a bispecific or multispecific antibody) that binds to one (or more) exemplary targets described elsewhere herein.

[0098] In some embodiments, the "original amino acid" refers to the amino acid present in V immediately before substitution with an aromatic amino acid (e.g., W, F, and Y). L In some embodiments, the term "non-aromatic amino acid" or "non-aromatic residue" refers to an amino acid that does not contain an aromatic ring. In some embodiments, "non-aromatic residue" refers to an amino acid residue that is not W, F or Y.

[0099] In some embodiments, the amino acid at position 91 (e.g., the original amino acid) is substituted with Y. In some embodiments, the amino acid at position 94 (e.g., the original amino acid) is substituted with Y. In some embodiments, the amino acid at position 96 (e.g., the original amino acid) is substituted with W. In some embodiments, the amino acid at position 91 (e.g., the original amino acid) is substituted with Y, and the amino acid at position 94 (e.g., the original amino acid) is substituted with Y. In some embodiments, the amino acid at position 91 (e.g., the original amino acid) is substituted with Y, and the amino acid at position 96 (e.g., the original amino acid) is substituted with W. In some embodiments, the amino acid at position 91 (e.g., the original amino acid) is substituted with Y, the amino acid at position 94 (e.g., the original amino acid) is substituted with Y, and the amino acid at position 96 (e.g., the original amino acid) is substituted with W.

[0100] In some embodiments, the method further comprises converting the heavy chain variable domain (V H ) from an uncharged residue to a charged residue selected from aspartic acid (D), arginine (R), glutamic acid (E) and lysine (K), wherein the amino acid numbering is according to Kabat. In some embodiments, the method further comprises replacing the heavy chain variable domain (V H ) is substituted from a non-aromatic residue to an aromatic residue selected from the group consisting of tryptophan (W), phenylalanine (F) and tyrosine (Y).

[0101] In some embodiments, one or more of the above substitutions are introduced into an antibody fragment, e.g., comprising V L Domain and V H Such antibody fragments include, but are not limited to, Fab, Fab', monospecific F(ab')2, bispecific F(ab')2, one-armed antibodies, ScFv, Fv, etc.

[0102] In some embodiments, the antibody into which one or more of the above substitutions are introduced is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody comprises a kappa light chain. In some embodiments, the antibody comprises a lambda light chain. In certain embodiments, V L In some embodiments, V HIn some embodiments, the antibody comprises a mouse Fc region. In some embodiments, the antibody comprises a human Fc region, such as a human IgG Fc region (e.g., a human IgG1, human IgG2, human IgG3m, or human IgG4 Fc region). In some embodiments, the antibody is a monospecific antibody. In some embodiments, the antibody is a multispecific antibody, such as a bispecific antibody.

[0103] In certain embodiments, the antibody in which one or more of the above substitutions are introduced is a bispecific antibody comprising a first V H (V L 1) The first V of the pair L (V L 1) and the second V H (V H 2) The second V of the pair L (V L 2), where V L 1 contains Q38K substitution mutation, V H 1 contains Q39E substitution mutation, V L 2 contains Q38E substitution mutation, V H 2 comprises a Q39K substitution mutation, wherein the amino acid numbering is according to Kabat. In some embodiments, V L 1 contains Q38E substitution mutation, V H 1 contains Q39K substitution mutation, V L 2 contains Q38K substitution mutation, V H 2 comprises a Q39E substitution mutation, wherein the amino acid numbering is according to Kabat. It will be apparent to one of ordinary skill in the art that the term "V L 1", "V H 1", "V L 2” and “V H 2" is an arbitrary name, and in any of the embodiments herein, for example, "V L 1" and "V L 2” can be reversed.

[0104] Additionally or alternatively, in some embodiments, the antibody into which one or more of the above substitutions are introduced is a bispecific antibody comprising a first heavy chain (HC1), a first light chain (LC1), a second heavy chain (HC2) and a second light chain (LC2), wherein the first heavy chain comprises a first C1; H 1 domain (C H 11), the first light chain comprises a first C L Domain (C L1 ), the second heavy chain comprises a second C H 1 domain (C H12), the second light chain comprises a first C L Domain (C L2 ). It is obvious to those skilled in the art that the terms "HC1", "HC2", "LC1", "LC2", etc. are arbitrary names, and that, for example, "HC1" and "HC2" in any embodiment herein may be reversed. H 1 domain and L1 C L Any of the above mutations in the domain may alternatively be in the C H Domain 1 and C of L2 L In some embodiments, the method further comprises replacing C with E H S183 in 11, C is replaced by K L1 In V133, replace C with K H S183 in 12, and substitution of E for C L2 V133 in, wherein the amino acid numbering is according to the EU index. In some embodiments, the method further comprises replacing C with K H S183 in 11, replace C with E L1 In V133, replace C with E H S183 in 12, and substitution of K for C L2 V133 in, wherein amino acid numbering is according to the EU index. See, e.g., Dillon et al., (2017) MABS 9(2):213-230 and WO2016 / 172485. In some embodiments, HCl further comprises a first C H 2(C H 21) Domain and / or first C H 3(C H Additionally or alternatively, in some embodiments, HC2 further comprises a second C H 2(C H 22) domain and / or second C H 3(C H 32) domain. In some embodiments, C H 32 is changed so that in C H 31 / C H 32 interface, one or more amino acid residues are replaced by one or more amino acid residues with larger side chain volume, thereby H The surface of 32 is generated with C H 31 interacting protrusions, and C H 31 is changed so that in C H 31 / C H 32 interface, one or more amino acid residues are replaced by amino acid residues with smaller side chain volumes, therebyH 31 on the surface produced with C H 32 interacting cavities. In some embodiments, C H 31 is changed so that in C H 31 / C H 32 interface, one or more amino acid residues are replaced by one or more amino acid residues with larger side chain volume, thereby H 31 on the surface produced with C H 32 interacting protrusions, and C H 32 is changed so that in C H 31 / C H 32 interface, one or more amino acid residues are replaced by amino acid residues with smaller side chain volumes, thereby H The surface of 32 is generated with C H 31 interacts with a cavity. In some embodiments, the protrusion is a knob mutation, such as a knob mutation comprising T366W, wherein the amino acid numbering is according to the EU index. In some embodiments, the cavity is a hole mutation, such as a hole mutation comprising at least one, at least two, or all three of T366S, L368A, and Y407V, wherein the amino acid numbering is according to the EU index. Additional details about knob-hole structural mutations are provided, such as US 5,731,168, US 5,807,706, US 7,183,076, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the HC1 / LC1 pair of the bispecific antibody binds to a first antigen, and the HC2 / LC2 pair of the bispecific antibody binds to a second antigen. In some embodiments, the HC1 / LC1 pair of the bispecific antibody binds to a first epitope of the first antigen, and the HC2 / LC2 pair of the bispecific antibody binds to a second epitope of the first antigen.

[0105] A method of preparing (such as modifying or engineering) an antibody (e.g., a bispecific antibody) to obtain a modified antibody (e.g., a modified bispecific antibody) with improved preferential heavy chain / light chain pairing is provided, the method comprising modifying a light chain variable domain (V L ) at position 94 and / or V LThe amino acid at position 96 (e.g., the original amino acid) is substituted from an uncharged residue to a charged residue to obtain a modified antibody (e.g., a modified bispecific antibody), the charged residue being selected from aspartic acid (D), arginine (R), glutamic acid (E) and lysine (K), wherein the amino acid numbering is according to Kabat. In some embodiments, the method includes at least replacing the amino acid at position 94 and position 96 (e.g., the original amino acid) from an uncharged residue to a charged residue (e.g., D, R, E or K) to obtain a modified antibody (e.g., a bispecific antibody). In some embodiments, the modified antibody (e.g., a bispecific or multispecific antibody) is combined with an exemplary target described elsewhere herein. In many cases, the sequences of the heavy and light chains of the antibodies combined with these targets are publicly available, and can be aligned and mapped to the Kabat numbering scheme, and then scanned against the Kabat sequence database to identify the position to be replaced.

[0106] In some embodiments, the amino acid at position 94 (e.g., the original amino acid) is substituted with D to obtain a modified antibody (e.g., a modified bispecific antibody). In some embodiments, the amino acid at position 96 (e.g., the original amino acid) is substituted with R to obtain a modified antibody (e.g., a modified bispecific antibody). In some embodiments, the amino acid at position 94 (e.g., the original amino acid) is substituted with D, and the amino acid at position 96 (e.g., the original amino acid) is substituted with R to obtain a modified antibody (e.g., a modified bispecific antibody).

[0107] In some embodiments, the method further comprises converting the heavy chain variable domain (V H ) is substituted from an uncharged residue to a charged residue selected from aspartic acid (D), arginine (R), glutamic acid (E) and lysine (K) to obtain a modified antibody (e.g., a modified bispecific antibody), wherein the amino acid numbering is according to Kabat. In some embodiments, the amino acid at position 95 (e.g., the original amino acid) is substituted with D to obtain a modified antibody (e.g., a modified bispecific antibody). In some embodiments, V L The amino acid at position 94 (e.g., the original amino acid) is replaced by D, V L The amino acid at position 96 of (eg, the original amino acid) is substituted with R, and V H The amino acid at position 95 of (eg, the original amino acid) is substituted with D to obtain a modified antibody (eg, a modified bispecific antibody).

[0108] Also provided is a method of preparing (such as modifying or engineering) an antibody (e.g., a bispecific antibody) to obtain a modified antibody (e.g., a modified bispecific antibody) with improved preferential heavy chain / light chain pairing, the method comprising modifying a heavy chain variable domain (V H ) is substituted from an uncharged residue to a charged residue selected from aspartic acid (D), arginine (R), glutamic acid (E) and lysine (K) to obtain a modified antibody (e.g., a modified bispecific antibody), wherein the amino acid numbering is according to Kabat. In some embodiments, the amino acid at position 95 (e.g., the original amino acid) is substituted with D to obtain a modified antibody (e.g., a modified bispecific antibody).

[0109] Also provided is a method of preparing (such as modifying or engineering) an antibody (e.g., a bispecific antibody) to obtain a modified antibody (e.g., a modified bispecific antibody) with improved preferential heavy chain / light chain pairing, the method comprising modifying a light chain variable domain (V L ) at position 91, V L Position 94 and / or V L The amino acid at position 96 (e.g., the original amino acid) is substituted from a non-aromatic residue to an aromatic residue to obtain a modified antibody (e.g., a modified bispecific antibody), wherein the amino acid numbering is according to Kabat. In some embodiments, the method comprises the step of substituting at least two amino acids at position 91, position 94, or position 96 (e.g., the original amino acid) from a non-aromatic residue to an aromatic residue to obtain a modified antibody (e.g., a modified bispecific antibody), wherein the aromatic residue is selected from W, F, and Y. In some embodiments, the method comprises the step of substituting the amino acids at positions 94 and 96 (e.g., the original amino acid) from a non-aromatic residue to an aromatic residue to obtain a modified antibody (e.g., a modified bispecific antibody), wherein the aromatic residue is selected from W, F, and Y. In some embodiments, the method comprises the step of substituting each of the amino acids at positions 91, 94, and 96 (e.g., the original amino acids) from a non-aromatic residue to an aromatic residue selected from W, F, and Y to obtain a modified antibody (e.g., a modified bispecific antibody). In some embodiments, the modified antibody (e.g., a bispecific or multispecific antibody) binds to an exemplary target described elsewhere herein.

[0110] In some embodiments, the amino acid at position 91 (e.g., the original amino acid) is replaced by Y to obtain a modified antibody (e.g., a modified bispecific antibody). In some embodiments, the amino acid at position 94 (e.g., the original amino acid) is replaced by Y to obtain a modified antibody (e.g., a modified bispecific antibody). In some embodiments, the amino acid at position 96 (e.g., the original amino acid) is replaced by W to obtain a modified antibody (e.g., a modified bispecific antibody). In some embodiments, the amino acid at position 91 (e.g., the original amino acid) is replaced by Y, and the amino acid at position 94 (e.g., the original amino acid) is replaced by Y to obtain a modified antibody (e.g., a modified bispecific antibody). In some embodiments, the amino acid at position 91 (e.g., the original amino acid) is replaced by Y, and the amino acid at position 96 (e.g., the original amino acid) is replaced by W to obtain a modified antibody (e.g., a modified bispecific antibody). In some embodiments, the amino acid at position 94 (e.g., the original amino acid) is replaced by Y, and the amino acid at position 96 (e.g., the original amino acid) is replaced by W to obtain a modified antibody (e.g., a modified bispecific antibody). In some embodiments, the amino acid at position 91 (e.g., the original amino acid) is substituted with Y, the amino acid at position 94 (e.g., the original amino acid) is substituted with Y, and the amino acid at position 96 (e.g., the original amino acid) is substituted with W to obtain a modified antibody (e.g., a modified bispecific antibody).

[0111] In some embodiments, the method further comprises converting the heavy chain variable domain (V H ) is substituted from an uncharged residue to a charged residue selected from aspartic acid (D), arginine (R), glutamic acid (E) and lysine (K) to obtain a modified antibody (e.g., a modified bispecific antibody), wherein the amino acid numbering is according to Kabat. In some embodiments, the method further comprises replacing the heavy chain variable domain (V H ) is substituted from a non-aromatic residue to an aromatic residue selected from the group consisting of tryptophan (W), phenylalanine (F) and tyrosine (Y) to obtain a modified antibody (e.g., a modified bispecific antibody).

[0112] In some embodiments, a method of making (such as modifying or engineering) an antibody (e.g., a bispecific antibody) comprises modifying V H and / or V L , for example by introducing one or more of the above substitutions into V H and / or V L To obtain the modified V H and / or modified V L , and the modified VH and / or modified V L Grafting onto an antibody (eg, a bispecific antibody) to obtain a modified antibody (eg, a modified bispecific antibody).

