Engineered multispecific antibodies and other multimeric proteins having mutations in the asymmetric CH2-CH3 region

Abstract

The present invention relates to engineered multispecific antibodies and other multimeric proteins with asymmetric CH2-CH3 region mutations, and methods of making and using them.

Description

Technical Field

[0001] The present invention relates to engineered multispecific antibodies and other multimeric proteins having mutations in the asymmetric CH2-CH3 region, and methods for their manufacture and use.

[0002] (Sequence Listing) This application includes a sequence listing submitted via EFS-Web, the entire contents of which are incorporated herein by reference. The ASCII text file was created on May 29, 2018, has the file name JBI5124WOPCT_ST25.txt, and is 164 kilobytes in size.

Background Art

[0003] Therapeutic biologics programs are increasingly directed towards bispecific antibodies for dual targeting, attempts to redirect cells, and modulation of immune checkpoints. Indeed, many bispecific therapeutics are currently in clinical trials (Jachimowicz et al. BioDrugs. 2014(4):331-43). The development of bispecific antibodies is limited by the difficulty of both upstream and downstream processes, i.e., being able to generate high-titer and pure products in a reproducible and scalable manner, and separating bispecific molecules from excess parental or intermediate molecules. Methods have been developed for specifically pairing IgG heavy chains or half molecules, such as knob-in-hole, controlled Fab arm exchange, CrossMAb, and common light chain and orthogonal Fab interfaces. The production of Fv-based molecules (i.e., BiTE, diabody) and non-IgG scaffolds (i.e., DARPin, adnectin, finomer, and centyrin) is of increasing interest in developing these molecules as therapeutics.

[0004] As a disadvantage of Fv alone or alternative scaffold-based molecules, their serum half-life may generally be short due to urinary excretion or degradation in lysosomes because they are not recycled by FcRn. Therefore, IgG-based multispecific molecules with a complete Fc domain are attractive due to their longer serum half-life, ability to promote effector functions, and induction of the apoptotic pathway.

[0005] Purification of bispecific antibodies can be difficult because multiple steps are required to remove residual parental antibodies and other intermediate mAb and Ab fragment molecules. Since such molecules can have biophysical properties similar to those of the induced bispecific antibodies, they cannot be easily separated by chromatography methods. This difficulty in purification can lead to a decrease in the yield or purity of the bispecific molecule.

Summary of the Invention

Problems to be Solved by the Invention

[0006] Therefore, alternative bispecific and multispecific formats, as well as methods for purifying bispecific and multispecific molecules such as antibodies, are still in demand.

Means for Solving the Problems

[0007] The present invention provides an isolated multispecific antibody comprising a first CH2-CH3 region having mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R, or T307P / V309Q / Q311R, and a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309, and 311, wherein the residue numbering follows the EU index.

[0008] The present invention also provides an isolated multispecific antibody comprising a first CH2-CH3 region having the mutation Q311R and a second CH2-CH3 region having a wild-type amino acid residue at position 311, wherein the residue numbering follows the EU index.

[0009] The present invention also provides an isolated multispecific antibody comprising a first CH2-CH3 region having the mutation Q311K and a second CH2-CH3 region having a wild-type amino acid residue at position 311, wherein the residue numbering follows the EU index.

[0010] The present invention also provides an isolated multispecific antibody comprising a first CH2-CH3 region having the mutation T307P / L309Q and a second CH2-CH3 region having wild-type amino acid residues at positions 307 and 309, wherein the residue numbering follows the EU index.

[0011] The present invention also provides an isolated multispecific antibody comprising a first CH2-CH3 region having the mutation T307P / V309Q and a second CH2-CH3 region having wild-type amino acid residues at positions 307 and 309, wherein the residue numbering follows the EU index.

[0012] The present invention also provides an isolated multispecific antibody comprising a first CH2-CH3 region having the mutation T307P / L309Q / Q311R and a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, wherein the residue numbering follows the EU index.

[0013] The present invention also provides an isolated multispecific antibody comprising a first CH2-CH3 region having the mutation T307P / V309Q / Q311R and a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, wherein the residue numbering follows the EU index.

[0014] The present invention also provides an isolated polynucleotide that comprises a polynucleotide encoding a first CH2-CH3 region having mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R, comprises a polynucleotide encoding a first CH2-CH3 region having mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R and a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, or also provides an isolated polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 87, 88 or 91.

[0015] The present invention also provides a vector that comprises an isolated polynucleotide encoding a first CH2-CH3 region having mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R, comprises an isolated polynucleotide having a polynucleotide sequence of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 87, 88 or 91, comprises an isolated polynucleotide encoding a first CH2-CH3 region having mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R and a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, or SEQ ID NO: 27 and 47, respectively, SEQ ID NO: 28 and 47, respectively, Array numbers 29 and 47, respectively, Array numbers 30 and 47, respectively, Array numbers 31 and 48, respectively, Array numbers 32 and 48, respectively, Array numbers 33 and 48, respectively, Array numbers 34 and 48, respectively, Array numbers 35 and 49, respectively, Array numbers 36 and 49, respectively, Array numbers 37 and 49, respectively, Array numbers 38 and 49, respectively, Array numbers 39 and 50, respectively, Array numbers 40 and 50, respectively, Array numbers 41 and 50, respectively, Array numbers 42 and 50, respectively, Array numbers 43 and 51, respectively, Array numbers 44 and 51, respectively, Array numbers 45 and 51, respectively, Array numbers 46 and 51, respectively, Array numbers 87 and 89, respectively, Array numbers 87 and 90, respectively, Array numbers 88 and 89, respectively, Array numbers 88 and 90, respectively, Array numbers 92 and 89, respectively, or Array numbers 92 and 90, respectively, also provides a vector comprising an isolated polynucleotide having.

[0016] The present invention also provides a host cell comprising the vector of the present invention.

[0017] The present invention also provides a method for producing the isolated multispecific antibody of the present invention, culturing the host cell of the present invention under conditions under which the multispecific antibody is expressed; purifying the multispecific antibody using protein A ligand affinity chromatography, also provides a method comprising.

[0018] The present invention also provides a method for producing an isolated multispecific antibody comprising a first heavy chain having the mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R and a second heavy chain having wild-type amino acid residues at positions 307, 309 and 311, providing a first parental antibody comprising a first heavy chain having the mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R and a first light chain, providing a second parental antibody comprising a second heavy chain having wild-type amino acid residues at positions 307, 309 and 311 and a second light chain, contacting the first parental antibody and the second parental antibody in a sample, incubating the sample, and purifying the multispecific antibody using protein A ligand affinity chromatography.

[0019] The present invention also provides an isolated antibody comprising two heavy chains or fragments thereof having the same amino acid sequence and two light chains or fragments thereof, wherein the two heavy chains have the mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R and the residue numbering follows the EU index.

[0020] The present invention also provides a polynucleotide encoding an antibody heavy chain comprising the CH2-CH3 region of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 52, 53 or 56, or a polynucleotide having the polynucleotide sequence of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 87, 88 or 91.

[0021] The present invention also provides a multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region having mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R, the second polypeptide comprises a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, and the residue numbering follows the EU index.

[0022] The present invention also provides a pharmaceutical composition comprising the multimeric protein of the present invention.

[0023] The present invention also provides a method for producing an isolated multimeric protein comprising a first CH2-CH3 region having mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R and a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, comprising: providing a first parent protein comprising a first CH2-CH3 region having mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R; providing a second parent protein comprising a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311; contacting the first parent protein and the second parent protein in a sample; incubating the sample; and purifying the multispecific protein using protein A ligand affinity chromatography. BRIEF DESCRIPTION OF THE DRAWINGS

[0024]

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DETAILED DESCRIPTION OF THE INVENTION

[0025] All publications, including but not limited to patents and patent applications cited herein, are hereby incorporated by reference in their entirety as if fully set forth herein.

[0026] It should be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0027] Any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, but exemplary materials and methods are described herein. The following terms are used in the description and claims of the present invention.

[0028] Definitions As used in this specification and the appended "claims", the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes combinations of two or more cells.

[0029] "Multimeric protein" refers to a protein composed of two or more separate polypeptide chains that associate to form a single protein. The polypeptide chains can be associated by non-covalent bonds or by covalent bonds, for example, via disulfide bonds.

[0030] "Binding" refers to the specific binding of two proteins, such as the binding of an antibody to its antigen or the binding of a multispecific protein to its ligand. "Specific binding" generally refers to the selective binding of two proteins with an equilibrium dissociation constant (K -8 M or less, for example, about 1×10 -9 M or less, about 1×10 -10 M or less, about 1×10 -11 M or less, or about 1×10 -12 M or less, typically at least 100-fold lower K D than that in the binding to non-specific antigens (e.g., BSA, casein), referring to the selective binding at K D . D

[0031] "Decrease in binding" refers to a measurable decrease in the binding of the antibody or multispecific protein of the present invention having at least one mutation in the CH2-CH3 region to the Protein A ligand when compared to the binding of the parent molecule without the mutation.

[0032] "Modulating binding" refers to a measurable difference in the binding of the antibody or multispecific protein of the present invention having at least one mutation in the CH2-CH3 region to FcγR or FcRn.

[0033] "Antigen" refers to a molecule such as a protein or a fragment of a protein that can initiate an immune response in a subject.

[0034] "Asymmetric stabilizing mutation" refers to mutations in the first and second CH2-CH3 regions at different positions, which are advantageous (e.g., stabilizing) for the formation of heterodimers between the first and second CH2-CH3 regions rather than the formation of homodimers within the first CH2-CH3 region or between the first and second CH2-CH3 regions.

[0035] ​"Heterologous protein" refers to a polypeptide or protein that is not a natural part or portion of a polypeptide containing the CH2-CH3 region of an endogenous cell.

[0036] "Fibronectin type III (FN3) domain" (FN3 domain) refers to a domain often present in proteins including fibronectin, tenascin, intracellular cytoskeletal proteins, cytokine receptors, and prokaryotic enzymes (Bork and Doolittle, Proc Nat Acad Sci USA 89:8990-8994, 1992; Meinke et al., J Bacteriol 175:1910-1918, 1993; Watanabe et al., J Biol Chem 265:15659-15665, 1990). Representative FN3 domains include 15 different FN3 domains present in human tenascin C, 15 different FN3 domains present in human fibronectin (FN), and non-natural synthetic FN3 domains described, for example, in U.S. Patent No. 8,278,419. Individual FN3 domains are referred to by domain number and protein name (e.g., the third FN3 domain of tenascin (TN3), or the tenth FN3 domain of fibronectin (FN10)). FN3 domains can be engineered to bind antigens with high specificity and affinity.

[0037] "Fynomer" refers to an antigen-binding protein derived from the SH3 domain of human Fyn that can be engineered to bind antigens with high specificity and affinity.

[0038] "Antibody" has a broad meaning and includes monoclonal antibodies, antigen-binding fragments, monospecific, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single-chain antibodies, domain antibodies, and any other modified forms of immunoglobulin molecules including an antigen-binding site with the required specificity, including immunoglobulin molecules. A "full-length antibody" is composed of two heavy chains (HCs) and two light chains (LCs) linked to each other by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain is composed of a heavy-chain variable region (VH) and a heavy-chain constant region (composed of the domains CH1, hinge CH2, and CH3). Each light chain is composed of a light-chain variable region (VL) and a light-chain constant region (CL). VH and VL can be further divided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with framework regions (FRs). Each VH and VL is composed of three CDRs and four FR fragments arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus.

[0039] "Complementary determining region (CDR)" is the region in an antibody that binds to an antigen. There are three CDRs in VH (HCDR1, HCDR2, HCDR3) and three CDRs in VL (LCDR1, LCDR2, LCDR3). CDRs can be defined using various representations such as Kabat (Wu and Kabat, J Exp Med 132:211-250, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia And Lesk, J Mol Biol 196:901-917, 1987), and IMGT (Lefranc Et al., Dev Comp Immunol 27:55-77, 2003). The correspondence between different representations and the numbering of variable regions is described (see, for example, Lefranc et al., Dev Comp Immunol 27:55-77, 2003; Honegger and Pluckthun, J Mol Biol 309:657-70, 2001; International ImMunoGeneTics (IMGT) database; web resource, http: / / www_imgt_org). Available programs such as abYsis by UCL Business PLC can be used to represent CDRs. As used herein, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2", and "LCDR3" include CDRs defined by any of the methods described by Kabat, Chothia, or IMGT as described above, unless otherwise stated herein.

[0040] Immunoglobulins can be assigned to five major classes, IgA, IgD, IgE, IgG, and IgM, according to the amino acid sequence of the heavy chain constant region. IgA and IgG are further subclassified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Antibody light chains of all vertebrate species can be assigned to one of two distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequence of their constant region.

[0041] "Antigen-binding fragment" refers to a portion of an immunoglobulin molecule that retains the antigen-binding properties of the original full-length antibody. Representative antigen-binding fragments include heavy chain complementarity-determining regions (HCDR) 1, 2, and / or 3, light chain complementarity-determining regions (LCDR) 1, 2, and / or 3, VH, VL, VH and VL, Fab, F(ab’)2, Fd, and Fv fragments, and domain antibodies (dAbs) consisting of either one VH domain or one VL domain. The VH and VL domains can be linked to each other via a synthetic linker such that the VH / VL domains pair either intramolecularly or intermolecularly when the VH and VL domains are expressed on separate chains, forming the design of various types of single-chain antibodies such as single-chain Fv (scFv) or diabodies that form monovalent antigen-binding sites. For example, it is described in WO 98 / 44001, WO 88 / 01649, WO 94 / 13804, and WO 92 / 01047.

[0042] "CH2-CH3 region" refers to a part of the human antibody constant domain and includes amino acid residues 231 to 446 (residue numbering according to the EU index). The CH2-CH3 region may lack the C-terminal lysine at position 447.

[0043] "Monoclonal antibody" refers to a population of antibodies having a single amino acid composition in each heavy chain and each light chain, except for possible well-known modifications such as removing the C-terminal lysine from the antibody heavy chain. Monoclonal antibodies typically bind specifically to one antigenic epitope, although bispecific or multispecific monoclonal antibodies bind specifically to two or more different antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies can be monospecific or multispecific, or monovalent, bivalent, or multivalent. Bispecific antibodies are included within the term "monoclonal antibody".

[0044] "Isolated" refers to a homogeneous population of molecules (e.g., a protein such as a synthetic polynucleotide or an antibody) that is substantially separated and / or purified from other components related to the system in which the molecule, such as a recombinant cell, is produced, and also refers to a protein that has been subjected to at least one purification or isolation step. "Isolated antibody" refers to an antibody that is substantially free of other cellular materials and / or chemical substances, and includes antibodies isolated to a higher purity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity.

[0045] "Humanized antibody" refers to an antibody in which the CDR sequences are derived from non-human species and the framework is derived from human immunoglobulin sequences. Since the framework may contain substitutions within the framework, the framework need not be an exact copy of the sequence of the expressed human immunoglobulin or human immunoglobulin germline gene.

[0046] "Human antibody" refers to an antibody that is optimized such that the immune response is minimal when administered to a human subject. The variable region of a human antibody is derived from human germline immunoglobulin sequences. If the antibody includes a constant region or a portion of the constant region, the constant region is also derived from human germline immunoglobulin sequences.

[0047] A human antibody comprises a heavy chain variable region or a light chain variable region that is "derived from" a human germline immunoglobulin sequence when the variable region of the antibody is obtained from a system that uses human germline immunoglobulin genes. Such representative systems include human immunoglobulin gene libraries displayed on phage or mammalian cells, and non-human transgenic animals such as mice or rats having a human immunoglobulin locus. A "human antibody" typically has amino acid differences when compared to immunoglobulin sequences expressed in humans, for example, due to the introduction of somatic mutations, the introduction of intentional substitutions in the framework or CDRs, and amino acid changes introduced during cloning and VJD recombination in non-human animals. A "human antibody" usually has an amino acid sequence that is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence encoded by a human germline immunoglobulin sequence. In some cases, a "human antibody" may contain a consensus framework sequence obtained from human framework sequence analysis as described, for example, in Knappik et al., J Mol Biol 296:57-86,2000, or a synthetic HCDR3 incorporated into a human immunoglobulin gene library displayed on phage as described, for example, in Shi et al., J Mol Biol 397:385-396,2010 and International Publication No. WO 2009 / 085462. Antibodies in which the CDRs are derived from non-human species are not included in the definition of "human antibody".

[0048] "Recombinant" refers to antibodies and other proteins prepared, expressed, produced, or isolated by recombinant means.

[0049] "Multispecificity" refers to proteins such as antibodies that specifically bind to two or more different antigens or two or more different epitopes within the same antigen. Multispecific proteins can have cross-reactivity with the same antigen from other related species (homologs), such as other humans or primates, such as Macaca fascicularis (cynomolgus monkey, cyno), Pan troglodytes (chimpanzee, chimp), or Callithrix jacchus (common marmoset, marmoset), or can bind to epitopes shared between two or more different antigens.