[0113] In some embodiments, V has been substituted, modified, and / or engineered according to the methods described herein. H / V L The pair is subjected to at least one affinity maturation step (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 affinity maturation steps). Affinity maturation is a process by which the heavy chain / light chain pairs of antibodies obtained, for example, by the methods described herein, are subjected to a regimen selected to increase affinity for a target (e.g., a target ligand or target antigen, as described in further detail below) (see Wu et al. (1998) Proc Natl Acad Sci USA. 95, 6037-42). Details on affinity maturation of antibodies are also described in, for example, Merchant et al. (2013) Proc Natl Acad Sci US A. 110(32):E2987-96; Julian et al. (2017) Scientific Reports. 7:45259; Tiller et al. (2017) Front. Immunol. 8:986; Koenig et al. (2017) Proc Natl Acad Sci US A. 114(4):E486-E495; Yamashita et al. (2019) Structure. 27, 519 527; Payandeh et al. (2019) J. Cell Biochem. 120: 940-950; Richter et al. (2019) mAbs. 11 (1): 166-177; Cisneros et al. (2019) Mol. Syst. Des. Eng. 4: 737-746. In certain embodiments, the V of the heavy chain / light chain pair obtained by the method herein is H and / or V L One or more amino acid positions in are randomized (i.e., at positions other than those described above, i.e., V L 91, 94 and / or 96, and optionally V H 95 in) to generate a heavy chain / light chain variant library. Then screen V H / V LA library of variants is prepared to identify those variants with the desired affinity for the target. Therefore, in certain embodiments, the methods described herein further comprise the following steps: (a) mutagenizing or randomizing the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and / or CDR-L3 of the heavy chain / light chain pair obtained by the methods herein at one or more positions to generate V H / V L variant library, (b) contacting V with a target (e.g., a target ligand or a target antigen) H / V L Variant library, (c) detection of target and V H / V L variants, and (d) obtaining a V variant that specifically binds to the target H / V L Variants. As described above, V L 91, 94 and / or 96 and optionally V H Position 95 in is not further targeted for randomization. Methods for mutagenizing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and / or CDR-L3 of an antibody (or its fragment antigen-binding fragment) are known in the art and discussed elsewhere herein. Details on libraries and library screening are provided elsewhere herein.

[0114] In certain embodiments, the methods described herein further comprise the steps of: (e) determining a V H / V L Variants (i.e., affinity matured V H / V L In some embodiments, the methods described herein further comprise the following steps: (f) converting the affinity matured V H / V L The modified antibody (e.g., affinity matured modified bispecific antibody) grafted onto an antibody (e.g., a bispecific antibody) to become affinity matured. In some embodiments, the methods described herein further comprise the following steps: (g) evaluating the affinity matured V H / V L The degree of preferential pairing / preferential assembly is demonstrated, for example, using the method described below.

[0115] Also provided herein are antibodies (eg, monospecific, bispecific, or multispecific antibodies) or antibody fragments produced according to any one of the methods or a combination of methods described above.

[0116] Preferential pairing / preferential assembly of antibody heavy and light chains

[0117] As described above, preferential pairing describes the pairing pattern of a first polypeptide (e.g., a heavy chain) with a second polypeptide (e.g., a light chain) when pairing occurs between the first polypeptide and the second polypeptide in the presence of one or more additional different polypeptides (e.g., additional heavy chains and / or light chains). When HC1 is co-expressed with at least LC1 and LC2, if the amount of HC1 / LC1 heavy chain-light chain pairing is greater than the amount of HC1 / LC2 pairing, preferential pairing (e.g., homologous pairing) occurs between, for example, HC1 and LC1 of an antibody (e.g., a bispecific antibody). Similarly, when HC2 is co-expressed with at least LC1 and LC2, if the amount of HC2 / LC2 heavy chain-light chain pairing is greater than the amount of HC2 / LC1 pairing, preferential pairing (e.g., homologous pairing) occurs between, for example, HC2 and LC2 of a multispecific antibody (e.g., a bispecific antibody). HC1 / LC1, HC1 / LC2, HC2 / LC1, and HC2 / LC2 pairing can be measured by methods known in the art (e.g., liquid chromatography mass spectrometry (LCMS)), as described in further detail elsewhere herein.

[0118] In certain embodiments, the methods provided herein are used to generate (e.g., produce) antibodies (e.g., bispecific antibodies), wherein HC1 preferentially pairs with LC1. Additionally or alternatively, the methods provided herein are used to generate (e.g., produce) antibodies (e.g., bispecific antibodies), wherein HC2 preferentially pairs with LC2. In certain embodiments, the methods provided herein are used to generate (e.g., produce) antibodies (e.g., bispecific antibodies), wherein HC1 preferentially pairs with LC1, and HC2 preferentially pairs with LC2. In certain embodiments, when HC1 of the antibody (e.g., bispecific antibody) produced by the methods provided herein is co-expressed with HC2, LC1, and LC2, a bispecific antibody comprising a desired pairing (e.g., HC1 / LC1 and HC2 / LC2) is produced at the following relative yields: at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 71%, at least about 71%, at least about 72%, at least about 73%, at least about 74% %, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 99%, or more than about 99%, including any ranges between these values. The relative yields of bispecific antibodies comprising the desired pairings (e.g., HC1 / LC1 and HC2 / LC2) can be determined using, for example, mass spectrometry, as described in the Examples.

[0119] In certain embodiments, the expressed polypeptides of the antibodies (e.g., bispecific antibodies) produced using the methods provided herein are assembled with improved specificity to reduce the generation of mispaired heavy and light chains. In certain embodiments, during production, the C H 1V H Domain V L The domains assemble (e.g., preferentially assemble).

[0120] Methods for assessing correct pairing / preferential pairing / preferential assembly

[0121] The preferential pairing, correct pairing and / or preferential assembly of HCl and LC1 of the modified antibodies (e.g., modified bispecific antibodies) prepared according to the methods described herein can be determined using any of a variety of methods known to those of ordinary skill in the art. For example, the degree of preferential pairing of HCl and LC1 in the modified antibodies (e.g., modified bispecific antibodies) can be determined via a light chain competition assay (LCCA). International patent application PCT / US2013 / 063306, filed on October 3, 2013, describes various embodiments of LCCA, and is incorporated herein by reference in its entirety for all purposes. The method allows for quantitative analysis of the pairing of heavy chains with specific light chains within a co-expressed protein mixture, and can be used to determine whether a particular immunoglobulin heavy chain is selectively associated with either of the two immunoglobulin light chains when the heavy chain and light chain are co-expressed. The method is briefly described as follows: at least one heavy chain and two different light chains are co-expressed in a cell at a ratio such that the heavy chain is the limiting pairing reactant; the secretory protein is optionally separated from the cells; the immunoglobulin light chain polypeptide associated with the heavy chain is separated from the remaining secretory protein to produce a separated heavy chain pairing portion; the amount of each different light chain in the separated heavy chain portion is detected; and the relative amount of each different light chain in the separated heavy chain portion is analyzed to determine the ability of at least one heavy chain to selectively pair with one of the light chains.

[0122] In certain embodiments, the preferential pairing of HCl and LC1 of a modified antibody (such as a modified bispecific or multispecific antibody) prepared according to the methods provided herein is measured via mass spectrometry (such as liquid chromatography-mass spectrometry (LC-MS) native mass spectrometry, acid mass spectrometry, etc.). Mass spectrometry is used to quantify the relative heterodimer population, including each light chain, using the molecular weight difference of the relative heterodimer population to identify each different species. In certain embodiments, the correct or preferential pairing is determined by LC-MS as described herein. In certain embodiments, the correct or preferential pairing of Fv or Fab is measured.

[0123] Multispecific antibody formats

[0124] Modified antibodies (e.g., modified bispecific antibodies) prepared according to the methods provided herein can be used together with any of a variety of bispecific or multispecific antibody formats known in the art. Various formats have been developed in the art to address the therapeutic opportunities provided by molecules with a variety of binding specificities. Several methods have been described to prepare bispecific antibodies in which a specific antibody light chain or fragment is paired with a specific antibody heavy chain or fragment.

[0125] For example, C H 1 / C LMutations in the interface that promote selective pairing of homologous Fabs or pairing of HC and LC are described in Dillon et al. (2017) MABS 9(2):213-230 and WO2016 / 172485, the contents of which are incorporated herein by reference in their entirety.

[0126] The knob-and-hole structure is used for the C H Heterodimerization technology of 3 domains. Previously, the knob-in-hole structure technology has been applied to the production of human full-length bispecific antibodies with a single public light chain (LC) (Merchant et al., "An efficient route to human bispecific IgG." Nat Biotechnol. 1998; 16: 677 81; Jackman et al., "Development of a two-part strategy to identify a therapeutic human bispecific antibody that inhibits IgE receptor signaling." J Biol Chem. 2010; 285: 20850 9.) See also WO1996027011, which is incorporated herein by reference in its entirety for all purposes.

[0127] Antibodies (e.g., bispecific antibodies) produced using the methods provided herein can be further modified to include additional heterodimerization domains that have a strong preference for forming heterodimers relative to homodimers. Illustrative examples include, but are not limited to, for example, WO2007147901 ( et al. Novo Nordisk: describing ionic interactions); WO 2009089004 (Kannan et al. Amgen: describing electrostatic steering effects); WO 2010 / 034605 (Christensen et al. - Genentech; describing coiled coils). See also, for example, Pack, P. & Plückthun, A., Biochemistry 31, 1579-1584 (1992), describing leucine zippers, or Pack et al., Bio / Technology 11, 1271-1277 (1993), describing helix-turn-helix helix motifs. The phrases "heteromultimerization domain" and "heterodimerization domain" are used interchangeably herein. In certain embodiments, the antibodies (e.g., bispecific antibodies) produced using the methods provided herein comprise one or more heterodimerization domains.

[0128] U.S. Patent Publication No. 2009 / 0182127 (Novo Nordisk, Inc.) describes a method for modifying the Fc interface and the C-terminal region of the light-heavy chain pair. H 1:C L Bispecific antibodies are produced by removing amino acid residues at the position of the light chain of one pair of chains that reduce the ability of the light chain of one pair of chains to interact with the heavy chain of the other pair of chains.

[0129] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature, 305:537 (1983)) and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26-35 (1997)). Multispecific antibodies can also be prepared by engineering electrostatic steering effects for preparing antibody Fc-heterodimer molecules (see, e.g., WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); and using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605).

[0130] Multispecific antibodies can also be provided in an asymmetric format, where domains are crossed in one or more binding arms with the same antigen specificity, i.e., by exchanging V H / V L domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), C H 1 / C) domain (see, e.g., WO 2009 / 080253) or a complete Fab arm (see, e.g., WO2009 / 080251, WO 2016 / 016299, see also Schaefer et al., PNAS, 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-20). In one aspect, the multispecific antibody comprises a crossover Fab fragment. The term "crossover Fab fragment" or "xFab fragment" or "exchange Fab fragment" refers to a Fab fragment in which the variable regions or constant regions of the heavy and light chains are exchanged. The crossover Fab fragment comprises a light chain variable region (V L ) and heavy chain constant region 1 (C H 1) composed of polypeptide chains, and the heavy chain variable region (VH ) and the light chain constant region (C L ) composed of polypeptide chains. Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations into the domain interface to guide correct Fab pairing. See, for example, WO 2016 / 172485.

[0131] An overview of various bispecific and multispecific antibody formats is provided in Klein et al. (2012) mAbs 4:6, 653-663 and Spiess et al. (2015) "Alternative molecular formats and therapeutic applications for bispecific antibodies." Mol. Immunol. 67 (2015) 95-106.

[0132] In some embodiments, modified antibodies (eg, modified bispecific antibodies) prepared by the methods provided herein are reformatted into any of the multispecific antibody formats described above to further ensure correct heavy / light chain pairing.

[0133] Antibody production and purification

[0134] Cultivation of host cells

[0135] In certain embodiments, the modified antibodies (e.g., modified bispecific or multispecific antibodies) prepared according to the methods provided herein can be provided by the following method: (a) introducing a set of polynucleotides encoding HC1, HC2, LC1, and LC2 into a host cell; and (b) culturing the host cell to produce antibodies (e.g., bispecific or multispecific antibodies). In certain embodiments, the polynucleotides encoding LC1 and LC2 are introduced into the host cell at a predetermined ratio (e.g., molar ratio or weight ratio). In certain embodiments, polynucleotides encoding LC1 and LC2 are introduced into host cells such that the ratio (e.g., molar ratio or weight ratio) of LC1:LC2 is about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or about 5.5:1, including any range between these values. In certain embodiments, the ratio is a molar ratio. In certain embodiments, the ratio is a weight ratio. In certain embodiments, polynucleotides encoding HC1 and HC2 are introduced into host cells at a predetermined ratio (e.g., molar ratio or weight ratio). In certain embodiments, polynucleotides encoding HCl and HC2 are introduced into host cells such that the ratio of HCl:HC2 (e.g., a molar ratio or a weight ratio) is about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or about 5.5:1, including any range between these values. In certain embodiments, the ratio is a molar ratio. In certain embodiments, the ratio is a weight ratio. In certain embodiments, polynucleotides encoding HCl, HC2, LC1, and LC2 are introduced into host cells at a predetermined ratio (e.g., a molar ratio or a weight ratio). In certain embodiments, polynucleotides encoding HC1, HC2, LC1, and LC2 are introduced into the host cell such that the ratio (e.g., molar ratio or weight ratio) of HC1+HC2:LC1,+LC2 is about 5:1, about 5:2, about 5:3, about 5:4, about 1:1, about 4:5, about 3:5, about 2:5, or about 1:5, including any ranges between these values.In certain embodiments, polynucleotides encoding LC1, LC2, HC1, and HC2 are introduced into a host cell such that the ratio (e.g., molar ratio or weight ratio) of LC1+LC2: HC1, + HC2 is about 1:1:1:1, about 2.8:1:1:1, about 1.4:1:1:1, about 1:1.4:1:1, about 1:2.8:1:1, about 1:1:2.8:1, about 1:1:1.4:1, about 1:1:1:2.8, or about 1:1:1:1.4, including any range between these values. In certain embodiments, the ratio is a molar ratio. In certain embodiments, the ratio is a weight ratio.

[0136] In certain embodiments, the modified antibody (e.g., modified bispecific or multispecific antibody) prepared according to the method provided herein is produced and further includes determining the optimal ratio of the polynucleotides for introducing into the cell. In certain embodiments, mass spectrometry is used to determine the antibody yield (e.g., bispecific antibody yield), and the optimal chain ratio is adjusted to maximize the protein yield (e.g., bispecific antibody yield). In certain embodiments, the antibody (e.g., bispecific or multispecific antibody) generated according to the method provided herein is further included in harvesting or recovering the antibody from the cell culture. In certain embodiments, the antibody (e.g., bispecific or multispecific antibody) generated according to the method provided herein is further included in purifying the antibody harvested or recovered.

[0137] Host cells for producing modified antibodies (e.g., modified bispecific antibodies) prepared according to the methods provided herein can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing host cells. In addition, Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655 or 5,122,469; WO 90 / 03430; WO Any of the media described in 87 / 00195 or U.S. Reissued Patent 30,985 can be used as culture medium for the host cells. Any of these media can be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as Drugs), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, and the like are those previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan.