[0050] "Bispecificity" refers to proteins such as antibodies that specifically bind to two different antigens or two different epitopes within the same antigen. Bispecific proteins can have cross-reactivity with the same antigen from other related species (homologs), such as other humans or primates, such as Macaca fascicularis (cynomolgus monkey, cyno), Pan troglodytes (chimpanzee, chimp), or Callithrix jacchus (common marmoset, marmoset), or can bind to epitopes shared between two or more different antigens.

[0051] "Monospecificity" refers to proteins such as antibodies that specifically bind to one distinct antigen or distinct epitope. Monospecific proteins can have cross-reactivity with the same antigen from other related species (homologs), such as other humans or primates, such as Macaca fascicularis (cynomolgus monkey, cyno), Pan troglodytes (chimpanzee, chimp), or Callithrix jacchus (common marmoset, marmoset), or can bind to epitopes shared between two or more different antigens.

[0052] "Vector" refers to a polynucleotide that can be replicated within a biological system or can move between such systems. Vector polynucleotides usually contain elements such as an origin of replication, a polyadenylation signal, or a selectable marker that have the function of promoting the replication or maintenance of these polynucleotides in biological systems (such as cells, viruses, animals, plants, etc.) and reconstituted biological systems.

[0053] "Protein A ligand affinity chromatography" refers to an affinity chromatography method that utilizes the affinity of the IgG binding domain of a protein A ligand for the Fc region of an immunoglobulin molecule. This Fc region includes the human or animal immunoglobulin constant domains CH2 and CH3, or immunoglobulin domains substantially similar thereto. Protein A ligands include native protein A derived from the cell wall of Staphylococcus aureus, protein A produced by recombinant or synthetic methods, and variants that retain the ability to bind to the Fc region. In practice, protein A ligand chromatography involves the use of a protein A ligand immobilized on a solid support. See Gagnon, Protein A Affinity Chromatography, Purification Tools for Monoclonal Antibodies, pp. 155-198, Valiated Biosystems, 1996. The solid support is a non-aqueous matrix to which the protein A ligand adheres. Such well-known supports include agarose, sepharose, glass, silica, polystyrene, nitrocellulose, carbon, sand, cellulose, and any other suitable material. Any suitable well-known method can be used to attach a second protein to the solid support. Such solid supports with and without an immobilized protein A ligand are readily available from many commercial suppliers such as Vector Laboratory (Burlingame, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), BioRad (Hercules, Calif.), Amersham Biosciences (a division of GE Healthcare, Uppsala, Sweden), Pall (Port Washington, N.Y.), and EMD Millipore (Billerica, Mass.). A protein A ligand immobilized on a porous glass matrix is commercially available as PROSEP® A (Millipore). The solid phase may also be an agarose-based matrix.The Protein A ligand immobilized on the agarose matrix is commercially available as MABSELECT™ (Amersham Biosciences).

[0054] An "expression vector" refers to a vector that can be used in a biological or reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0055] A "polynucleotide" refers to a synthetic molecule containing a nucleotide chain covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a synthetic polynucleotide.

[0056] A "polypeptide" or "protein" refers to a molecule containing at least two amino acid residues linked by peptide bonds to form a polypeptide. A small polypeptide consisting of less than 50 amino acids may be referred to as a "peptide".

[0057] A "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications, such as one or more substitutions, insertions, or deletions.

[0058] "Valency" refers to the number of antigen-specific binding sites present in a molecule. Thus, the terms "monovalent", "divalent", "tetravalent", and "hexavalent" refer to the presence of 1, 2, 4, and 6 antigen-specific binding sites, respectively, in a molecule.

[0059] "Protein A ligand" refers to naturally occurring or modified Staphylococcus aureus protein A, and includes engineered protein A domains. Engineered protein A may be, for example, a Z domain, a Z domain, a mutant of the Y domain, or an engineered protein A lacking the D and E domains. Engineered protein A domains cannot bind (or bind with extremely low affinity if they do bind) to the VH3 domain of immunoglobulins, but can still bind to the CH2-CH3 regions of IgG1, IgG2, and IgG4.

[0060] "Z domain" is a synthetic engineered mutant of the B domain of Staphylococcus aureus protein A having mutations A1V and G29A when compared to the wild-type B domain of protein A. The Z domain has the amino acid sequence of SEQ ID NO: 1. Further Z domain mutants include the amino acid sequences of SEQ ID NOs: 99, 100, and 101, and the mutants described in US Patent Application Publication No. 2006 / 0194950.

[0061] SEQ ID NO: 1 VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK

[0062] SEQ ID NO: 99 FNMQCQRRFYEALHDPNLNEEQRNAKIKSIRDDC

[0063] SEQ ID NO: 100 VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK

[0064] SEQ ID NO: 101 FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD

[0065] Throughout this specification, unless otherwise indicated, the numbering of amino acid residues in antibody constant regions follows the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Correlations between the numbering systems of different constant domains are available in the International ImMunoGeneTics (IMGT) database; web resource, http: / / www_imgt_org).

[0066] In this specification, the conventional one-letter and three-letter amino acid codes as shown in Table 1 are used.

[0067] [Table 1]

[0068] Composition of the substance: Bispecific antibody The present invention provides bispecific antibodies that facilitate purification using protein A ligand chromatography and other multimeric CH2-CH3 region-containing proteins having asymmetric mutations in the CH2-CH3 region, polynucleotides encoding them, vectors, host cells, and methods for producing and using them.

[0069] To produce and purify full-length bispecific therapeutic antibodies, it is required to efficiently separate the bispecific antibody from excess parental and / or intermediate molecules. In this specification, Fc mutations that reduce the binding of the mutated heavy chain to the protein A ligand are identified. Thus, bispecific antibodies having these Fc mutations in an asymmetric form (e.g., in only one of the heavy chains) can be purified from the parental antibody based on the differential elution profile from a protein A ligand affinity column.

[0070] A variety of methods have been developed for specifically pairing IgG heavy chains or half - molecules, including knob - in - hole (see, e.g., U.S. Patent No. 7,695,936), CrossMAb (Schaefer et al., Proc Natl Acad Sci U S A 108:11187 - 11192, 2011), controlled Fab - arm exchange (Labrijn et al., Proc Natl Acad Sci U S A 110:5145 - 5150, 2013), common light chain (see, e.g., U.S. Patent No. 7,951,917), and orthogonal Fab interfaces (see Lewis et al., Nat Biotechnol 32:191 - 198, 2014). The compositions and methods described herein provide further improved methods for making and purifying bispecific antibodies.

[0071] FcRn is involved in the transfer of maternal IgG to the fetus and the protection of serum IgG from lysosomal degradation. Both of these processes involve IgG binding to FcRn at acidic pH (<6.5) in recycling endosomes with a K of approximately 600 nM DIt depends on the property of FcRn that binds at neutral pH and dissociates at neutral pH to release IgG back into the serum (Roopenian and Akilesh, Nat Rev Immunol 7:715-725, 2007). Since IgG binds to FcRn at the CH2-CH3 interface, one Fc has two identical FcRn binding sites. Structural and biochemical studies have shown that one Fc binds to two FcRn heterodimers, but intracellular trafficking may involve multimerization of FcRn itself on the membrane surface. In several studies, it has been shown that modulating the interaction between Fc and FcRn strongly affects the serum half-life (Dall’Acqua et al., J Immunol 169:5171-5180, 2002; Hinton et al., J Biol Chem. 279(8):6213-6216, 2004 Hinton et al., J Immunol, 176:346-356, 2006, Vaccaro et al., Nat Biotechnol. 23:1283-1288, 2005, Yeung et al., J Immunol, 182:7663-7671, 2009, Stapleton et al., Nat Commun 2:599, 2011), leading to the conclusion that FcRn plays a major role in determining the serum half-life of IgG in adults.

[0072] Attempts to modulate the protein A ligand binding properties of Abs often result in a significant decrease in serum half-life because both protein A and the neonatal Fc receptor (FcRn) share binding sites on Fc. The mutations introduced herein do not reduce the binding of Fc to FcRn and thus do not reduce the serum half-life of the engineered antibody. One of the mutations introduced, Q311R, slightly enhanced the binding to FcRn, leading to an increase in the serum half-life of the antibody.

[0073] The examples describe the efficient genetic manipulation and purification of multispecific full-length antibodies from parental antibodies, but the techniques described herein are applicable to any multimeric protein containing two CH2-CH3 regions.

[0074] The present invention provides an isolated multispecific antibody comprising a first CH2-CH3 region having mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R, or T307P / V309Q / Q311R, and a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309, and 311, with residue numbering according to the EU index.