[0138] Harvesting or recovering and purifying antibodies

[0139] In related aspects, producing a modified antibody (e.g., a modified bispecific antibody) prepared according to the methods described herein comprises culturing the above-described host cell under conditions that allow expression of the modified antibody; and recovering (e.g., harvesting) the modified antibody. In certain embodiments, producing a modified antibody (e.g., a modified bispecific antibody) prepared according to the methods described herein further comprises purifying the recovered modified antibody (e.g., a modified bispecific antibody) to obtain a substantially homogeneous preparation, e.g., for further assays and uses.

[0140] The modified antibodies (e.g., modified bispecific antibodies) prepared according to the methods described herein can be produced intracellularly, or directly secreted into the culture medium. If such modified antibodies are produced intracellularly, then as a first step, the particulate debris of the host cell or cleavage fragments are removed, for example, by centrifugation or ultrafiltration. In the case where the modified antibodies (e.g., modified bispecific antibodies) prepared according to the methods described herein are secreted into the culture medium, commercially available protein concentration filters (e.g., Amicon or Millipore Pellicon ultrafiltration units) are usually first used to concentrate the supernatant from such expression systems. Protease inhibitors such as PMSF can be included in any of the aforementioned steps to inhibit proteolysis and antibiotics can be included to prevent the growth of exogenous contaminants.

[0141] Substantially homogeneous preparations of modified antibodies (e.g., modified bispecific antibodies) prepared according to the methods described herein can be obtained from cells using standard protein purification methods known in the art. The following procedures are examples of suitable purification procedures: fractionation on an immunoaffinity column or an ion exchange column, ethanol precipitation, reversed-phase HPLC, chromatography on silica gel or on a cation exchange resin (such as DEAE), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration, for example, using Sephadex G-75.

[0142] Additionally or alternatively, modified antibodies (eg, modified bispecific antibodies) prepared using the methods described herein can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a preferred purification technique.

[0143] In some aspects, the preparation derived from the cell culture medium as described above is applied to a solid phase fixed with protein A so that the modified antibody (e.g., modified bispecific antibody) specifically binds to protein A. The solid phase is then washed to remove pollutants that are non-specifically bound to the solid phase. The modified antibody (e.g., modified bispecific antibody) is recovered from the solid phase by eluting into a solution comprising a chaotropic agent or a mild detergent. Exemplary chaotropic agents and mild detergents include, but are not limited to, guanidine hydrochloride, urea, lithium perchlorate, arginine, histidine, SDS (sodium dodecyl sulfate), Tween, Triton, and NP-40, all of which are commercially available.

[0144] The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody (e.g., bispecific antibody). Protein A can be used to purify antibodies based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is mostly agarose, but other matrices can also be used. Mechanically stable matrices, such as controlled pore glass or poly (styrene divinyl), have faster flow rates and shorter processing times than agarose. When the modified antibody (e.g., modified bispecific antibody) contains C H When there are 3 domains, Bakerbond Resins (JT Baker, Phillipsburg, NJ) can be used for purification. Other protein purification techniques such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica gel, chromatography on heparin, chromatography on anion or cation exchange resins (such as polyaspartic acid columns) can also be used depending on the antibody to be recovered (e.g., bispecific antibodies). Chromatography, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0145] After any preliminary purification steps, the mixture comprising the modified antibody (e.g., modified bispecific antibody) and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH between about 2.5-4.5, preferably at low salt concentrations (e.g., about 0-0.25 M salt). The production of the modified antibody (e.g., modified bispecific antibody) can alternatively or additionally (for any of the aforementioned specific methods) comprise dialysis of the solution comprising the polypeptide mixture.

[0146] Libraries and library screening

[0147] Also provided herein are libraries that exhibit preferential pairing of heavy chain / light chain pairs (or antigen-binding fragments thereof).

[0148] For example, provided herein are libraries comprising a plurality of antigen binding domain variants, each antigen binding domain variant comprising a different antibody heavy chain domain (V H ) and different antibody light chain domains (V L ), where each V H All of them contain different CDR-H1, CDR-H2 and CDR-H3 sequences, wherein each V L All contain different CDR-L1, CDR-L2 and CDR-L3 sequences, and each V L 94 positions or each V L At least one amino acid at position 96 of is a charged residue selected from aspartic acid (D), arginine (R), glutamic acid (E) and lysine (K), wherein the amino acid numbering is according to Kabat. L The two amino acids at positions 94 and 96 of V are both charged residues independently selected from D, R, E and K. In some embodiments, each V L The amino acid at position 94 of is D. In some embodiments, each V L The amino acid at position 96 of is R. In some embodiments, each V L The amino acids at position 94 of L The amino acid at position 96 of is R. In some embodiments, each V H The amino acid at position 95 of each V is a charged residue selected from D, R, E and K. In some embodiments, each V H The amino acid at position 95 of is D. In some embodiments, each V L The amino acids at position 94 are all D, and each V L The amino acids at position 96 of H The amino acid at position 95 is all D.

[0149] Also provided herein are libraries comprising a plurality of antigen binding domain variants, each antigen binding domain variant comprising a different antibody heavy chain domain (V H ) and different antibody light chain domains (V L ), where each V H All of them contain different CDR-H1, CDR-H2 and CDR-H3 sequences, wherein each V L All contain different CDR-L1, CDR-L2 and CDR-L3 sequences, and each V L At position 91, each V L94 positions or each V L At least one amino acid at position 96 of is an aromatic residue selected from tryptophan (W), phenylalanine (F) and tyrosine (Y), wherein the amino acid numbering is according to Kabat. L At least two amino acids at positions 91, 94, or 96 (e.g., positions 91 and 94, 91 and 96, or 94 and 96) of the amino acid sequence are aromatic residues selected from W, F, and Y. In some embodiments, each V L The amino acid at position 91 of is Y. In some embodiments, each V L The amino acid at position 94 of is Y. In some embodiments, each V L The amino acid at position 96 of is W. In some embodiments, each V L The amino acid at position 91 is Y, and each V L The amino acid at position 94 of is Y. In some embodiments, each V L The amino acid at position 91 is Y, and each V L The amino acid at position 96 of is W. In some embodiments, each V L The amino acid at position 94 is Y, and each V L The amino acid at position 96 of is W. In some embodiments, each V L The amino acid at position 91 is Y, and each V L The amino acid at position 94 is Y, and each V L The amino acid at position 96 of is W. In some embodiments, each V H The amino acid at position 95 of is a charged residue selected from aspartic acid (D), arginine (R), glutamic acid (E) and lysine (K), wherein the amino acid numbering is according to Kabat. H The amino acid at position 95 of is an aromatic residue selected from the group consisting of tryptophan (W), phenylalanine (F) and tyrosine (Y).

[0150] In certain embodiments, the library is a polypeptide library (e.g., a plurality of any polypeptides described herein). In certain embodiments, the polypeptide library provided herein is a polypeptide display library. Such polypeptide display libraries can be screened to select and / or evolve binding proteins with desired properties for a variety of uses, including but not limited to treatment, prevention, veterinary, diagnostic, reagent or material applications. In certain embodiments, the library is a nucleic acid library (e.g., a plurality of any nucleic acids described herein), wherein each nucleic acid (or a group of nucleic acids) encodes different antigenic domain binding variants described herein. In some embodiments, the library is a plurality of host cells (e.g., prokaryotic or eukaryotic host cells), each of which contains (and, for example, expresses) different nucleic acids (or a group of nucleic acids), wherein each different nucleic acid (or a group of nucleic acids) encodes different antigenic domain binding variants described herein

[0151] In certain embodiments, the libraries provided herein comprise at least 2, 3, 4, 5, 10, 30, 100, 250, 500, 750, 1000, 2500, 5000, 7500, 10000, 25000, 50000, 75000, 100000, 250000, 500000, 750000, 1000000, 250000, 500000, 750000, 10000000 or more than 10000000 different antigen binding domains, including any range between these values. In certain embodiments, the libraries provided herein have about 2, about 5, about 10, about 50, about 100, about 250, about 500, about 750, about 10 3 , about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , about 10 12 , about 10 13 , about 10 14 or more than about 10 14 (such as about 10 15 or about 10 16 ), including any ranges between these values.

[0152] In certain embodiments, the library provided herein is produced via genetic engineering. A variety of methods for mutagenesis and subsequent library construction (as well as appropriate methods for screening or selection) have been previously described. Such mutagenesis methods include, but are not limited to, other methods such as error-prone PCR, ring shuffling or oligonucleotide-directed mutagenesis, random nucleotide insertion or recombination. More details about these methods are described in, for example, Abou-Nadler et al. (2010) Bioengineered Bugs 1, 337-340; Firth et al. (2005) Bioinformatics 21, 3314-3315; Cirino et al. (2003) Methods Mol Biol 231, 3-9; Pirakitikulr (2010) Protein Sci 19, 2336-2346; Steffens et al. (2007) J. BiomolTech 18, 147-149 and other documents. Therefore, in certain embodiments, a multi-specific antigen-binding protein library produced via genetic engineering technology is provided.

[0153] In certain embodiments, the libraries provided herein are produced via in vitro translation. In short, in vitro translation requires cloning a protein-coding sequence into a vector containing a promoter, producing mRNA by transcribing the cloned sequence with an RNA polymerase, and, for example, using a cell-free extract, synthesizing the protein by in vitro translation of the mRNA. The desired mutant protein can be produced simply by changing the cloned protein-coding sequence. Many mRNAs can be effectively translated in wheat germ extracts or rabbit reticulocyte lysates. More details about in vitro translation are described in, for example, Hope et al. (1985) Cell 43, 177-188; Hope et al. (1986) Cell 46, 885-894; Hope et al. (1987) EMBO J.6, 2781-2784; Hope et al. (1988) Nature 333, 635-640; and Melton et al. (1984) Nucl. Acids Res. 12, 7057-7070.

[0154] Therefore, multiple nucleic acid molecules encoding polypeptide display libraries described herein are provided. Also provided herein are expression vectors operably linked to multiple nucleic acid molecules. Also provided are methods for making libraries provided herein by providing multiple nucleic acids encoding multiple antigen binding domains described herein and expressing the nucleic acids.

[0155] In certain embodiments, the libraries provided herein are produced via chemical synthesis. Methods for solid phase and liquid phase peptide synthesis are well known in the art and are described, for example, in Methods of Molecular Biology, 35, Peptide Synthesis Protocols, (M. W. Pennington and B. M. Dunn, eds.), Springer, 1994; Welsch et al. (2010) Curr Opin Chem Biol 14, 1-15; Methods of Enzymology, 289, Solid Phase Peptide Synthesis, (G. B. Fields, ed.), Academic Press, 1997; Chemical Approaches to the Synthesis of Peptides and Proteins, (P. Lloyd-Williams, F. Albericio, and E. Giralt, eds.), CRC Press, 1997; Fmoc Solid Phase Peptide Synthesis, A Practical Approach, (W. C. Chan, P. D. White, eds.), Oxford University Press, 2000; Solid Phase Synthesis, A Practical Guide, (S. F. Kates, F. White, eds.), Oxford University Press, 2001; Edited by Albericio), Marcel Dekker, 2000; P. Seneci, Solid-Phase Synthesis and Combinatorial Technologies, John Wiley & Sons, 2000; Synthesis of Peptides and Peptidomimetics (M. Goodman, editor-in-chief, edited by A. Felix, L. Moroder, C. Tmiolo), Thieme, 2002; NLBenoiton, Chemistry of Peptide Synthesis, CRCPress, 2005; Methods in Molecular Biology, 298, Peptide Synthesis and Applications, (J.Howl (ed.) Humana Press, 2005; and Amino Acids, Peptides and Proteins in Organic Chemistry, Vol. 3, Building Blocks, Catalysts and Coupling Chemistry, (AB Hughs (ed.)) Wiley-VCH, 2011. Therefore, in certain embodiments, a multi-specific antigen-binding protein library produced via chemical synthesis technology is provided. .

[0156] In certain embodiments, the library provided herein is a display library. In certain embodiments, the display library is a phage display library, a phagemid display library, a virus display library, a bacteria display library, a yeast display library, a λgt11 library, a CIS display library, and an in vitro compartmentalization library, or a ribosome display library. Methods for preparing and screening such display libraries are well known to those skilled in the art and are described, for example, in Molek et al. (2011) Molecules 16, 857-887; Boder et al., (1997) Nat Biotechnol 15, 553-557; Scott et al. (1990) Science 249, 386-390; Brisette et al. (2007) Methods Mol Biol 383, 203-213; Kenrick et al. (2010) Protein Eng Des Sel 23, 9-17; Freudl et al. (1986) J Mol Biol 188, 491-494; Getz et al. (2012) Methods Enzymol 503, 75-97; Smith et al. (2014) Curr Drug Discov Technol 11, 48-55; Hanes et al. (1997) Proc Natl Acad Sci USA 94, 4937-4942; Lipovsek et al., (2004) J Imm Methods 290, 51-67; Ullman et al., (2011) Brief. Funct. Genomics, 10, 125 134; Odegrip et al. (2004) Proc Natl Acad Sci USA 101, 2806-2810; and Miller et al. (2006) Nat Methods 3, 561-570.

[0157] In certain embodiments, the libraries provided herein are RNA-protein fusion libraries generated, for example, by the techniques described in Szostak et al., US 6258558, US 6261804, US 5643768, and US 5658754. In certain embodiments, the libraries provided herein are DNA-protein libraries, such as described in, for example, US 6416950.

[0158] Screening methods

[0159] Libraries provided herein can be screened to identify antigen binding variants with high affinity to a target (e.g., an antigen). Therefore, a method for obtaining antigen binding variants in conjunction with a target (e.g., a target described elsewhere herein) is provided herein.

[0160] In certain embodiments, the method comprises: a) contacting the library described herein under conditions that allow the target of interest to bind to the antigen binding domain variants in the library that specifically bind to the target, (b) detecting the binding of the target to the antigen binding domain variant that specifically binds to the target (e.g., detecting a complex comprising the target and the antigen binding domain variant that specifically binds to the target), and (c) obtaining the antigen binding domain variant that specifically binds to the target. In some embodiments, the method further comprises subjecting the antigen binding domain variants thus identified to at least one affinity maturation step, wherein V L The amino acid at position 91, position 94 and / or position 96 is used for randomization. In some embodiments, V is not selected H The amino acid at position 95 was used for randomization.

[0161] In some embodiments, the method further comprises generating an antibody (such as a bispecific antibody or a multispecific antibody) comprising an antigen binding domain variant that binds a target of interest (e.g., an affinity matured antigen binding domain variant that binds a target of interest).

[0162] In certain embodiments, a complex comprising a target and an antigen binding domain variant that specifically binds to the target is provided. In certain embodiments, the method further comprises determining the V H and / or V L of the nucleic acid sequence.

[0163] Affinity maturation is a process in which antigen binding domain variants are subjected to a regimen that selects for increased affinity for a target (e.g., a target ligand or target antigen) (see Wu et al. (1998) Proc Natl Acad Sci USA. 95, 6037-42). In certain embodiments, after identification from library screening, antigen binding domain variants that specifically bind to a first target ligand are further randomized (i.e., at positions other than those described above, i.e., V L 91, 94 and / or 96, and optionally V H 95 in). For example, in certain embodiments, the method of obtaining an antigen binding domain variant that specifically binds to a first target ligand further comprises: (e) mutagenizing or randomizing previously identified antigen binding domain variants CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and / or CDR-L3 to produce further antigen binding domain variants, (f) contacting the first target ligand with the further randomized antigen binding domain variants, (g) detecting binding of the target to the further randomized antigen binding domain variants, and (h) obtaining further randomized antigen binding domain variants that specifically bind to the target. As described above, V in the antigen binding domain variants L 91, 94 and / or 96 and optionally V HPosition 95 in is not further targeted for randomization. Methods for mutagenizing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and / or CDR-L3 of an antigen binding domain are known in the art and may include, for example, random mutagenesis, CDR walking mutagenesis or sequential and parallel optimization, mutagenesis by structure-based rational design, site-specific mutagenesis, enzyme-based mutagenesis, chemical-based mutagenesis, and gene synthesis methods for synthetic antibody gene production. See, for example, Yang et al. 1995, CDR Walking Mutagenesis for the Affinity Mutation of a Potent Human Anti-HIV-1 Antibody into the Picomolar Range, J. Mol. Biol. 254: 392-40, and Lim et al., 2019, Review: Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation, Int. J. Mol. Sci, 20: 1861, the contents of which are incorporated herein by reference in their entirety.

[0164] In certain embodiments, the method further comprises (i) determining the nucleic acid sequence of an antigen binding domain variant that specifically binds to the target.

[0165] In certain embodiments, the further randomized antigen binding domain variants comprise at least one or at least two randomized CDRs that were not previously randomized in the first library. Multiple rounds of randomization (i.e., in addition to randomization in V L At position 91, at position 94 and / or at position 96 and optionally V H 95 in the ), screening and selection until an antigen-binding domain variant with sufficient affinity for the target is obtained. Therefore, in certain embodiments, steps (e)-(h) or steps (e)-(i) are repeated one, two, three, four, five, six, seven, eight, nine, ten or more times to identify an antigen-binding domain variant that specifically binds to the first target ligand. In some embodiments, the antigen-binding domain variant that has undergone two or more rounds of randomization, screening and selection binds to the target with an affinity that is at least as high as the affinity of the antigen-binding domain variant that has undergone one round of randomization, screening and selection.

[0166] Libraries of antigen binding domain variants described herein can be screened by any technique known in the art for evolving new or improved binding proteins that specifically bind to a target ligand. In certain embodiments, the target ligand is immobilized on a solid support (such as a column resin or a microtiter plate well) and the target ligand is contacted with a library of candidate multispecific antigen binding proteins (e.g., any library described herein). The selection technology may be, for example, phage display (Smith (1985) Science 228, 1315-1317), mRNA display (Wilson et al. (2001) Proc Natl Acad Sci USA 98:3750-3755), bacterial display (Georgiou et al. (1997) Nat Biotechnol 15:29-34), yeast display (Boder and Wittrup (1997) Nat. Biotechnol. 15:553-5577), or ribosome display (Hanes and Plückthun (1997) Proc Natl Acad Sci USA 94:4937 4942 and WO2008 / 068637).

[0167] In certain embodiments, the library of antigen binding domain variants is a phage display library. In certain embodiments, phage particles displaying antigen binding domain variants described herein are provided. In certain embodiments, phage particles displaying antigen binding domain variants described herein are provided, which antigen binding domain variants are capable of binding to a target ligand.

[0168] Phage display is a technique by which multiple multispecific antigen-binding protein variants are displayed as fusion proteins fused to a coat protein on the surface of a phage particle (Smith, GP (1985) Science, 228: 1315-7; Scott, JK and Smith, GP (1990) Science 249: 386; Sergeeva, A. et al. (2006) Adv. Drug Deliv. Rev. 58: 1622-54). The utility of phage display lies in the fact that large libraries of selectively randomized protein variants (or randomly cloned cDNAs) can be rapidly and efficiently sorted to find those sequences that bind to a target molecule with high affinity.

[0169] Display of polypeptide (Cwirla, SE et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378) or protein (Lowman, HB et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352:624; Marks, JD et al. (1991), J. Mol. Biol., 222:581; Kang, AS et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage has been used to screen millions of polypeptides or oligopeptides for those with specific binding properties (Smith, GP (1991) Current Opin. Biotechnol., 2:668; Wu et al. (1998) Proc Natl Acad Sci USA. May 2003). 95, 6037-42). Multivalent phage display methods have been used to display small random peptides and small proteins by fusion with gene III or gene VIII of filamentous phage. (Wells and Lowman, Curr. Opin. Struct. Biol., 3: 355-362 (1992), and references cited therein.) In monovalent phage display, a protein or peptide library is fused to gene III or a portion thereof and expressed at low levels in the presence of wild-type gene III protein, so that the phage particle displays one copy of the fusion protein or no copy of the fusion protein. Relative to multivalent phage, the activity effect is reduced, allowing sorting based on intrinsic ligand affinity and the use of phagemid vectors, which simplifies DNA manipulation. (Lowman and Wells, Methods: A companion to Methods in Enzymology, 3: 205-0216 (1991).)

[0170] Sorting phage libraries of antigen binding domain variants requires the construction and propagation of large numbers of variants, a procedure for affinity purification using the target ligand, and a means to assess the results of the binding enrichment (see, e.g., US 5223409, US 5403484, US 5571689, and US 5663143).

[0171] Most phage display methods use filamentous phage (eg, M13 phage). Also known are the lambda phage display system (see WO 1995 / 34683, US 5627024), the T4 phage display system (Ren et al. (1998) Gene 215:439; Zhu et al. (1998) Cancer Research, 58:3209-3214; Jiang et al. (1997) Infection & Immunity, 65:4770-4777; Ren et al. (1997) Gene, 195:303-311; Ren (1996) Protein Sci., 5:1833; Efimov et al. (1995) Virus Genes, 10:173) and the T7 phage display system (Smith and Scott (1993) Methods in Enzymology, 217:228-257; US.5766905).

[0172] Many other improvements and variants of the basic phage display concept have now been developed. These improvements enhance the ability of the display system to screen peptide libraries for binding to a selected target molecule, as well as the ability to display functional proteins with the following potential: the desired properties of these proteins have been screened. A combined reaction device for phage display reactions has been developed (WO1998 / 14277), and phage display libraries have been used to analyze and control bimolecular interactions (WO 1998 / 20169; WO1998 / 20159) and the properties of constrained helical peptides (WO 1998 / 20036). WO 1997 / 35196 describes a method for separating affinity ligands, wherein a phage display library is contacted with a solution (wherein the ligand will bind to the target molecule) and another solution (wherein the affinity ligand will not bind to the target molecule) to selectively separate the binding ligand. WO 1997 / 46251 describes a method for bio-panning a random phage display library with affinity purified antibodies, then isolating the binding phage, followed by micro-panning using microplate wells to isolate high affinity binding phage. Such methods can be applied to libraries of antigen binding domain variants disclosed herein. The use of Staphylococcus aureus protein A as an affinity tag has also been reported (Li et al. (1998) Mol Biotech.9:187). WO 1997 / 47314 describes the use of a combinatorial library (which may be a phage display library) to distinguish enzyme specific substrate subtraction libraries. Other methods for selecting specific binding proteins are described in US 5498538, US 5432018 and WO1998 / 15833. Methods for generating peptide libraries and screening these libraries are also disclosed in US 5723286, US 5432018, US 5580717, US 5427908, US 5498530, US 5770434, US 5734018, US 5698426, US 5763192 and US 5723323.

[0173] Exemplary Antigens / Target Molecules

[0174] Examples of molecules that can be targeted by antibodies (eg, bispecific or multispecific antibodies) generated using the methods provided herein include, but are not limited to, soluble serum proteins and their receptors and other membrane-bound proteins (eg, adhesins). In another embodiment, the multispecific antigen-binding proteins provided herein are capable of binding to one, two or more cytokines, cytokine-related proteins and cytokine receptors selected from the group consisting of 8MPI, 8MP2, 8MP38 (GDFIO), 8MP4, 8MP6, 8MP8, CSFI (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (αFGF), FGF2 (βFGF), FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10, FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFN-γ, IFN-γ, IFN-γ, IFN-γ, IFN-γ, IFN-γ, IFN-γ, IFN-γ, IFN-γ, IFN-γ, IFN-γ, IFN-γ, IFN-γ, IFN-γ, IFN-γ, IFN-γ, IFN-γ, IFN-γ, IFN-γ, IFN-γ

[0175] IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFN81, IFNG, IFNWI, FEL1, FEL1(EPSELON), FEL1(ZETA), IL 1A, IL 1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL1 0, IL 11, IL 12A, IL12B, IL 13, IL 14, IL 15, IL 16, IL 17, IL 17B、IL 18、IL 19, IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL 30. PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFBb3, LTA(TNF-β), LTB, and TNF (TNF-α), TNFSF4(OX40-dependent), TNFSF5 (CD40-dependent), TNFSF6(FasL), TNFSF7 (CD27-like), TNFSF8 (CD30-like), TNFSF9 (4-1BB-like), TNFSF10(TRAIL), TN FSF11(TRANCE), TNFSF12(APO3L), TNFSF13(April), TNFSF13B, TNFSF14(HVEM-L), TNFSF15(VEGI), TNFSF18, HGF(VEGFD), VEGF, VEFGA EGFB, VEGFC, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL 3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, IL 11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R, IL22R, IL1HY1, IL 1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AIF1, HGF, LEP(processor), PTN, and THPO.

[0176] In another embodiment, the target molecule is a chemokine, chemokine receptor or chemokine-related protein selected from the group consisting of CCLI (1-309), CCL2 (MCP-1 / MCAF), CCL3 (MIP-Iα), CCL4 (MIP-Iβ), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCL11 (eotaxin), CCL 13 (MCP-4), CCL 15 (MIP-Iδ), CCL 16 (HCC-4), CCL 17 (TARC), CCL 18 (PARC), CCL 19 (MDP-3b), CCL20 (MIP-3α), CCL21 (SLC / exodus-2), CCL22 (MDC / STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2 / eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL27 (CTACK / ILC), CCL28, CXCLI (GROI), CXCL2 (GR02), CXCL3 (GR03), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCL 10 (IP 10), CXCL 11 (1-TAC), CXCL 12 (SDFI), CXCL 13, CXCL14, CXCL 16, PF4 (CXCL4), PPBP (CXCL7), CX3CL1(SCYDI), SCYEI, XCLI(lymphocyte chemoattractant), XCL2(SCM-Iβ), BLRI(MDR15), CCBP2(D6 / JAB61), CCRI(CKRI / HM145), CCR2(mcp-IRBIRA), CCR3(CKR3 / CMKBR3), CCR4, CCR5(CMKBR5 / ChemR13), CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7(CKR7 / EBII), CCR8(CMKBR8 / TER1 / CKR-L1), CCR9(GPR-9-6), CCRL1(VSHK1), CCRL2(L-CCR), XCR1(GPR5 / CCXCR1), CMKLR1, CMKOR1(RDC1), CX3CR1( V28), CXCR4, GPR2(CCR10), GPR31, GPR81(FKSG80), CXCR3(GPR9 / CKR-L2), CXCR6(TYMSTR / S TRL33 / Bonzo), HM74, IL8RA (IL8Rα), IL8RB (IL8Rβ), LTB4R (GPR16), TCP10, CKLFSF2, CKLFSF 3. CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, BDNF, C5R1, CSF3, GRCC10(C10), EPO, FY(DARC), GDF5, HDF1, HDF1α, DL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2 and VHL.