[0075] Isolated multispecific antibodies having asymmetric Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R, or T307P / V309Q / Q311R mutations can be efficiently purified from parental antibodies using protein A ligand affinity chromatography. The introduced Q311K, T307P / L309Q, and T307P / L309Q / Q311R mutations are not expected to alter the antibody's half-life or effector function because they do not reduce the binding of the engineered antibody to FcRn or FcγR. The introduced Q311R mutation increased the antibody's binding to FcRn and serum half-life.

[0076] The present invention also provides an isolated multispecific antibody comprising a first CH2-CH3 region having mutation Q311R and a second CH2-CH3 region having a wild-type amino acid residue at position 311, with residue numbering according to the EU index.

[0077] The present invention also provides an isolated multispecific antibody comprising a first CH2-CH3 region having mutation Q311K and a second CH2-CH3 region having a wild-type amino acid residue at position 311, with residue numbering according to the EU index.

[0078] The present invention also provides an isolated multispecific antibody comprising a first CH2-CH3 region having the mutations T307P / L309Q and a second CH2-CH3 region having wild-type amino acid residues at positions 307 and 309, wherein the residue numbering follows the EU index.

[0079] The present invention also provides an isolated multispecific antibody comprising a first CH2-CH3 region having the mutations T307P / V309Q and a second CH2-CH3 region having wild-type amino acid residues at positions 307 and 309, wherein the residue numbering follows the EU index.

[0080] The present invention also provides an isolated multispecific antibody comprising a first CH2-CH3 region having the mutations T307P / L309Q / Q311R and a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, wherein the residue numbering follows the EU index.

[0081] The present invention also provides an isolated multispecific antibody comprising a first CH2-CH3 region having the mutations T307P / V309Q / Q311R and a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, wherein the residue numbering follows the EU index.

[0082] In some embodiments, the first CH2-CH3 region has a reduced binding to Protein A ligand as compared to the second CH2-CH3 region.

[0083] Binding to the Protein A ligand can be determined experimentally using any suitable method. Such methods can use ProteOn XPR36, Biacore 3000 or KinExA instruments. The measured affinity may vary when measured under different conditions (e.g., osmolality, pH). Thus, the affinity and other binding parameters (e.g., K D 、kon , k off The measurement of off is generally performed using standardized conditions and a standardized buffer such as the buffer described in this specification. Alternatively, the binding to the Protein A ligand can also be directly evaluated using Protein A ligand chromatography with a pH gradient. Molecules with reduced binding to the Protein A ligand elute at a higher pH. In an exemplary Protein A ligand chromatography, an mAbSelect Sure column (GE Healthcare) can be used, and the sample can be eluted in three steps using a buffer containing 50 mM citrate at a pH of about 4.7, 4.2, or 3.4.

[0084] In some embodiments, the Protein A ligand comprises Protein A of Staphylococcus aureus.

[0085] In some embodiments, the Protein A ligand comprises a Z domain.

[0086] In some embodiments, the Z domain has the amino acid sequence of SEQ ID NO: 1.

[0087] In some embodiments, the Protein A ligand comprises a Y domain.

[0088] In some embodiments, the Protein A ligand has the amino acid sequence of SEQ ID NO: 99.

[0089] In some embodiments, the Protein A ligand has the amino acid sequence of SEQ ID NO: 100.

[0090] In some embodiments, the Protein A ligand has the amino acid sequence of SEQ ID NO: 101.

[0091] Staphylococcus aureus protein A (spA) has five homologous helical IgG-binding domains designated E, D, A, B, and C (Uhlen, Guss et al. 1984). Each of these domains is sufficient to bind to the Fc region, but spA also binds to the VH region of human VH3 family members (Romagnani et al., J Immunol 129:596-602, 1982; Sasso et al., J Immunol, 147:1877-1883, 1991). Introduction of mutations that enhance the stability of the B or C domain of SpA resulted in synthetic Z and Y domains that are resistant to high pH treatment and bind only to Fc, respectively. Tandem or tetrameric Z domains, tetrameric Y domains, or native spA are incorporated into commercially available affinity resins such as MabSelect SuRe (GE), TOYOPEARL AF-rProtein A HC-650F, and MabSelect Xtra.

[0092] In some embodiments, the multispecific antibody is of the IgG1 isotype.

[0093] In some embodiments, the multispecific antibody is of the IgG2 isotype.

[0094] In some embodiments, the multispecific antibody is of the IgG4 isotype.

[0095] The examples provide experimental data regarding the efficient production and purification of IgG1 multispecific antibodies, but residues 307 and 311 are conserved in all three isotypes, position 309 is conserved between IgG1 and IgG4, and Leu is conservatively substituted with Val in IgG2, so the identified mutations are expected to function in the IgG2 and IgG4 isotypes as well.

[0096] In some embodiments, the binding of the multispecific antibody to FcγR is equivalent to that of the parental antibody without mutations.

[0097] In some embodiments, the FcγR is FcγRI, FcγRIIa, FcγRIIb, and / or FcγRIIIa.

[0098] In some embodiments, the FcγR is FcγRI.

[0099] In some embodiments, the FcγR is FcγRIIa.

[0100] In some embodiments, the FcγR is FcγRIIb.

[0101] In some embodiments, the FcγR is FcγRIIIa.

[0102] Exemplary bispecific antibodies having equivalent binding to FcγR include bispecific antibodies having the Q311R or T307P / L309Q / Q311R mutations.

[0103] In some embodiments, the binding of the bispecific antibody to FcRn is equivalent to the binding of the parental antibody without the mutation.

[0104] Exemplary bispecific antibodies having equivalent binding to FcRn include bispecific antibodies having the Q311K or T307P / L309Q / Q311R mutations.

[0105] In some embodiments, the binding of the bispecific antibody to FcRn is improved compared to the binding of the parental antibody without the FcRn mutation.

[0106] Exemplary bispecific antibodies having improved binding to FcRn include antibodies having the Q311R mutation.

[0107] The present invention also provides an isolated multispecific antibody comprising a first CH2-CH3 region having mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R, or T307P / V309Q / Q311R, and a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, with residue numbering according to the EU index, wherein the multispecific antibody further has asymmetric stabilizing mutations within the first CH2-CH3 region and the second CH2-CH3 region.

[0108] In some embodiments, the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are F405L and K409R, respectively, wild-type and F405L / R409K, respectively, T366W and T366S / L368A / Y407V, respectively, T366Y / F405A and T394W / Y407T, respectively, T366W / F405W and T394S / Y407A, respectively, F405W / Y407A and T366W / T394S, respectively, L351Y / F405A / Y407V and T394W, respectively, T366I / K392M / T394W and F405A / Y407V, respectively, T366L / K392M / T394W and F405A / Y407V, respectively, L351Y / Y407A and T366A / K409F, respectively, L351Y / Y407A and T366V / K409F, respectively, Y407A and T366A / K409F, respectively, D399K / E356K and K409D / K392D, respectively, or D399K / E356K / E357K and K409D / K392D / K370, respectively.

[0109] In some embodiments, the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are F405L and K409R, respectively.

[0110] In some embodiments, the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are wild type and F405L / R409K, respectively.

[0111] In some embodiments, the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are T366W and T366S / L368A / Y407V, respectively.

[0112] In some embodiments, the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are T366Y / F405A and T394W / Y407T, respectively.

[0113] In some embodiments, the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are T366W / F405W and T394S / Y407A, respectively.

[0114] In some embodiments, the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are F405W / Y407A and T366W / T394S, respectively.

[0115] In some embodiments, the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are L351Y / F405A / Y407V and T394W, respectively.

[0116] In some embodiments, the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are T366I / K392M / T394W and F405A / Y407V, respectively.

[0117] In some embodiments, the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are T366L / K392M / T394W and F405A / Y407V, respectively.

[0118] In some embodiments, the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are L351Y / Y407A and T366A / K409F, respectively.

[0119] In some embodiments, the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are L351Y / Y407A and T366V / K409F, respectively.

[0120] In some embodiments, the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are Y407A and T366A / K409F, respectively.

[0121] In some embodiments, the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are D399K / E356K and K409D / K392D, respectively.

[0122] In some embodiments, the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are D399K / E356K / E357K and K409D / K392D / K370, respectively.

[0123] Introducing asymmetric stabilizing mutations into bispecific or multispecific antibodies can facilitate downstream processes for separating them from excess parental or intermediate molecules.