[0177] In another embodiment, the antibodies (e.g., bispecific or multispecific antibodies) produced using the methods provided herein are capable of binding to one or more targets selected from the group consisting of: ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; ANGPTL; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF (BLys); B AG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLRI (MDR15); BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (netrin); BRCA1; C19orf10 (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6 / JAB61); CCL1 (1-309); CCL11 (eotaxin); CCL13 (MCP-4); CCL15 (MIP1δ); CCL16 (H CC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3β); CCL2 (MCP-1); MCAF; CCL20 (MIP-3α); CCL21 (MTP-2); SLC; exodus-2; CCL22 (MDC / STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2 / eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK / ILC); CCL28; CCL3 (MTP-Iα); CCL4 (MDP-Iβ); CCL5 (RANT ES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKRI / HM145); CCR2 (mcp-IRβ / RA); CCR3 (CKR / CMKBR3); CCR4; CCR5 (CMKBR 5 / ChemR13); CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6); CCR7 (CKBR7 / EBI1); CCR8 (CMKBR8 / TER1 / CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR);CD164; CD19; CD1C; CD20; CD200; CD22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CDS; CD80; CD81; CD83; CD86; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p21 / WAF1 / Cip1); CDKN1B (p27 / Kip1); CDKN1C; CDKN2A (P16INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); CMKLR1; CMKOR1 (RDC1); CNR1; COL 18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSFI (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (b-catenin); CTSB (cathepsin B); CX3CL1 (SCYDI); CX3CR1 (V28); CXCL1 (GRO1); CXCL10 (IP-10); CXCL11 (I-TAC / IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GRO3); CXCL5 (ENA-78 / LIX); C XCL6(GCP-2); CXCL9(MIG); CXCR3(GPR9 / CKR-L2); CXCR4; CXCR6(TYMSTR / STRL33 / Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP; DES; DKFZp451J0118; DNCLI; DP P4; E2F1; ECGF1; EDG1; EFNA1; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EPHB4; EPO; ERBB2(Her-2); EREG; ERK8; ESR1; ESR2; F3(TF); FADD;FasL; FASN; FCER1A; FCER2; FCGR3A; FGF; FGF1 (αFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF 20;FGF21;FGF22;FGF23;FGF3(int-2);FGF4(HST);FGF5;FGF6(HST-2);FGF7(KGF);FGF8;FGF9;FGFR3;FIGF(VEGFD);FEL1(EPSILON);FIL1(ZET A); FLJ12584; FLJ25530; FLRTI (fibronectin); FLT1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1; GM-CSF; GNASI; GNRHI; GPR2 (CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCCIO (C10); GRP; GSN (gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIF1A; HOP1; histamine and histamine receptors; HLA-A; HLA-DRA; HM74; HMOXI; HUMCYT2A; ICEBERG; ICOSL; 1D2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNγ; DFNW1; IGBP1; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12 RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1; IL18RAP; IL19; IL1A; IL1B; ILIF10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2, ILIRN; IL2; IL20; IL20RA; IL21 R;IL22;IL22R;IL22RA2;IL23;IL24;IL25;IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); EL7; EL7R; EL8; IL8RA; DL8RB; IL8RB; DL9; DL9R; DLK; INHA; INHBA; INSL3; INSL4; IRAK1; ERAK2; ITGA1; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC Box BP); KLF6; KLKIO; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (keratin 19); KRT2A; KHTHB6 (hair-specific H-type keratin); LAMAS; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); L TB4R2; LTBR; MACMARCKS; MAG or OMgp; MAP2K7 (c-Jun); MDK; MIB1; midkine; MEF; MIP-2; MKI67; (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionein-111); MTSS1; MUC1 (mucin); MYC; MY088; NCK2; neuromucin; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66(Nogo); NgR-p75; NgR-Troy; NME1(NM23A); NOX5; NPPB; NR0B1; NR0B2; NR1D1 ;NR1D2;NR1H2;NR1H3;NR1H4;NR112;NR113;NR2C1;NR2C2;NR2E1;NR2E3;NR2F1;NR2F2;NR2F6;NR3C1;NR3C 2;NR4A1;NR4A2;NR4A3;NR5A1;NR5A2;NR6A1;NRP1;NRP2;NT5E;NTN4;ODZI;OPRD1;P2RX7;PAP;PART1;PATE;PAWR;PCA3;PCNA;POGFA;POGFB;PECAM1;PF4(CXCL4);PGF;PGR;phosphoproteoglycan;PIAS2;PIK3CG;PLAU(uPA);PLG;PLXDC1; PPBP (CXCL7); PPID; PRI; PRKCQ; PRKDI; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21 Rac2); RARB; RGSI; RGS13; RGS3; RNF110 (ZNF144); ROBO2; S100A2; SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobulin 2); SCGB2A2 (mammaglobulin 1); SCYEI (endothelial cell-monocyte activating cytokine); SDF2; SERPINA1; SERPINA3; SERP1NB5 (mammaglobulin); SERPINE1 (PAI-1); SERPDMF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPPI; SPRR1B (Sprl); ST6GAL1; STABI; STAT6; STEAP; STEAP2; TB4R2; TBX21; TCPIO; TOGFI; TEK; TGFA; TGFBI; TGFB1II; TGFB2; TGFB3; TGFBI; TGFBRI; TGFBR2; TGFBR3; THIL; THBSI (thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TMP3; tissue factor; TLR1; TLR2; TLR3; TLR4; TL R5;TLR6;TLR7;TLR8;TLR9;TLR10;TNF;TNF-a;TNFAEP2(B94);TNFAIP3;TNFRSFIIA;TNFRSF1A;TNFRSF1B;TNFRSF21;TNFRSF5;TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF11 (TRANCE); TNFSF12 (AP03L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM -L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSFS (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptor; TOP2A (Topoisomerase Ea); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP;TWEAK; VEGF; VEGFB; VEGFC; Versican; VHL C5; VLA-4; XCL1 (lymphocyte chemoattractant factor); XCL2 (SCM-1b); XCRI (GPR5 / CCXCRI); YY1; and ZFPM2.

[0178] Preferred molecular target molecules for antibodies (e.g., bispecific or multispecific antibodies) generated using the methods provided herein include: CD proteins, such as CD3, CD4, CDS, CD16, CD19, CD20, CD34, CD64, CD200; members of the ErbB receptor family, such as EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules, such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, α4 / β7 integrin and αv / β3 integrin, including α or β subunits thereof (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors, such as VEGF-A, VEGF-C; tissue factor (TF); alpha interferon (αIFN); TNFα, interleukins, such as IL-1β, IL-3, IL-4, IL-5, IL-S, IL-9, IL-13, IL 17 AF, IL-1S, IL-13Rα1, IL13Rα2, IL-4R, IL-5R, IL-9R, IgE; blood group antigens; flk2 / flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; RANKL, RANK, RSV F protein, protein C, etc.

[0179] In one embodiment, the antibodies (e.g., bispecific or multispecific antibodies) generated using the methods provided herein bind to low-density lipoprotein receptor-related protein (LRP)-1 or LRP-8 or transferrin receptor and at least one target selected from the group consisting of: 1) β-secretase (BACE1 or BACE2), 2) α-secretase, 3) γ-secretase, 4) τ-secretase, 5) amyloid precursor protein (APP), 6) death receptor 6 (DR6), 7) amyloid beta peptide, 8) α-synuclein, 9) parkin, 10) huntingtin, 11) p75 NTR, and 12) caspase-6

[0180] In one embodiment, the antibodies (e.g., bispecific or multispecific antibodies) generated using the methods provided herein bind to at least two target molecules selected from the group consisting of IL-1α and IL-1β, IL-12 and IL-1S; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-5 and IL-4; IL-13 and IL-1β; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MEF; IL-13 and TGF-~; IL-13 and LHR agonist; IL-12 and TWEAK, IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAMS, IL-13 and PED2, IL17A and IL 17F, CD3 and CD19, CD138 and CD20; CD138 and CD40; CD19 and CD20; CD20 and CD3; CD3S and CD13S; CD3S and CD20; CD3S and CD40; CD40 and CD20; CD-S and IL-6; CD20 and BR3, TNFα and TGF-β, TNFα and IL-1β; TNFα and IL-2, TNFα and IL-3, TNFα and IL-4, TNFα and IL-5, TNFα and IL6, TNFα and IL8, TNFα and IL-9, TNFα and IL-10, TNFα and IL-11, TNFα and IL-12, TNFα and IL-13, TNFα and IL-14, TNFα and IL-15, TNFα and IL-16, TNFα and IL-17, TNFα and IL-18, TNFα and IL-19, TNFα and IL-20, TNFα and IL-23, TNFα and IFNα, TNFα and CD4, TNFα and VEGF, TNFα and MIF, TNFα and ICAM-1, TNFα and PGE4, TNFα and PEG2, TNFα and RANK ligand, TNFα and Te38, TNFα and BAFF, TNFα and CD22, TNFα and CTLA-4, TNFα and GP130, TNF a and IL-12p40, VEGF and HER2, VEGF-A and HER2, VEGF-A and PDGF, HER1 and HER2, VEGFA and ANG2, VEGF-A and VEGF-C, VEGF-C and VEGF-D, HER2 and DR5, VEGF and IL-8, VEGF and MET, VEGFR and MET receptor, EGFR and MET, VEGFR and EGFR, HER2 and CD64, HER2 and CD3, HER2 and CD16, HER2 and HER3;EGFR (HER1) and HER2, EGFR and HER3, EGFR and HER4, IL-14 and IL-13, IL-13 and CD40L, IL4 and CD40L, TNFR1 and IL-1R, TNFR1 and IL-6R and TNFR1 and IL-18R, EpCAM and CD3, MAPG and CD28, EGFR and CD64, CSPGs and RGM A; CTLA-4 and BTN02; IGF1 and IGF2; IGF1 / 2 and Erb2B; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGMA; POL-1 and CTLA-4; and RGM A and RGM B. ;

[0181] Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens to generate antibodies. For transmembrane molecules, for example, receptors, fragments thereof (e.g., the extracellular domain of receptors) can be used as immunogens. Alternatively, cells expressing transmembrane molecules can be used as immunogens. Such cells can be derived from natural sources (e.g., cancer cell lines), or can be cells that have been transformed by recombinant technology to express transmembrane molecules. Other antigens and forms thereof that can be used to prepare antibodies will be apparent to those skilled in the art.

[0182] Activity assay

[0183] Antibodies (e.g., bispecific or multispecific antibodies) produced using the methods provided herein can be characterized by various assays known in the art to characterize their physical / chemical properties and biological functions. Such assays include, but are not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.

[0184] In certain embodiments, the biological activity of the antibody (e.g., bispecific or multispecific antibody) produced using the methods provided herein is analyzed. In certain embodiments, the antigen binding activity of the antibody (e.g., bispecific or multispecific antibody) produced using the methods provided herein is tested. Antigen binding assays known in the art and useful herein include, but are not limited to, any direct or competitive binding assays, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assays), "sandwich" immunoassays, immunoprecipitation assays, fluorescent immunoassays, and protein A immunoassays using, for example, Western blots.

[0185] The foregoing written description is considered sufficient to enable those skilled in the art to implement the present invention. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. In fact, in addition to those shown and described herein, various modifications will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0186] Examples

[0187] Example 1: Methods and Materials

[0188] Antibody construct design and synthesis

[0189] All antibodies in the following examples use the Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest." Bethesda, MD: NIH, 1991) and EU (Edelman et al., "The covalent structure of an entire gammaG immunoglobulin molecule." Proc Natl Acad Sci USA 1969; 63: 78-85) numbering systems to number the variable domains and constant domains, respectively. Antibody constructs were synthesized by gene To generate, and whenever applicable, the antibody construct was subcloned into an expression plasmid (pRK5) as previously described (Dillon et al., "Efficient production of bispecific IgG of different isotypes and species of origin in single mammalian cells." MAbs 2017; 9: 213-30). All antibody HCs in this study were deglycosylated (N297G mutation) and the carboxyl terminal lysine (ΔK447) was deleted to reduce product heterogeneity, thereby facilitating accurate quantification of BsIgG by LCMS (Dillon et al., hereinafter the same; Yin et al. "Precise quantification of mixtures of bispecific IgG produced insingle host cells by liquid chromatography-Orbitrap high-resolution massspectrometry." MAbs 2016; 8: 1467-76). The two component HCs of all BsIgGs in this study were engineered to contain a "knob" mutation in the first listed antibody (e.g., T366W) or a "hole" mutation in the second listed antibody (e.g., T366S:L368A:Y407V) to promote HC heterodimerization (Atwell et al. "Stable heterodimers from remodeling the domain interface of ahomodimer using a phage display library. J Mol Biol 1997; 270: 26-35).

[0190] For some BsIgG in this study, FR mutations were carefully performed to provide sufficient mass difference between correctly paired and incorrectly paired BsIgG species for more accurate quantification by LCMS analysis. The mass difference required for accurate quantification of bispecific IgG yield is ≤118Da (Yin et al., the same below). Specifically, when combined with anti-CD3 or variants (in Table A), anti-IL-1β or anti-GFRα (Table B), the antibodies and mutations are anti-HER2 V L R66G; when combined with anti-ANG2 or variants (in Table F), antibodies and mutations are anti-VEGFA V L F83A; when combined with anti-Factor D 25D7 v1 or anti-IL-33 or anti-HER2 (in Table G2), the antibody and mutations are anti-CD3 VL N34A:F83A; when combined with anti-CD3, the antibody and mutations are anti-RSPO3 V L F83A; Antibody and mutated to anti-EGFR V when combined with anti-SIRPα or anti-Factor D 20D12 v1 L F83A; plus when combined with anti-GFRα1 (Table B or Figures 1A-1F ) when combined, the antibody and the mutation are anti-IL-4V L N31A:F83A. Based on comparison with the parental antibody, the selected residues had no detectable effect on BsIgG yield.

[0191] Antibody expression and purification

[0192] All BsIgGs derived from HEK293 The four plasmids corresponding to the two LCs and two HCs were co-transfected into cells (ThermoFisher Scientific). The LC DNA was different for each experiment, and the highest bispecific yields were reported with the optimal HC:LC ratio, as previously described (Dillon et al., supra). The ratio of the two HCs was fixed at 1:1. Transfected cell cultures (30 mL) were grown at 37°C with shaking for 7 days. BsIgG from filtered cell culture supernatants were purified by protein A affinity chromatography ( AF-rProtein A, Tosoh Bioscience) was purified in a high-throughput manner. Size exclusion chromatography on a SEC-300 column (10 mm×300 mm, 3 μm particle size, Sepax Technology) was used to remove impurities such as aggregates and half IgG1. An extinction coefficient A of 1.5 was used. 0.1％ 280nm To calculate the IgG1 concentration, multiply the protein concentration by the elution volume to estimate the purification yield after Protein A chromatography.

[0193] Analysis and Characterization of BsIgG by SECHPLC

[0194] BsIgG samples (20 μL) were purified by size exclusion chromatography under isocratic conditions on a column connected to an HPLC column ( UltiMate 3000, Thermo Fisher Scientific) Chromatographic separation was performed on a SuperSW3000 column (4.6×150 mm, 4 μm) (Tosoh Bioscience). The mobile phase was 200 mM potassium phosphate and 250 mM potassium chloride at pH 7.2, with a flow rate of 0.3 mL / min, and absorbance was measured at a wavelength of 280 nm.

[0195] Determination of BsIgG yield by high-resolution LCMS

[0196] As previously described, the chromatograms were determined by mass spectrometry (Thermo Fisher Scientific). Plus Extended MassRange ) was used to perform quantification of BsIgG yield (intensity of the correctly paired LC species relative to all three mispaired IgG1 species) and assuming no response bias between different mass peaks (see Yin et al., hereinafter).

[0197] For denaturing mass spectrometry, samples (3 μg) were injected into a Dionex 3000 Rapid Separation Liquid Chromatography (RSLC) System Reversed Phase HPLC Column heated to 80°C ( Thermo Fisher Scientific, 2.1 mm × 50 mm). A binary gradient pump was used to deliver solvent A (99.88% water containing 0.1% formic acid and 0.02% trifluoroacetic acid) and solvent B (90% acetonitrile containing 9.88% water plus 0.1% formic acid and 0.02% trifluoroacetic acid) as a gradient from 20% to 65% solvent B at a rate of 300 μL / min in 4.5 min. The solvent was stepped to 90% solvent B in 0.1 min and held at 90% for 6.4 min to clean the column. Finally, the solvent was stepped to 20% solvent B in 0.1 min and held for 3.9 min for re-equilibration. The samples were analyzed online via electrospray ionization into the mass spectrometer using the following parameters for data acquisition: 3.90 kV spray voltage; 325°C capillary temperature; 200 S-lens RF level; 15 sheath gas flow rate and 4 auxiliary gas flow rate in the ESI source; scan range from 1,500 to 6,000 m / z; desolvation, in-source CID 100 eV, CE 0; resolution of 17,500 at m / z 200; positive polarity; 10 microscans; 3E6 AGC target; fixed AGC mode; 0 averaging; 25 V source DC bias; 8 V injection flat electrode DC; 7 V flat electrode inter-lens; 6 V bent flat electrode DC; 0 V transfer multipole DC adjustment bias; 0 V C-Trap entrance lens adjustment bias; trap gas pressure set to 2.

[0198] For native mass spectrometry, samples (10 μg) were injected into a Dionex 3000 RSLC system heated to 30°C Acquity BEH size exclusion chromatography column (Waters, 4.6 mm×150 mm). Isocratic chromatography runs (10 min) utilized an aqueous mobile phase containing 50 mM ammonium acetate (pH 7.0, flow rate 300 μL / min).

[0199] The samples were analyzed online via electrospray ionization into the mass spectrometer using the following parameters for data acquisition: 4.0 kV spray voltage; 320°C capillary temperature; 200 S-lens RF level; 4 sheath gas flow rates and 0 auxiliary gas flow rates in the ESI source; scan range from 300 to 20,000 m / z; desolvation, in-source CID 100 eV, CE 0; resolution of 17,500 at m / z 200; positive polarity; 10 microscans; 1E6 AGC target; fixed AGC mode; 0 averaging; 25 V source DC bias; 8 V injection flat electrode DC; 7 V flat electrode inter-lens; 6 V bent flat electrode DC; 0 V transfer multipole DC adjustment bias; 0 V C-Trap entrance lens adjustment bias; trap gas pressure set to 2.

[0200] Using Protein Metrics Intact Software and Thermo Fisher BIOPHARMA The mass spectrometry data collected was analyzed using the 3.0 software. The signal intensity of the correctly paired LC species from the deconvoluted spectrum of each sample was used for quantification relative to the three mispaired IgG1 species. HC homodimers and half IgG were either undetectable or present in trace amounts and excluded from the calculation. The correct LC-paired BsIgG was estimated from a homogenous mixture of BsIgG and double LC mispaired IgG1 using the algebraic formula described previously (see Yin et al., the same below).

[0201] SDS-PAGE gel analysis of BsIgG

[0202] BsIgG purified by protein A and size exclusion chromatography were analyzed by SDS-PAGE. Samples were prepared in the presence and absence of DTT for analysis of electrophoretic mobility under reducing and non-reducing conditions, respectively. The samples mixed with the sample dye were heated at 95°C for 5 min with DTT or for 1 min without DTT and then electrophoresed at 120 V on a 4-20% Tris-glycine gel (Bio-Rad). The gel was then washed with GELCODE TMBlue protein stain (Thermo Fisher Scientific) was used for staining and destained in water. An equal amount of protein (6 μg) was loaded for each sample.

[0203] Kinetic binding experiments

[0204] Kinetic binding experiments were performed using surface plasmon resonance on a BIAcore T200 instrument (GE Healthcare). Anti-Fab (GE Healthcare) was immobilized [approximately 12000 resonance units (RU)] on a CM5 sensor chip. Parental and mutant Fabs were captured on the immobilized surface and the binding of the analyte was evaluated. Sensorgrams were generated using an injection time of 3 min, a flow rate of 50 μl / min at 25°C with the following analyte concentrations: 0, 0.293, 1.17, 4.6875, 18.75, 75, 300 nM HER2-ECD (in-house) and VEGF-C (Cys156Ser) (R&D Systems, Cat. No. 752-VC); 0, 0.0195, 0.0781, 0.3125, 1.25, 5, 20 mM VEGF165 (R&D Systems, Cat. No. 293-VE) and IL-13 (in-house); 0, 0.0732, 0.293, 1.17, 4.6875, 18.75, 75 nM MET-R Fc (R&D Systems, Cat. No. 8614-MT), IL-1β (R&D Systems, catalog number 201-LB / CF), EGFR Fc (R&D Systems, catalog number 344-ER); 0, 0.976, 3.906, 15.625, 62.5, 250nM biotinylated CD3 (in-house). Dissociation was monitored for 900 seconds after injection of the analyte. The running buffer used was 10mM HEPES, pH 7.4, 150mM NaCl, 0.003% EDTA, 0.05% Tween (HBS-EP+, GE Healthcare). The chip surface was regenerated after each injection of 10mM glycine (pH 2.1). The sensorgram was calibrated using a double blank reference (a substation with zero analyte concentration and a blank reference cell). The sensorgram was then analyzed using a 1:1 Langmuir model using the software provided by the manufacturer.

[0205] Example 2: Elucidating heavy chain / light chain pairing preferences to facilitate assembly of bispecific IgG in single cells

[0206] Preface

[0207] In the studies described here, high-throughput production and high-resolution LCMS analysis (Dillon et al. "Efficient production of bispecific IgG of different isotypes and species of origin in single mammalian cells." MAbs 2017;9:213-30; Yin et al. Precise quantification of mixtures of bispecific IgG produced in single host cells by liquid chromatography-Orbitrap high-resolution mass spectrometry." MAbs 2016;8:1467-76) were used to investigate 99 different antibody pairs containing a knob-in-hole HC but no Fab mutations for BsIgG yield. One-third of the antibody pairs showed high (>65%) BsIgG yields, consistent with strong intrinsic cognate HC / LC chain pairing preferences. For such antibody pairs, the yield of BsIgG was higher in the presence of two C H 1 / C L Previously determined charge mutations (Dillon et al., "Efficient production of bispecific IgG of different isotypes and species of origin in single mammalian cells." MAbs 2017; 9: 213-30) were installed at the domain interface to enhance the production of BsIgG. Next, we investigated whether high yields of BsIgG require homologous chain pairing preferences in one or both arms. Mutational analysis was used to identify specific residues in CDR H3 and L3 that contribute to high BsIgG yields. CDR H3 and L3 and the identified specific residues were then inserted into other available, unrelated antibodies (which showed random HC / LC chain pairing) to determine their effects on BsIgG yields. Finally, mutational analysis was used to study the effects of interchain disulfide bonds on the yield of BsIgG.

[0208] Effects of constituent antibody pairs on BsIgG yield

[0209] Previously, high yields (>65%) of BsIgG were observed for two bispecific antibodies (i.e., anti-EGFR / MET and anti-IL-13 / IL-4) in the presence of knob-in-hole heavy chain (HC) mutations but without Fab arm mutations (Dillon et al., hereinafter the same). In order to study the strength and frequency of cognate heavy chain / light chain (HC / LC) pairing preferences, larger antibody pair combinations (n=99) were used to generate BsIgG. For simplicity, all bispecific antibodies in this study were constructed using human IgG1 HC constant domains. Six antibodies that bind to IL-13, IL-4, MET, EGFR, HER2, or CD3 (Dillon et al., hereinafter the same) were used to construct matrices for all 15 possible BsIgG1s. Next, these six antibodies were replaced by 14 additional antibodies, primarily of the κLC isotype, of which the LC isotype was three λLC isotypes (anti-DR5, anti-α5β1, anti-RSPO2) (see Table A below). In Table A, germline gene families were identified by comparing LC and HC sequences to the human antibody germline gene repertoire using a proprietary alignment tool. The closest matches to germline gene segments are reported. All antibodies used in this study were humanized except for three fully human antibodies (anti-CD33, anti-PDGF-C, anti-Flu B).

[0210] Table A: Germline gene family and LC isotype analysis of different antibodies evaluated for LC / HC pairing preference.

[0211]

[0212]

[0213] KV = kappa variable; LV = lambda variable, HV = heavy chain variable; na = not available.

[0214] 1. Merchant M, Ma

[0215] 2. Schaefer G, Haber L, Crocker LM, Shia S, Shao L, Dowbenko D, Totpal K, Wong A, Lee CV, Stawicki S et al., A two-in-one antibody against HER3 and EGFR has superior inhibitory activity compared with monospecific antibodies. Cancer Cell 2011;20:472-86.

[0216] 5. Ultsch M, Bevers J, Nakamura G, Vandlen R, Kelley RF, Wu LC, Eigenbrot C. Structural basis of signaling blockade by anti-IL-13 antibody lebrikizumab. J Mol Biol 2013;425:1330-9.

[0217] 6. Spiess C, Bevers J, 3rd, Jackman J, Chiang N, Nakamura G, Dillon M, Liu H, Molina P, Elliott JM, Shatz W et al., Development of a human IgG4 bispecific antibody for dual targeting of interleukin-4(IL-4) and interleukin-13(IL-13) cytokines. J Biol Chem 2013;288:26583-93.

[0218] 8. Carter P, Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL, Rowland AM, Kotts C, Carver ME, Shepard HM. Humanization of an anti-p185 HER2 antibody for human cancer therapy. Proc Natl Acad Sci U S A 1992;89:4285-9.

[0219] 9. Rodrigues ML, Shalaby MR, Werther W, Presta L, Carter P. Engineering a humanized bispecific F(ab')2 fragment for improved binding to T cells. Int JCancer Suppl 1992;7:45-50.

[0220] 10. Bhakta S, Crocker LM, Chen Y, Hazen M, Schutten MM, Li D, Kuijl C, OhriR, Zhong F, Poon KA, et al., An anti-GDNF family receptor alpha 1(GFRA1)antibody-drug conjugate for the treatment of hormone receptor-positive breastcancer. Mol Cancer Ther 2018;17:638-49.

[0221] 11. Adams C, Totpal K, Lawrence D, Marsters S, Pitti R, Yee S, Ross S, Deforge L, Koeppen H, Sagolla M, et al., Structural and functional analysis of the interaction between the agonistic monoclonal antibody Apomab and the proapoptotic receptor DR5. Cell Death Differ 2008; 15:751-61.

[0222] Next, the antibody pairs shown in Table B below were expressed in HEK293 derived The cells were co-expressed, and the yield of BsIgG was determined using a modified version of a previously described method (see Dillon et al., Yin et al., hereinafter the same). No antibody pairs contained the Fab mutations described by Dillon et al. (hereinafter the same). All bispecific antibody pairs contained knob-and-hole mutations for heavy chain heterodimerization.

[0223] After the co-expression of antibody pairs and protein A chromatography, the purified IgG1 pool is further purified by size exclusion chromatography (SEC) to remove any small amount of aggregates and half IgG1 present before being quantified by high-resolution LCMS. The yield of BsIgG correctly assembled in a homogeneous mass (i.e., same molecular mass) mixture also comprising LC- missense IgG1 is estimated using a previously developed algebraic formula (see Yin et al., hereinafter the same). The data shown in Table B are the yields of BsIgG from the LC DNA ratio of optimization. BsIgG yields>65% are represented in bold. The HC of mAb-1 includes a "hole" mutation (T366S:S368A:Y407V), and the HC of mAb-2 includes a "knob" mutation (T366W) (Atwell et al. "Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library." J Mol Biol 1997; 270:26-35).

[0224] Table B: Half-antibody pairs used to study BsIgG yield

[0225]

[0226]

[0227] NA = not applicable; monospecific antibody.

[0228] The yields of BsIgG1 for 99 unique antibody pairs varied over a wide range: 22%-95% (see Table B). Strikingly, for most (>80%) of the antibody pairs, non-random HC / LC pairing was observed (>30% yield of BsIgG1); for 33 and 48 antibody pairs, high (>65%) and intermediate (30%-65%) yields of BsIgG1 were seen, respectively. Nearly quantitative (>90%) formation of BsIgG1 was measured for two antibody pairs (anti-MET / DR5 and anti-IL-13 / DR5).

[0229] Figures 1A-1F Low yields (<30%, e.g., anti-LGR5 / IL-4, see Figure 1A and 1B ), intermediate yield (30%-65%, for example, anti-SIRPα / IL-4, see Figure 1C and 1D ) and high yield (>65%, e.g., anti-MET / DR5, see Figure 1E and 1F). The corresponding antibody pairs were transiently co-transfected into HEK293 derived Before quantification of BsIgG1 yield by high-resolution LCMS, IgG1 species were purified by protein A chromatography and size exclusion chromatography as described by Dillon et al. (hereinafter) and Yin et al. (hereinafter). Figure 1A , 1C The data shown in 1E are the mass envelopes for charge states 38+ and 39+, and Figure 1B , 1D and 1F show the corresponding deconvoluted data and provide a schematic representation of the presence of different IgG1 species.

[0230] The BsIgG1 yield of each antibody studied varied over a wide range, depending on its partner antibody. For example, the BsIgG1 yield of the anti-MET antibody varied from a low of about 21% (when paired with anti-IL-33) to a high of about 95% (when paired with anti-DR5) (Table B). In order to investigate any effects of the "knob" and "hole" mutations on cognate HC / LC pairing preferences, BsIgG1 was produced using HCs containing the "knob" mutation in mAb1 and the "hole" mutation in mAb2, and vice versa (Table B). In all cases tested (n=15), the yield of BsIgG1 was least affected by which HC contained the "knob" and "hole" mutations (Table B). The IgG species recovered from 30 mL of culture by protein A chromatography varied by more than about 5-fold (1.5 mg to 8.0 mg)

[0231] The above results indicate that high yields of BsIgG1 without Fab mutations are common, depending on the composition of the antibody pair.

[0232] C H 1 / C L Effect of interface charge mutations on the yield of BsIgG1 from antibody pairs with cognate HC / LC pairing preference

[0233] Previously, combinations of mutations at all four domains / domain interfaces in conjunction with the knob-to-hole HC mutation (i.e., V H / V L Both and C H 1 / C L Both) were used for near quantitative assembly of BsIgG of different isotypes in a single mammalian host cell (see Dillon et al., hereinafter). Here, antibody pairs that gave high yields of BsIgG1 without any Fab mutations were identified (Table B). These antibody pairs differed in their variable domain sequences, while the constant domains (i.e., IgG1 C H 1 and k C L ) are identical in most cases. Assume that for such antibody pairs, only two C H 1 / C LMutations at the interface may be sufficient to enhance the yield of correctly assembled bispecific antibodies to about 100%. Eleven different antibody pairs were selected and expressed in the presence or absence of the previously reported C H 1 / C L The yield of BsIgG1 was compared with that of the case where the domain interface charge was mutated (see Dillon et al., hereinafter the same). L V133E and C H 1 S183K mutation was engineered, and the “mortise” arm was replaced with C L V133K and C H 1 S183E mutation was engineered (see Dillon et al., hereinafter). H 1 / C L Charge mutations at the interface increased BsIgG1 yields by approximately 12%-34% for all antibody pairs, increasing to ≥90% BsIgG1 yield in most cases (9 / 11). Figure 2 ).for Figure 2 The first listed antibody in the pair comprises a C L V133E and C H 1S183K mutation, and the second listed antibody contains C L V133K and C H 1 S183E mutation (see Dillon et al., hereinafter the same). The 90% yield of BsIgG1 is Figure 2 The dashed horizontal line in C L V133E and C H 1 The S183K mutation did not affect the affinity of the antibody for its target antigen (data not shown).