[0124] Exemplary asymmetric stabilizing mutations are those that facilitate Fab-arm exchange between two parental antibodies (e.g., half-molecule exchange, exchange on the heavy-chain / light-chain pair). In this technique, mutations that favor heterodimer formation of two parental antibody half-molecules are introduced into the heavy-chain CH3 interface of each parental antibody in vitro in a cell-free environment or using co-expression. For example, Fab-arm exchange of IgG1 can be facilitated using the mutation F405L of the first parental antibody and K409R of the second parental antibody. For IgG4 antibodies, the F405L / R409K mutations of the wild-type first and second parental antibodies can be used.

[0125] Additional asymmetric stabilizing mutations are the knob-in-hole mutations (Genentech) or mutations that introduce electrostatically complementary residues (Chugai, Amgen, NovoNordisk, Oncomed). Exemplary knob-in-hole mutations (represented as the mutation position in the first parental antibody / the mutation position in the second parental antibody) are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V. Exemplary mutations that introduce electrostatically complementary residues include those described in U.S. Patent Application Publication Nos. 2010 / 0015133, 2009 / 0182127, 2010 / 028637, or 2011 / 0123532. Additional asymmetric stabilizing mutations include L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W described in U.S. Patent Application Publication Nos. 2012 / 0149876 or 2013 / 0195849.

[0126] Mutations are typically introduced at the DNA level into molecules such as the constant domain of an antibody using standard methods.

[0127] In some embodiments, the multispecific antibody comprises a Q311R / F405L mutation in the first CH2-CH3 region and a K409R mutation in the second CH2-CH3 region.

[0128] In some embodiments, the multispecific antibody comprises a Q311K / F405L mutation in the first CH2-CH3 region and a K409R mutation in the second CH2-CH3 region.

[0129] In some embodiments, the multispecific antibody comprises a T307P / L309Q / F405L mutation in the first CH2-CH3 region and a K409R mutation in the second CH2-CH3 region.

[0130] In some embodiments, the multispecific antibody comprises a T307P / L309Q / Q311R / F405L mutation in the first CH2-CH3 region and a K409R mutation in the second CH2-CH3 region.

[0131] In some embodiments, the multispecific antibody comprises a Q311R / K409R mutation in the first CH2-CH3 region and an F405L mutation in the second CH2-CH3 region.

[0132] In some embodiments, the multispecific antibody comprises a Q311K / K409R mutation in the first CH2-CH3 region and an F405L mutation in the second CH2-CH3 region.

[0133] In some embodiments, the multispecific antibody comprises a T307P / L309Q / K409R mutation in the first CH2-CH3 region and an F405L mutation in the second CH2-CH3 region.

[0134] In some embodiments, the multispecific antibody comprises a T307P / L309Q / Q311R / K409R mutation in the first CH2-CH3 region and an F405L mutation in the second CH2-CH3 region.

[0135] In some embodiments, the multispecific antibody comprises a Q311R mutation in the first CH2-CH3 region and an F405L / R409K mutation in the second CH2-CH3 region.

[0136] In some embodiments, the multispecific antibody comprises a Q311K mutation in the first CH2-CH3 region and an F405L / R409K mutation in the second CH2-CH3 region.

[0137] In some embodiments, the multispecific antibody comprises a T307P / V309Q mutation in the first CH2-CH3 region and an F405L / R409K mutation in the second CH2-CH3 region.

[0138] In some embodiments, the multispecific antibody comprises a T307P / V309Q / Q311R mutation in the first CH2-CH3 region and an F405L / R409K mutation in the second CH2-CH3 region.

[0139] In some embodiments, the multispecific antibody comprises a Q311R / T366W mutation in the first CH2-CH3 region and a T366S / L368A / Y407V mutation in the second CH2-CH3 region.

[0140] In some embodiments, the multispecific antibody comprises a Q311K / T366W mutation in the first CH2-CH3 region and a T366S / L368A / Y407V mutation in the second CH2-CH3 region.

[0141] In some embodiments, the multispecific antibody comprises a T307P / L309Q / T366W mutation in the first CH2-CH3 region and a T366S / L368A / Y407V mutation in the second CH2-CH3 region.

[0142] In some embodiments, the multispecific antibody comprises a T307P / L309Q / Q311R / T366W mutation in the first CH2-CH3 region and a T366S / L368A / Y407V mutation in the second CH2-CH3 region.

[0143] In some embodiments, the multispecific antibody comprises a Q311R / T366S / L368A / Y407V mutation in the first CH2-CH3 region and a T366W mutation in the second CH2-CH3 region.

[0144] In some embodiments, the multispecific antibody comprises a Q311K / T366S / L368A / Y407V mutation in the first CH2-CH3 region and a T366W mutation in the second CH2-CH3 region.

[0145] In some embodiments, the multispecific antibody comprises the T307P / L309Q / T366S / L368A / Y407V mutations in the first CH2-CH3 region and the T366W mutation in the second CH2-CH3 region.

[0146] In some embodiments, the multispecific antibody comprises the T307P / L309Q / Q311R / T366S / L368A / Y407V mutations in the first CH2-CH3 region and the T366W mutation in the second CH2-CH3 region.

[0147] Exemplary amino acid sequences of the CH2-CH3 regions of the multispecific antibodies of the present invention are shown in Tables 2 and 3.

[0148] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 22, respectively.

[0149] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 22, respectively.

[0150] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 22, respectively.

[0151] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region each comprise the amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 22.

[0152] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 6 and SEQ ID NO: 23, respectively.

[0153] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 23, respectively.

[0154] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 8 and 23, respectively.

[0155] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 9 and 23, respectively.

[0156] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 10 and 24, respectively.

[0157] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 11 and 24, respectively.

[0158] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 12 and 24, respectively.

[0159] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 13 and 24, respectively.

[0160] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 14 and 25, respectively.

[0161] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 15 and 25, respectively.

[0162] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 16 and 25, respectively.

[0163] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 25, respectively.

[0164] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 18 and SEQ ID NO: 26, respectively.

[0165] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 26, respectively.

[0166] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 20 and SEQ ID NO: 26, respectively.

[0167] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 26, respectively.

[0168] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 52 and SEQ ID NO: 54, respectively.

[0169] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 52 and SEQ ID NO: 55, respectively.

[0170] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 53 and SEQ ID NO: 54, respectively.

[0171] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 53 and SEQ ID NO: 55, respectively.

[0172] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 56 and 54, respectively.

[0173] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region have the amino acid sequences of SEQ ID NO: 56 and 55, respectively.

[0174] The multispecific antibody of the present invention may further include a common light chain to further facilitate the downstream process of separating the multispecific antibody from excess parent or intermediate molecules.

[0175] In some embodiments, the multispecific antibody includes a first light chain and a second light chain.

[0176] In some embodiments, the first light chain and the second light chain have the same amino acid sequence.

[0177] In some embodiments, the multispecific antibody is a bispecific antibody.

[0178] [Table 2]

[0179] [Table 3-1]

[0180] [Table 3-2]

[0181] [Table 3-3]

[0182] [Table 3-4]

[0183] Except that IgG2 has valine at position 309, positions 307, 309, and 311 are conserved across isotypes, so the mutations can transfer to the IgG2 and IgG4 isotypes. Positions 366, 368, and 407 are also conserved across antibody isotypes. F405L is conserved, but IgG4 has an R at position 409. To promote Fab arm exchange of human IgG4 antibodies, one parental antibody is engineered to have the F405L / R409K mutations and the other parental antibody is wild type.

[0184] In some embodiments, the multispecific antibody binds to at least two antigens.

[0185] In some embodiments, the antigen is ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, aggrecan, AGR2, AICDA, AIF1, AIG1, AKAP1, AKAP2, albumin, AMH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOC1, APOE, AR, AZGP1 (zinc-binding a-glycoprotein), B7.1, B7.2, BAD, BAFF, BAG1, BAI1, BCL2, BCL6, BDNF, BLNK, BLR1 (MDR15), BlyS, BMP1, BMP2, BMP3B (GDF10), BMP4, BMP6, BMP8, BMPR1A, BMPR1B, BMPR2, BPAG1 (plectin), BRCA1, BTLA, C19orf10 (IL27w), C3, C4A, C5, C5R1, CANT1, CASP1, CASP4, CAV1, CCBP2 (D6 / JAB61), CCL1 (1-309), CCL11 (eotaxin), CCL13 (MCP-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3b), CCL2 (MCP-1), MCAF, CCL20 (MIP-3a), CCL21 (MIP-2), SLC, exodus-2, CCL22 (MDC / STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2 / eotaxin-2), CCL25 (TECT), CCL26 (eotaxin-3), CCL27 (CTACK / ILC), CCL28, CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CCR1 (CKR1 / HM145), CCR2 (mcp-1RB / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCR5 (CMKBR5 / ChemR13), CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7 (CKR7 / EBI1), CCR8 (CMKBR8 / TER1 / CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), CD123, CD137, CD164, CD16a, CD16b, CD19, CD1C,CD20, CD200, CD-22, CD24, CD28, CD3, CD30, CD32a, CD32b, CD37, CD38, CD39, CD3E, CD3G, CD3Z, CD4, CD40, CD40L, CD44, CD45RB, CD47, CD52, CD69, CD72, CD73, CD74, CD79A, CD79B, CD8, CD80, CD81, CD83, CD86, CD89, CD96, CDH1 (E-cadherin), CDH10, CDH12, CDH13, CDH18, CDH19, CDH20, CDH5, CDH7, CDH8, CDH9, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, CDKN1A (p21Wap1 / Cip1), CDKN1B (p27Kip1), 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, COL18A1, COL1A1, COL4A3, COL6A1, CR2, CRP, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTLA4, CTNNB1 (b-catenin), CTSB (Cathepsin B), CX3CL1 (SCYD1), 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), CXCL6 (GCP-2), CXCL9 (MIG), CXCR3 (GPR9 / CKR-L2), CXCR4, CXCR6 (TYMSTR / STRL33 / Bonzo), CYB5, CYC1, CYSLTR1, DAB2IP, DES, DKFZp451J0118, DNAM-1, DNCL1, DPP4, 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 (aFGF), FGF10, FGF11, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2 (bFGF), FGF20, FGF21, FGF22, FGF23, FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF8, FGF9, FGFR, FGFR3, FIGF (VEGFD), FIL1 (EPSILON), FIL1 (ZETA), FLJ12584, FLJ25530, FLRT1 (fibronectin), FLT1, FOS, FOSL1 (FRA-1), FY (DARC), GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GDF5, GFI1, GGT1, GITR, GITRL, GM-CSF, GNAS1, GNRH1, GPR2 (CCR10), GPR31, GPR44, GPR81 (FKSG80), GRCC10 (C10), GRP, GSN (gelsolin), GSTP1, HAVCR2, HDAC4, HDAC5, HDAC7A, HDAC9, HGF, HIF1A, HIP1, histamine and histamine receptor, HLA, HLA-A, HLA-DRA, HM74, HMOX1, HUMCYT2A, HVEM, ICEBERG, ICOS, ICOS-L, IDO, ID2, IFN-α, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNB1, IFN-γ, IFN-ω1, IGBP1, IGF1, IGF1R, IGF2, IGFBP2, IGFBP3, IGFBP6, IL-1, IL10, IL10RA, IL10RB, IL11, IL11RA, IL-12, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL14, IL15, IL15RA, IL16, IL17, IL17B, IL17C, IL17R, IL18, IL18BP, IL18R1, IL18RAP, IL19, IL1A, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL1HY1, IL1R1, IL1R2, IL1RAPIL1RAPL1, IL1RAPL2, IL1RL1, IL1RL2, IL1RN, IL2, IL20, IL20RA, IL21R, 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), IL7, IL7R, IL8, IL8RA, IL8RB, IL8RB, IL9, IL9R, ILK, INHA, INHBA, INSL3, INSL4, Insulin, Insulin Receptor, IRAK1, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 (a6 Integrin), ITGAV, ITGB3, ITGB4 (b4 Integrin), JAG1, JAK1, JAK3, JUN, K6HF, KAI1, KDR, KITLG, KIR, KLF5 (GCBoxBP), KLF6, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, KRT1, KRT19 (Keratin 19), KRT2A, KRTHB6 (Hair-Specific Type II Keratin), LAG-3, LAMA5, LDL, LEP (Leptin), LFA, Lingo-p75, Lingo-Troy, LPS, LTA (TNF-b), LTB, LTB4R (GPR16), LTB4R2, LTBR, MACMARCKS, MAG or Omgp, MAP2K7 (c-Jun), MDK, Mesothelin, MIB1, Midkine, MIF, MIP-2, MKI67 (Ki-67), MMP2, MMP9, MS4A1, MSMB, MT3 (Metallothionein-III), MTSS1, MUC1 (Mucin), MYC, MYD88, NCK2, Neurocan, NFKB1, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, NgR-Nogo66 (Nogo), NgR-p75, NgR-Troy, NKG2D, NKp46, NME1 (NM23A), NOX5, NPPB, NR0B1, NR0B2, NR1D1, NR1D2, NR1H2, NR1H3, NR1H4, NRII2, NRII3, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3,NR5A1, NR5A2, NR6A1, NRP1, NRP2, NT5E, NTN4, ODZ1, OPRD1, OX-40, OX-40L, P2RX7, PAP, PART1, PATE, PAWR, PCA3, PCNA, PD-1, PDGFA, PDGFB, PECAM1, PF4(CXCL4), PGF, PGR, Phosphacan, PIAS2, PIK3CG, PLAU(uPA), PLG, PLXDC1, PPBP(CXCL7), PPID, PR1, PRKCQ, PRKD1, PRL, PROC, PROK2, PSAP, PSCA, PTAFR, PTEN, PTGS2(COX-2), PTN, RAC2(p21Rac2), RARB, RGS1, RGS13, RGS3, RNF110(ZNF144), ROBO2, ROR1, SI00A2, SCGB1D2(Lipophilin B), SCGB2A1(Mammaglobin 2), SCGB2A2(Mammaglobin 1), SCYE1(Endothelial monocyte activating cytokine), SDF2, SERPINA1, SERPINA3, SERPINB5(Maspin), SERPINE1(PAI-1), SERPINF1, SHBG, SLA2, SLC2A2, SLC33A1, SLC43A1, SLIT2, SPP1, SPRR1B(Spr1), ST6GAL1, STAB1, STAT6, STEAP, STEAP2, TB4R2, TBX21, TCP10, TDGF1, TEK, TF(Transferrin receptor), TGFA, TGFB1, TGFB111, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, THBS1(Thrombospondin-1), THBS2, THBS4, THPO, TIE(Tie-1), TIGIT, TIM-3, TIMP3, Tissue factor, TLR10, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TNF, TNF-a, TNFAIP2(B94), TNFAIP3, TNFRSF11A, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF5, TNFRSF6(Fas), TNFRSF7, TNFRSF8, TNFRSF9, TNFSF10(TRAIL), TNFSF11(TRANCE), TNFSF12(APO3L), TNFSF13(April), TNFSF13B, TNFSF14(HVEM-L), TNFSF15(VEGI)TNFSF18, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TOLLIP, Toll-like receptor, TOP2A (topoisomerase Iia), TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TREM1, TREM2, TRPC6, TSLP, TWEAK, VEGF, VEGFB, VEGFC, versican, VHLC5, VISTA, VLA-4, XCL1 (lymphotactin), XCL2 (SCM-1b), XCR1 (GPR5 / CCXCR1), YY1, and ZFPM2. (topoisomerase Iia), TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TREM1, TREM2, TRPC6, TSLP, TWEAK, VEGF, VEGFB, VEGFC, versican, VHLC5, VISTA, VLA-4, XCL1 (lymphotactin), XCL2 (SCM-1b), XCR1 (GPR5 / CCXCR1), YY1, and ZFPM2.

[0186] In some embodiments, the multispecific antibody binds to CD3.

[0187] In some embodiments, the multispecific antibody binds to CD3 and a tumor antigen.

[0188] In some embodiments, the multispecific antibody binds to two antigens, and the two antigens are any two of PD1, CD27, CD28, NKP46, ICOS, GITR, OX40, CTLA4, LAG3, TIM3, KIRa, CD73, CD39, IDO, BTLA, VISTA, TIGIT, CD96, CD30, HVEM, DNAM-1, LFA, tumor antigen, EGFR, cMet, FGFR, ROR1, CD123, IL1RAP, FGFR, mesothelin, CD3, T cell receptor, CD32b, CD32a, CD16a, CD16b, NKG2D, NKP46, CD28, CD47, DLL, CD8, CD89, HLA, B cell receptor, or CD137.

[0189] Genetic engineering of the multispecific antibodies of the present invention By introducing further Fc mutations into the multispecific antibodies of the present invention, effector functions and pharmacokinetic properties can be regulated. In the conventional immune function, as a result of the interaction between the antibody-antigen complex and cells of the immune system, a wide range of responses occur, from effector functions such as antibody-dependent cell cytotoxicity and phagocytosis to immunomodulatory signals such as the regulation of lymphocyte proliferation and antibody secretion. All of these interactions are initiated through the binding of the Fc region of the antibody or immune complex to specialized cell surface receptors. The diversity of cellular responses induced by antibodies and immune complexes is due to the structural heterogeneity of Fc receptors. That is, FcγRI (CD64), FcγRIIa (CD32A), and FcγRIII (CD16) are activating Fcγ receptors (i.e., enhancing the immune system), and FcγRIIb (CD32B) is an inhibitory Fcγ receptor (i.e., attenuating the immune system). Binding to the FcRn receptor regulates the half-life of the antibody.