[0234] Effect of cognate HC / LC pairing preference in one arm of BsIgG on the yield of BsIgG

[0235] The mechanistic basis for the observed high yields of BsIgG1 for some antibody pairs was studied. For this study, two antibody pairs, anti-EGFR / MET and anti-IL-4 / IL-13, were selected based on their high BsIgG1 yields and the absence of Fab mutations (see Table B and Dillon et al., hereinafter the same). A priori, one or both Fabs may exhibit a preference for cognate HC / LC pairing, which contributes to the high yield of BsIgG1. Three chain coexpression experiments were performed to distinguish these possibilities. A single HC (HC1) with a "knob" or "hole" mutation was expressed in Expi293F. TM The cells were transiently co-expressed with their cognate LC (LC1) and a competing non-cognate LC (LC2). Figure 3 ). Figure 3The asterisks in the figure indicate the presence of a "knob" or "hole" mutation in the HC. (HCs for anti-EGFR, anti-IL13, and anti-HER2 contain a "knob" mutation (T366W), while HCs for anti-MET, anti-IL4, and anti-CD3 contain a "hole" mutation (T366S:S368A:Y407V) (see Atwell et al., "Stable heterodimers from remodeling the domain interface of ahomodimer using a phage display library. J Mol Biol 1997; 270:26-35).) The half-IgG species were purified from the corresponding cell culture supernatants by protein A affinity chromatography and the extent of homologous and non-homologous HC / LC pairing assessed by high-resolution LCMS (see Dillon et al., Yin et al., hereinafter the same). The percentage of homologous HC / LC pairing was calculated by quantifying the half-IgG1 species.

[0236] As shown in Table C below, anti-MET HC showed a strong preference for its cognate LC (about 71%) compared to non-cognate anti-EGFR LC, while anti-EGFR HC showed only a slight preference for its cognate LC (about 56%) compared to non-cognate anti-MET LC. Anti-IL-13 HC showed a strong preference for its cognate LC (81%) compared to non-cognate anti-IL-4 LC, while anti-IL-4 HC showed no preference for its cognate LC (49%). These data are consistent with the view that the high BsIgG1 yield of anti-EGFR / MET is generated by the strong and weak cognate HC / LC pairing preferences of anti-MET and anti-EGFR antibodies, respectively. In contrast, the high BsIgG1 yield of anti-IL-13 / IL-4 apparently reflects the strong cognate HC / LC pairing preference of the anti-IL-13 antibody alone. Therefore, the cognate HC / LC pairing preference in one or both arms is apparently sufficient for the high yield of BsIgG1 in a single cell without the need for Fab mutation.

[0237] Table C: Quantification of antibody cognate chain preferences after co-expression.

[0238]

[0239] Anti-HER2 / CD3 was selected as a control for this study based on its low yield of BsIgG1 (see Table B and Dillon et al., below). Anti-HER2 HCs did not show a pairing preference for their cognate LCs compared to non-cognate anti-CD3 LCs. Similarly, anti-CD3 HCs did not show a pairing preference for their cognate LCs compared to non-cognate anti-HER2 LCs (see Table C).

[0240] When co-expressed in a single host cell, the pairing of HC with its cognate light chain (LC) or non-cognate LC was also assessed. Briefly, each HC was co-transfected with its cognate LC or non-cognate LC into a HEK293-derived cells. IgG1 and half IgG1 species were purified from cell culture supernatants by protein A chromatography and analyzed by LC-MS. (Labrijn et al. “Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange.” Proc Natl Acad Sci USA 2013; 110: 5145-50; SpiessC et al. “Bispecific antibodies with natural architecture produced by co-cultureof bacteria expressing two distinct half-antibodies.” Nat Biotechnol 2013; 31: 753-8). The percentage of homologous HC / LC pairing was calculated by quantifying the half IgG1 species. Protein expression yield was estimated by multiplying the antibody concentration by the elution volume obtained from the high-throughput protein A chromatography step. The HCs for anti-EGFR, anti-IL-13, and anti-HER2 contain a "knob" mutation (T366W), while the HCs for anti-MET, anti-IL-4, and anti-CD3 contain a "hole" mutation (T366S:S368A:Y407V) (see Spiess et al. "Alternative molecular formats and therapeutic applications for bispecific antibodies." Mol Immunol 2015; 67:95-106). In the absence of competition, the HCs can be efficiently assembled with non-cognate LCs, as judged by all six different mismatched HC / LC pairs tested (see Table D below).

[0241] Table D: HC paired with its cognate light chain (LC) or non-cognate LC when co-expressed in a single host cell

[0242]

[0243] Contribution of anti-METCDRL3 and CDRH3 to the production of anti-EGFR / MET BsIgG1

[0244] Sequence determinants in anti-MET antibodies that contribute to the high bispecific yield of anti-EGFR / MET BsIgG1 were investigated. The amino acid sequence differences between anti-EGFR and anti-MET antibodies are entirely located within the CDRs plus one additional framework region (FR) residue, VH 94 (immediately adjacent to CDR H3) (Figure 4). The remaining FRs, plus the CDRs of these antibodies k and C H 1 constant domain sequence, are identical (Figure 4). CDR L3 and H3 are in the V H / V L The most extensively involved CDRs at the domain interface, as demonstrated by the X-ray crystallographic structure of an anti-MET Fab in complex with its antigen (Protein Data Bank (PDB) identification code 4K3J) (see Merchant et al. "Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent." Proc Natl Acad Sci USA 2013; 110: E2987-96). These observations led to the hypothesis that CDRs L3 and H3 of anti-MET antibodies may contribute to the high bispecific yield of anti-EGFR / MET BsIgG1. Consistent with this idea, replacing CDRs L3 and H3 of anti-MET antibodies with the corresponding sequences from anti-CD3 antibodies resulted in a significant loss in bispecific yield (approximately 85% to 33%, Figure 5A In contrast, replacing both CDRs L3 and H3 of the anti-EGFR arm of the anti-EGFR / MET bispecific antibody resulted in only a small decrease in BsIgG yield (about 85% to 75%). Figure 5A ). Replacing the CDR L3 and H3 of the anti-EGFR and anti-MET arms resulted in random HC / LC pairing. These data support the view that CDR L3 and H3 of anti-MET made a major contribution to the high bispecific yield of anti-EGFR / MET BsIgG1 observed, while CDR L3 and H3 of anti-EGFR made a minor contribution. Replacing CDR L1 and H1 or CDR L2 and H2 from the anti-MET antibody with the corresponding anti-CD3 antibody sequences had little effect on the bispecific yield of anti-EGFR / MET BsIgG ( Figure 6 ).

[0245] Contribution of residues within anti-METCDR L3 and CDR H3 to the yield of anti-EGFR / MET BsIgG1

[0246] Next, we investigated the residues within CDR L3 and H3 of the anti-MET antibody that contribute to the high bispecific yield of anti-EGFR / METBs IgG1. The X-ray crystallographic structure of the anti-MET Fab (PDB accession code 4K3J) revealed the contact residues between CDR L3 and H3 ( Figure 7 ) and used to guide the selection of residues for mutational analysis. Alanine scanning mutagenesis of anti-MET CDR L3 and H3 (Cunningham et al. "High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis." Science 1989; 244: 1081-5) was used to map residues that contribute to the high bispecific yield of anti-EGFR / MET BsIgG1.

[0247] Table E1: Alanine scanning mutagenesis of CDR L3 and H3 contact residues of anti-MET antibodies

[0248]

[0249]

[0250] As shown in Table E1 above, V in CDR L3 L The Y91A mutation caused the largest decrease in bispecific yield of any of the 12 single alanine mutants tested (84% to 57%). L Y91A:Y94A, eliminated the high bispecific yield (84% to 23%). L Y91 and Y94 seem to have made a crucial contribution to the high bispecific yield of anti-EGFR / MET BsIgG1. The expression titers of all mutants are comparable to parental BsIgG1, as estimated by the yields recovered from protein A chromatograms (data not shown). The data shown in Table E1 represent the ± standard deviation of two independent experiments using optimized HC / LC DNA ratios (see Table B).

[0251] The affinity of the parent anti-MET Fab, and a subset of anti-MET Fab variants in Table E1 for MET was determined via surface plasmon resonance (SPR). on ), dissociation rate (k off ) and binding affinity (K D) are shown in Table E2 (nd means no binding detected). The P95A substitution in CDR L3 did not affect the binding of the anti-MET Fab variants to MET. Other single alanine substitutions in CDRL3 reduced affinity to varying degrees. For the anti-Met Fab variants with Y91A:Y94A or Y91A:W96A double substitutions in CDR L3, no binding to the antigen was detected.

[0252] Table E2

[0253]

[0254] Contribution of anti-IL13 CDR L3 and CDR H3 to the yield of anti-IL13 / IL14 BsIgG1

[0255] Given that specific residues in CDR L3 of anti-MET antibodies were found to be important for high bispecific yields of anti-EGFR / MET BsIgG1, it was hypothesized that similar principles could be applied to anti-IL-13 antibodies to contribute to high bispecific yields of anti-IL-13 / IL-4 BsIgG1. A similar experimental strategy was used to investigate this possibility. A notable difference between these two antibody pairs is that anti-IL-13 and anti-IL-4 antibodies differ in both their CDR and FR sequences ( FIG. 8 ), whereas anti-MET and anti-EGFR antibodies have identical FR sequences (except for V H 94) but differ in their CDR sequences (Fig. 4).

[0256] Substitution of CDRs L3 and H3 of the anti-IL-13 antibody with the corresponding sequences from the anti-CD3 antibody resulted in a significant loss in the bispecific yield of anti-IL-13 / IL-4BsIgG1 (approximately 72% to 37%, Figure 5B In contrast, when CDRs L3 and H3 of the anti-IL-4 antibody were replaced in a similar manner, a slight increase was observed ( Figure 5B ). These results indicate that CDRs L3 and H3 of the anti-IL-13 antibody contribute to the high bispecific yield of anti-IL-13 / IL-4BsIgG1.

[0257] Alanine scanning mutation analysis of anti-IL-13 CDR L3 and H3 (Cunningham et al., hereinafter) was used to map residues that contribute to the high bispecific yield of anti-IL-13 / IL-4BsIgG1. The X-ray crystallographic structure of anti-IL-13 Fab in complex with IL-13 (PDB accession code 4I77, see Ultsch et al. "Structural basis of signaling blockade by anti-IL-13 antibody lebrikizumab." J Mol Biol 2013; 425: 1330-9) revealed contact residues between CDR L3 and H3 ( Fig. 9 ) and used to select residues for mutation analysis (Table F1 below). CDR L3 mutation V L R96A caused the greatest decrease in bispecific yield of any of the nine single alanine mutants tested for CDR L3 and H3 and eliminated the high bispecific yield (72% to 29%). H D95A:P99A, also eliminated the high bispecific yield (72% to 26%). The expression titers of all mutants were comparable to the parental BsIgG1, as estimated by the yield recovered from the protein A chromatography (data not shown). The data shown in Table F1 represent the ± standard deviation of two independent experiments using the optimized HC / LC DNA ratio (see Table B).

[0258] Table F1: Alanine scanning mutagenesis of CDR L3 and H3 contact residues of anti-IL13 antibodies

[0259]

[0260] Therefore, judging by both the anti-EGFR / MET and anti-IL-13 / IL-4 BsIgG1 studied here, CDR L3 and / or H3 may have made a crucial contribution to the high bispecific yield.

[0261] The affinity of the parent anti-IL-13 Fab, and a subset of anti-IL-13 Fab variants in Table F1 for IL-13 was determined via SPR. on ), dissociation rate (k off ) and binding affinity (K D) are shown in Table F2 (nd means no binding detected). Neither the N92A nor the D94A substitutions in CDR L3 affected the binding of the anti-IL-13 Fab variants to IL-13. The R96A substitution in CDR L3 resulted in an approximately 10-fold loss in binding affinity, as did the D94:R96A double substitution in CDR L3. Other single alanine substitutions in CDR H3 reduced affinity to varying degrees. For the D95A:P99A double substitution in CDR H3, no binding to the antigen was detected.

[0262] Table E2.

[0263]

[0264]

[0265] Effects of CDR L3 and CDR H3 on the yield of BsIgG1

[0266] Next, a series of experiments were performed to determine whether CDR L3 and H3 from these antibodies were sufficient to provide high bispecific yields for other antibody pairs. Two antibody pairs with low bispecific yields were selected, namely anti-HER2 / CD3 (22%-24%) and anti-VEGFA / ANG2 (24%) (see Table B and Dillon et al., see below), and the CDR L3 and H3 of one arm of each of these two BsIgG1 were replaced with the corresponding CDR sequences from anti-MET or anti-IL-13 antibodies. For anti-HER2 / CD3 ( Fig. 10A ) and anti-VEGFA / ANG2( Fig. 10B ), a significant increase in the yield of BsIgG1 was observed in three out of four CDR L3 and H3 recruitment situations (from approximately 24% to 40-65%). Fig. 10A and 10B The data presented in are from the optimized LC DNA ratio. Fig. 10A and 10B The data in suggest that recruiting CDR L3 and H3 from antibodies with cognate HC / LC pairing preferences can enhance the yield of BsIgG1s without pairing preferences, but this is not always the case.

[0267] The effect of recruiting a single key residue from an anti-IL-13 antibody into other antibodies on BsIgG1 yield was studied. See Table G1 below. Amino acid numbering is according to Kabat. Antibodies containing variable domain mutations are in bold. The data shown are from optimized LC DNA ratios. Anti-VEGFC with an aspartic acid residue at position 95 (D95) was not mutated.

[0268] Table G1: Recruitment of single key residues from anti-IL13 antibody into other antibodies to study the effect on BsIgG1 yield

[0269]

[0270] When two or more key residues of the pairing preference of anti-IL-13 were transplanted to the corresponding positions in anti-HER2, anti-VEGFA or anti-VEGFC antibodies, an increase in bispecific yield was observed, although less than that of the parent anti-IL-13 / IL-4BsIgG1 (see Table G2 below). In Table G2, antibodies containing variable domain mutations are represented in bold, and the amino acid numbering is according to Kabat. Antibodies containing variable domain mutations are represented in bold underlined text. The data shown represent the mean ± SD of two independent experiments using optimized LC DNA ratios. Anti-VEGFC with an aspartic acid residue at position 95 (D95) did not mutate.

[0271] Table G2: Key residues from anti-IL13 antibody were recruited into other antibodies to study the effect on BsIgG1 yield

[0272]

[0273] Taken together, these results suggest that charged residues (such as D and R) at positions 94 and 96 (Kabat numbering) of CDR L3 and 95 (Kabat numbering) of CDR H3 may confer pairing preferences to some but not all antibody pairs.