[0190] In some embodiments, the multispecific antibodies of the present invention further have at least one mutation that regulates the binding of the antibody to FcγR.

[0191] In some embodiments, the multispecific antibodies of the present invention further have at least one mutation that regulates the binding of the antibody to FcRn.

[0192] Typical mutations that extend the half-life of the multispecific antibody include mutations M428L / N434S, M252Y / S254T / T256E, T250Q / M428L, N434A, and T307A / E380A / N434A. Typical mutations that shorten the half-life of the multispecific antibody include mutations H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R.

[0193] In some embodiments, the multispecific antibodies of the invention reduce the binding of the antibodies to activating Fcγ receptors (FcγR) and / or comprise at least one mutation that reduces Fc effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or antibody-dependent cell phagocytosis (ADCP).

[0194] Exemplary mutations that reduce the binding of the multispecific antibodies of the invention to activating FcγR and / or minimize antibody effector functions include L234A / L235A in IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S in IgG2, F234A / L235A in IgG4, S228P / F234A / L235A in IgG4, N297A in all Ig isotypes, V234A / G237A in IgG2, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D365E / L358M in IgG1, H268Q / V309L / A330S / P331S in IgG2, S267E / L328F in IgG1, L234F / L235E / D265A in IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S in IgG1, S228P / F234A / L235A / G237A / P238S in IgG4, and S228P / F234A / L235A / G236 deletion / G237A / P238S in IgG4.

[0195] Exemplary mutations that increase the binding of the multispecific antibodies of the invention to activating Fcγ and / or enhance antibody effector functions include S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, and G236A / S239D / I332E, K326A / E333A, K326W / E333A, H268F / S324T, S267E / H268F, S267E / S324T, and S267E / H268F / S324T.

[0196] By introducing the well-known S228P into the IgG4 antibody, the stability of IgG4 can be enhanced.

[0197] "Antibody-dependent cell-mediated cytotoxicity", "antibody-dependent cell-mediated cytotoxicity", namely "ADCC", is a mechanism that induces cell death depending on the interaction between antibody-coated target cells and effector cells having lytic activity such as natural killer cells, monocytes, macrophages, and neutrophils by Fcγ receptors (FcγR) expressed on effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcγRIIIa. The death of target cells coated with an antibody occurs as a result of effector cell activity through the secretion of pore-forming proteins and proteases. To evaluate the ADCC activity of the antibody of the present invention, the antibody can be added to cells expressing a desired antigen together with immune effector cells, and the immune effector cells are activated by the antigen-antibody complex, and as a result, cell lysis of the target cells can occur. Cell lysis can be detected by the release of a label (for example, a radioactive substrate, a fluorescent dye, or a natural intracellular protein) from the lysed cells. Representative effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Exemplary target cells include cells that express a desired antigen either endogenously or recombinantly. In an exemplary assay, target cells are used at a ratio of effector cells 50 to target cells 1. The target cells are pre-labeled with BATDA (PerkinElmer) at 37°C for 20 minutes, washed twice, and resuspended in DMEM, 10% heat-inactivated FBS, 2 mM L-glutamine (all from Invitrogen). Target cells (1×10 4 cells) and effector cells (0.5×10 6Combine (the cells in number) together and add 100 μL of the cells to the wells of a 96-well U-bottom plate. Add an additional 100 μL with or without the test antibody. Centrifuge the plate at 200 g for 3 minutes, incubate at 37 °C for 2 hours, and then centrifuge again at 200 g for 3 minutes. A total of 20 μL of the supernatant is removed per well, and cell lysis is measured by the addition of 200 μL of DELPHIA Europium-based reagent (PerkinElmer). Normalize the data against the maximum cytotoxicity with 0.67% Triton X-100 (Sigma Aldrich), and determine the minimum control by the natural release of BATDA from the target cells in the absence of any antibody.

[0198] "Antibody-dependent cell phagocytosis" ("ADCP") refers to a mechanism by which antibody-coated target cells are eliminated by uptake by phagocytic cells such as macrophages or dendritic cells. ADCP can be evaluated using monocyte-derived macrophages as effector cells and, as target cells engineered to express GFP or other labeled molecules, Daudi cells (ATCC® CCL-213™), or cells of B-cell leukemia or lymphoma or tumor expressing the desired antigen. The effector:target cell ratio can be, for example, 4:1. The effector cells may be incubated with the target cells for 4 hours with or without the addition of the antibody of the present invention. After incubation, the cells can be detached using actinase. Macrophages can be identified by anti-CD11b antibody and anti-CD14 antibody conjugated to a fluorescent label, but the rate of phagocytosis is determined using standard methods based on the percentage of GFP fluorescence in CD11 + and CD14 + macrophages.

[0199] "Complement-dependent cytotoxicity", i.e., (CDC), refers to a mechanism for inducing cell death in which the Fc effector domain of a target-binding antibody binds to and activates complement component C1q, and such complement component C1q then activates the complement cascade to cause the death of the target cell. Activation of the complement can also result in the deposition of complement components on the surface of the target cell, facilitating ADCC by binding of the complement receptor (e.g., CR3) to leukocytes. CDC can be measured, for example, by seeding Daudi cells at 1×10 5 cells / well (50 μL / well) in RPMI-B (RPMI supplemented with 1% BSA), adding 50 μL of the test antibody to the well at a final concentration of 0 - 100 μg / mL, incubating the reaction for 15 minutes at room temperature, adding 11 μL of pooled human serum to the well, and incubating the reaction for 45 minutes at 37°C. The percentage of lysed cells can be detected as the % of propidium iodide-stained cells in a FACS assay using standard methods.

[0200] Additional mutations that increase the binding of the antibody to FcyRIIb can be further introduced into the multispecific antibodies of the present invention. Exemplary such mutations include mutations S267E, S267D, S267E / I332E, S267E / L328F, G236D / S267E, and E233D / G237D / H268D / P271G / A330R / P238D.

[0201] Generally, mutations that increase the binding to activating FcγRs and decrease the binding to inhibitory FcRγIIb can be introduced by genetic engineering into antibodies used to enhance the immune response in a subject, such as for the treatment of cancer and infectious diseases. Mutations that decrease the binding to FcγRs or increase the binding to inhibitory FcRγIIb can be introduced by genetic engineering into antibodies used to attenuate the immune response in a subject, such as for the treatment of inflammatory or autoimmune diseases. Mutations that increase the binding to inhibitory FcγRIIb can also be introduced into agonist antibodies that bind to members of the TNF receptor superfamily and enhance their agonist activity.

[0202] The ability of the multispecific antibodies of the present invention to induce ADCC can be enhanced by genetically engineering the oligosaccharide components of the antibodies. Human IgG1 is N-glycosylated at Asn297, and most of the glycans are in the known biantennary G0, G0F, G1, G1F, G2, or G2F forms. Antibodies produced by unengineered CHO cells typically have a glycan fucose content of at least about 85%. Removal of core fucose from biantennary complex-type oligosaccharides added to the Fc region enhances the ADCC of the antibody by improving FcγRIIIa binding without altering antigen binding or CDC activity. Such mAbs can be obtained using different methods that have been reported to lead to the effective expression of relatively highly defucosylated antibodies with a biantennary complex type of Fc oligosaccharide, such as control of the culture osmolality, application of the mutant CHO cell line Lec13 as the host cell line, application of the mutant CHO cell line EB66 as the host cell line, application of the rat hybridoma cell line YB2 / 0 as the host cell line, introduction of small interfering RNAs specific for the α1,6-fucosyltransferase (FUT8) gene, or co-expression of β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II or kifunensine, a potent α-mannosidase I inhibitor.

[0203] In some embodiments, the multispecific antibodies of the present invention have a biantennary glycan structure with a fucose content of from about 0% to about 15%, such as 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.

[0204] In some embodiments, the multispecific antibodies of the present invention have a biantennary glycan structure with a fucose content of from about 50%, 40%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.