[0274] The affinity of the parental anti-HER2, anti-VEGFA and anti-VEGFC Fabs and a subset of anti-HER2, anti-VEGFA and anti-VEGFC Fab variants in Tables G1 and G2 to their respective targets was determined via SPR. on ), dissociation rate (k off ) and binding affinity (K D ) are shown in Table G3 (nd means no binding detected). Transferring key residues from anti-IL13 to other antibodies resulted in a loss of binding affinity. Notably, the T94D substitution in the CDR-L3 of anti-HER2 increased the BsIgG1 yield of the anti-HER2 / anti-CD3 BsAb from 24% to almost 50%, but only reduced the affinity of anti-HER2 for HER2 by 20-fold. Similarly, the V94D:W96R double substitution in the VEGFA CDR-L3 increased the BsIgG1 yield of the anti-VEGFA / anti-ANG2 BsAb from approximately 22% to approximately 52%, but only reduced the affinity of anti-VEGFA for VEGFA by approximately 20-fold.

[0275] Table G3

[0276]

[0277] In contrast to the results shown in Tables G1 and G2, when the key residues of the pairing preference of anti-cMet were transplanted to the corresponding positions of anti-HER2, anti-VEGFA or anti-VEGFC antibodies, almost no increase in bispecific yield was observed in most cases. See Table G4 below. In Table G4, antibodies containing variable domain mutations are indicated in bold, and the amino acid numbering is according to Kabat.

[0278] Table G4: Recruitment of key residues from anti-cMet antibody into other antibodies to study the effect on BsIgG1 yield

[0279]

[0280]

[0281] Contribution of interchain disulfide bonds to the yield of BsIgG1

[0282] Previously, it was hypothesized that the formation of interchain disulfide bonds between HC and LC acts as a kinetic trap to prevent chain exchange (Dillon et al., hereinafter the same). Experiments were performed to investigate whether the disulfide bonds between HC and LC affect the bispecific yields of two BsIgG1s (anti-EGFR / MET and anti-IL-13 / IL-4) with obvious homologous chain preferences and the bispecific yields of two controls (anti-HER2 / CD3 and anti-VEGFA / VEGFC) with random HC / LC pairings. In brief, cysteine ​​to serine mutations were used to generate BsIgG1 variants lacking interchain disulfide bonds: LC C214S and HC C220S. The removal of interchain disulfide bonds in the engineered variants was verified by SDS PAGE. Samples were electrophoresed under reducing or non-reducing conditions, as Fig.11 As shown. Four different BsIgG1 were analyzed: anti-HER2 / CD3 (lane 1); anti-VEGFA / VEGFC (lane 2); anti-EGFR / MET (lane 3); and anti-IL13 / IL14 (lane 4). As shown in Table H below, according to native mass spectrometry, no clear evidence was found that the interchain disulfide bonds affected the BsIgG1 yield of any of the four antibody pairs tested. The yields of BsIgG1 of parental variants and disulfide bond engineered variants are similar. The data in Table H are the mean ± standard deviation of three biological replicates using optimized DNA light chain ratios.

[0283] Table H: Mutation analysis to determine the effect of the disulfide bond between HC and LC on BsIgG1 yield.

[0284]

[0285] In summary, this study shows that cognate HC / LC pairing preference in BsIgG production in single cells is a common phenomenon that depends largely on the specific antibody pair. Mechanistically, this chain pairing preference may be strongly influenced by residues in CDR H3 and L3. In fact, this pairing preference can be used to reduce the number of Fab mutations used to drive the production of BsIgG1 and possibly other isotypes of BsIgG in single cells.

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[0296] Schaefer W, HR, Lorenz S, Imhof-Jung S, Regula JT, Klein C, Analysis of heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies by UHR-ESI-QTOF mass spectrometry. MAbs 2016;8:49-55.

[0297] M, Sellmann C, Maresch D, Halbig C, Becker S, Toleikis L, Hock B, Ruker F. Novel CH1:CL interfaces that enhance correct light chain pairing in heterodimeric bispecific antibodies. Protein Eng Des Sel 2017;30:685-96.

[0298] Kitazawa T, Igawa T, Sampei Z, Muto A, Kojima T, Soeda T, Yoshihashi K, Okuyama-Nishida Y, Saito H, Tsunoda H et al., A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model. Nature Medicine 2012;18:1570-4.

[0299] Sampei Z, Igawa T, Soeda T, Funaki M, Yoshihashi K, Kitazawa T, Muto A, Kojima T, Nakamura S, Hattori K. Non-antigen-contacting region of an asymmetric bispecific antibody to factors IXa / X significantly affects factor VIII-mimetic activity. MAbs 2015;7:120-8.

[0300] Sampei Z, Igawa T, Soeda T, Okuyama-Nishida Y, Moriyama C, Wakabayashi T, Tanaka E, Muto A, Kojima T, Kitazawa T et al., Identification and multidimensional optimization of an asymmetric bispecific IgG antibody mimicking the function of factor VIII cofactor activity. PLoS One 2013;8:e57479.

[0301] Carter PJ. Introduction to current and future protein therapeutics: a protein engineering perspective. Exp Cell Res 2011.

[0302] Wu H, Pfarr DS, Johnson S, Brewah YA, Woods RM, Patel NK, White WI, Young JF, Kiener PA. Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract. J Mol Biol 2007;368:652 - 65.

[0303] Cooke HA, Arndt J, Quan C, Shapiro RI, Wen D, Foley S, Vecchi MM, Preyer M. EFab domain substitution as a solution to the light-chain pairing problem of bispecific antibodies. MAbs 2018;10:1248 - 59.

[0304] Tiller KE, Li L, Kumar S, Julian MC, Garde S, Tessier PM. Arginine mutations in antibody complementarity-determining regions display context-dependent affinity / specificity trade-offs. J Biol Chem 2017; 292: 16638-52。

[0305] Dashivets T, Stracke J, Dengl S, Knaupp A, Pollmann J, Buchner J, Schlothauer T. Oxidation in the complementarity-determining regions differentially influences the properties of therapeutic antibodies. MAbs 2016; 8: 1525-35.

[0306] Lamberth K, Reedtz-Runge SL, Simon J, Klementyeva K, Pandey GS, Padkjaer SB, Pascal V, Leon IR, Gudme CN, Buus S et al., Post hoc assessment of the immunogenicity of bioengineered factor VIIa demonstrates the use of preclinical tools. Sci Transl Med 2017; 9。

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[0310] Example 3: Affinity maturation of the modified antibodies produced in Example 2

[0311] The exemplary antibodies in Table 1 generated in Example 2 were affinity matured to improve their affinity for their respective target antigens.

[0312] Table I

[0313]

[0314] *Amino acid numbering is according to Kabat.

[0315] **See Table G3.

[0316] In short, mutation is introduced into the CDR of the antibody in Table 1, so that one or more polypeptide libraries (for example, phage display library or cell surface display library) are produced for each antibody. The amino acid replacement that is introduced into the CDR-L3 and / or CDR-H3 of each antibody to improve bispecific productive rate (referring to Table 1) remains fixed, and is not randomized during library construction. Each library is then screened by panning or cell sorting (for example, such as Wark et al. (2006) Adv Drug Deliv Rev.58:657 670; Rajpal et al. (2005) Proc Natl Acad Sci US A.102:8466 8471 described), to identify the antibody variants that bind to target antigen (i.e. HER2, VEGFA or VEGFC) with high affinity. Such variants are then isolated and their affinity for their target antigen is determined, for example, via surface plasmon resonance, and compared to the affinity of the antibodies shown in Table 1 and the affinity of the parental antibody from which the antibodies in Table 1 are derived (see, e.g., Table G3). At least one round (such as at least any one of 2, 3, 4, 5, 6, 7, 8, 9 or 10 rounds) of affinity maturation is performed to identify high affinity anti-HER2 variants, high affinity anti-VEGFA variants and high affinity anti-VEGFC variants. The sequences of antibody variants with high affinity for their respective target antigens are determined.

[0317] Next, the variants identified in the above-mentioned screening are further analyzed to assess their impact on the bispecific antibody yield. In short, high-affinity anti-HER2, anti-VEGFA and anti-VEGFC variants are reformatted as bispecific antibodies. Exemplary bispecific antibodies include, but are not limited to, for example, anti-HER2 / anti-CD3, anti-VEGFA / anti-ANG2 and anti-VEGFC / anti-CD3 (see Tables G1 and G2 above). For example, according to the method detailed in Example 1, express and purify bispecific antibodies. For example, via size exclusion chromatography, high-resolution LCMS and / or SDS-PAGE gel analysis, assess the yield of correctly assembled bispecific antibodies, as detailed in Example 1. Use, for example, the bispecific antibodies shown in Tables G1 and G2 to perform control experiments in parallel. The yield of the bispecific antibodies comprising high-affinity anti-HER2 antibody variants, high-affinity anti-VEGFA variants or anti-VEGFC variants identified via library screening is compared with the yield of the bispecific antibodies comprising anti-HER2, anti-VEGFA or anti-VEGFC antibodies shown in Table 1. Additional modified antibodies that were subjected to one or more affinity maturation steps and further assayed for improved affinity and BsAb productivity (ie, as described above) are shown in Table G3.

[0318] Other references

[0319] Merchant et al. (2013) Proc Natl Acad Sci US A.110(32):E2987-96

[0320] Julian et al. (2017) Scientific Reports. 7:45259

[0321] Tiller et al. (2017) Front. Immunol. 8:986

[0322] Koenig et al. (2017) Proc Natl Acad Sci US A. 114(4):E486-E495

[0323] Yamashita et al. (2019) Structure. 27, 519 527

[0324] Payandeh et al. (2019) J Cell Biochem. 120:940-950

[0325] Richter et al. (2019) mAbs. 11(1): 166-177

[0326] Cisneros et al. (2019) Mol. Syst. Des. Eng. 4:737-746

[0327] The foregoing examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing specification and fall within the scope of the appended claims. Sequence Listing <110> Hoffmann-La Roche GmbH <120> Methods for preparing antibodies <130> 14639-20477.40 <140> Not yet assigned <141> Also attach <150> US 62 / 845,594 <151> 2019-05-09 <160> 8 <170> FastSEQ for Windows, Version 4.0 <210> 1 <211> 113 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr 20 25 30 Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ala Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 2 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Pro Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 3 <211> 119 <212> PRT <213> artificial sequence <220> <223> Synthesize construction <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Met Ile Asp Pro Ser Asn Ser Asp Thr Arg Phe Asn Pro Asn Phe 50 55 60 Lys Asp Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Tyr Arg Ser Tyr Val Thr Pro Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 4 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Gly Asn 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Glu Ile Ser Pro Ser Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ser Arg Val Ser Tyr Glu Ala Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 5 Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ser Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Asp Ser Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 6 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ile Asn Asp 20 25 30 Ala Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser His Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Thr Ser Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 7 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 7 Glu Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr 20 25 30 Ser Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Ala Met Ile Trp Gly Asp Gly Lys Ile Val Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu 65 70 75 80 Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Gly Asp Gly Tyr Tyr Pro Tyr Ala Met Asp Asn Trp Gly Gln Gly Ser 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 8 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Val Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Leu Asp Asn Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Ile Phe Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115

Claims

1. A method for improving the preferential pairing of a first heavy chain and a first light chain of a bispecific antibody comprising a first heavy chain, a first light chain, a second heavy chain and a second light chain, wherein the first heavy chain and the first light chain form a pair that binds to a first antigen and wherein the second heavy chain and the second light chain form a pair that binds to a second antigen, and wherein the cognate pairing between the first heavy chain and the first light chain and between the second heavy chain and the second light chain of the bispecific antibody is preferentially weak or absent, The method comprises the following steps: The light chain variable domain (V L ) is substituted with aspartic acid (D), The V L The uncharged amino acid at position 96 of The heavy chain variable domain (V H ) is substituted with aspartic acid (D), The amino acid numbering is based on Kabat, Wherein V L 94 bits, the V L The 96 bits and the V H a substitution at position 95 of the anti-HER2 arm of the anti-HER2 / anti-CD3 bispecific antibody, an anti-VEGFA arm of the anti-VEGFA / anti-ANG2 bispecific antibody, or an anti-VEGFC arm of the anti-VEGFC / anti-CD3 bispecific antibody, Wherein the bispecific antibody generated by said method comprises CDR L3 according to Kabat as shown in QQNNEDPRT and CDR H3 according to Kabat as shown in DGYYPYAM--DN.

2. The method of claim 1, further comprising subjecting the bispecific antibody to at least one affinity maturation step, wherein the V L The substituted amino acid at position 94 of L The substituted amino acid at position 96 and / or the V H The substituted amino acid at position 95 of is not random.

3. The method of claim 1, wherein the bispecific antibody is a human bispecific antibody, a humanized bispecific antibody, or a chimeric bispecific antibody.

4. The method of claim 1, wherein the bispecific antibody comprises a human IgG Fc region. 5 . The method according to claim 4 , wherein the human IgG Fc region is a human IgG1 Fc region, a human IgG2 Fc region, a human IgG3 Fc region or a human IgG4 Fc region.

6. The method of claim 5, wherein the human IgG Fc region comprises a first C H 2 domain (C H 21) First C H 3 domains (C H 31) Second C H 2 domain (C H 22) and the second C H 3 domains; (a) Where C H 32 is changed so that in C H 31 / C H 32 interface, one or more amino acid residues are replaced by one or more amino acid residues with larger side chain volumes, thereby H The surface of 32 is generated with C H 31 interacting protrusions; and Among them C H 31 is changed so that in C H 31 / C H 32 In the interface, one or more amino acid residues are replaced by amino acid residues with smaller side chain volumes, thereby H 31 on the surface produced with C H 32 interacting cavities, or (b) where C H 31 is changed so that in C H 31 / C H 32 interface, one or more amino acid residues are replaced by one or more amino acid residues with larger side chain volumes, thereby H 31 on the surface produced with C H 32 interacting protrusions; and Among them C H 32 is changed so that in C H 31 / C H 32 In the interface, one or more amino acid residues are replaced by amino acid residues with smaller side chain volumes, thereby H The surface of 32 is generated with C H 31 interacting cavities. The method of claim 6 , wherein the protrusion is a knob mutation.

8. The method of claim 7, wherein the knob mutation comprises T366W, wherein amino acid numbering is according to the EU index.

9. The method of claim 6, wherein the cavity is a hole mutation.

10. The method of claim 9, wherein the hole mutation comprises at least one, at least two or all three of T366S, L368A and Y407V, wherein the amino acid numbering is according to the EU index.

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