[0205] The "fucose content" means the amount of fucose monosaccharide in the sugar chain at Asn297. The relative amount of fucose is the ratio to the total sugar structure of the fucose-containing structure. These can be determined and quantified by a plurality of methods, for example, 1) use of MALDI-TOF of N-glycosidase F-treated samples (e.g., complexes, hybrids, and oligo and high-mannose structures), 2) enzymatic release of the Asn297 glycan, subsequent derivatization, and detection / quantification by HPLC (UPLC) and / or HPLC-MS (UPLC-MS) with fluorescence detection, 3) intact protein analysis of native or reduced mAb with or without treatment of the Asn297 glycan with Endo S or other enzymes that cleave between the first GlcNAc monosaccharide and the second GlcNAc monosaccharide and leave the fucose attached to the first GlcNAc, 4) digestion of the mAb into component peptides by enzymatic digestion (e.g., trypsin or endopeptidase Lys-C) followed by separation, detection, and quantification by HPLC-MS (UPLC-MC), or 5) separation of the mAb oligosaccharide from the mAb protein by specific enzymatic deglycosylation with PNGase F at Asn297. The released oligosaccharides are labeled with fluorophores and separated, and can be characterized by various supplementary techniques that allow for detailed characterization of the glycan structure, namely, matrix-assisted laser desorption ionization (MALDI) mass spectrometry by comparison of the measured mass to the theoretical mass, determination of the degree of sialylation by ion-exchange HPLC (GlycoSep C), separation and quantification of oligosaccharide types according to hydrophilicity criteria by normal-phase HPLC (GlycoSep N), and separation and quantification of oligosaccharides by high-performance capillary electrophoresis-laser-induced fluorescence (HPCE-LIF).

[0206] "Low fucose" or "low fucose content" refers to an antibody having a fucose content of about 0% to about 15%.

[0207] "Normal fucose" or "normal fucose content" refers to an antibody having a fucose content of more than about 50%, usually more than about 60%, 70%, 80%, or more than 85%.

[0208] The multispecific antibodies of the present invention may be post-translationally modified by processes such as glycosylation, isomerization, deglycosylation, or non-naturally occurring covalent modifications (e.g., addition of polyethylene glycol moieties (PEGylation) and lipidation). Such modifications can be carried out in vivo or in vitro. For example, the antibodies of the present invention described herein can have their pharmacokinetic profiles improved by conjugating (PEGylating) with polyethylene glycol. The conjugation can be carried out by techniques known to those skilled in the art. It has been shown that conjugation of therapeutic antibodies with PEG enhances pharmacokinetics without reducing function.

[0209] Multispecific antibodies of the present invention that can be modified to improve stability, selectivity, cross-reactivity, affinity, immunogenicity, or other desirable biological or biophysical properties are within the scope of the present invention. Antibody stability is affected by several factors including (1) core packing of individual domains that affect intrinsic stability, (2) protein / protein interfacial interactions that affect pairing of HC and LC, (3) burial of polar and charged residues, (4) H-bond networks for polar and charged residues, and (5) distribution of surface charge and polar residues in other intramolecular and intermolecular forces (Worn and Pluckthun 2001). Residues that may destabilize the structure can be identified based on the crystal structure of the antibody or, in some cases, by molecular modeling, and the effect of residues on antibody stability can be tested by creating and evaluating mutants that carry mutations at the identified residues. One way to increase antibody stability is to increase the midpoint temperature of the heat transition (T m ) as measured by differential scanning calorimetry (DSC). In general, the T m of a protein correlates with its stability and is inversely correlated with its sensitivity to unfolding and denaturation in solution and the degradation process depending on the ease of unfolding of the protein. Studies of formulations have shown that the T mIt has been suggested that it is closely related to the long-term physical stability of the corresponding mAb.

[0210] The C-terminal lysine (CTL) can be removed from the injected antibody by endogenous circulating carboxypeptidase in the bloodstream. During production, as described in US Patent Application Publication No. 20140273092, extracellular Zn 2+ , EDTA or EDTA-Fe 3+ By controlling the concentration of, the removal of CTL can be controlled below the highest level. The CTL content of the antibody can be measured using known methods.

[0211] In some embodiments, the multispecific antibody of the present invention has a C-terminal lysine content of about 10% to about 90%, about 20% to about 80%, about 40% to about 70%, about 55% to about 70%, or about 60%.

[0212] In some embodiments, the multispecific antibody of the present invention has a C-terminal lysine content of about 0%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0213] The present invention also provides an isolated antibody comprising two heavy chains and two light chains having the same amino acid sequence, wherein the two identical heavy chains have the mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R, and the residue numbering follows the EU index.

[0214] The present invention also provides an isolated antibody comprising two heavy chains and two light chains having the same amino acid sequence, wherein the two identical heavy chains have the mutation Q311R, and the residue numbering follows the EU index.

[0215] The present invention also provides an isolated antibody comprising two heavy chains and two light chains having the same amino acid sequence, wherein the two identical heavy chains have the mutation Q311K and the residue numbering follows the EU index.

[0216] The present invention also provides an isolated antibody comprising two heavy chains and two light chains having the same amino acid sequence, wherein the two identical heavy chains have the mutations T307P / L309Q and the residue numbering follows the EU index.

[0217] The present invention also provides an isolated antibody comprising two heavy chains and two light chains having the same amino acid sequence, wherein the two identical heavy chains have the mutation T307P / V309Q and the residue numbering follows the EU index.

[0218] The present invention also provides an isolated antibody comprising two heavy chains and two light chains having the same amino acid sequence, wherein the two identical heavy chains have the mutations T307P / L309Q / Q311R and the residue numbering follows the EU index.

[0219] The present invention also provides an isolated antibody comprising two heavy chains and two light chains having the same amino acid sequence, wherein the two identical heavy chains have the mutations T307P / V309Q / Q311R and the residue numbering follows the EU index.

[0220] The isolated antibody is useful as a parental antibody for generating the multispecific antibodies of the present invention.

[0221] In some embodiments, the isolated antibody further has the mutations F405L, K409R, F405L / R409K, T366W or T366S / L368A / Y407V.

[0222] In some embodiments, the isolated antibody is of the IgG1, IgG2 or IgG4 isotype.

[0223] Method for producing engineered multispecific antibodies of the present invention Engineered multispecific antibodies of the present invention having an amino acid sequence that has changed compared to the parental multispecific antibody can be produced using standard cloning and expression techniques. For example, site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s), and the effect on antibody binding or other properties of interest can be evaluated using well-known methods and the methods described in the examples herein.

[0224] Allotype of antibody The immunogenicity of therapeutic antibodies is associated with an increased risk of infusion reactions and a shortened duration of the therapeutic response (Baert et al., (2003) N Engl J Med 348:602-08). The degree to which a therapeutic antibody induces an immune response in a host can be partially determined by the allotype of the antibody (Stickler et al., (2011) Genes and Immunity 12:213-21). The allotype of an antibody is related to mutations in the amino acid sequence at specific positions in the constant region sequence of the antibody.

[0225] Table 4 shows the allotypes of selected IgG1, IgG2, and IgG4.

[0226] In some embodiments, the multispecific antibodies of the present invention are of the G2m(n), G2m(n-), G2m(n) / (n-), nG4m(a), G1m(17), or G1m(17,1) allotype.

[0227] [Table 4]

[0228] Production and isolation of the multispecific antibodies of the present invention The multispecific antibodies of the present invention can be produced using standard molecular biology techniques by facilitating Fab arm exchange of the parental antibody. The multispecific antibodies of the present invention can be purified using protein A ligand affinity chromatography.

[0229] The present invention also provides a method for producing an isolated multispecific antibody comprising a first heavy chain or a fragment thereof having a mutation Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R and a second heavy chain or a fragment thereof having wild-type amino acid residues at positions 307, 309 and 311, comprising: providing a first parental antibody comprising a first heavy chain or a fragment thereof having a mutation Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R and a first light chain; providing a second parental antibody comprising a second heavy chain or a fragment thereof having wild-type amino acid residues at positions 307, 309 and 311 and a second light chain; contacting the first parental antibody and the second parental antibody in a sample; incubating the sample; and purifying the multispecific antibody using protein A ligand affinity chromatography. A method is also provided.

[0230] The present invention also provides a method for producing an isolated multispecific antibody comprising a first heavy chain or a fragment thereof having a mutation T307P / L309Q and a second heavy chain or a fragment thereof having wild-type amino acid residues at positions 307 and 309, comprising: providing a first parental antibody comprising a first heavy chain or a fragment thereof having a mutation T307P / L309Q and a first light chain; providing a second parental antibody comprising a second heavy chain or a fragment thereof having wild-type amino acid residues at positions 307 and 309 and a second light chain; contacting the first parental antibody and the second parental antibody in a sample; incubating the sample; and purifying the multispecific antibody using protein A ligand affinity chromatography. A method is also provided.

[0231] The present invention also provides a method for producing an isolated multispecific antibody comprising a first heavy chain having the mutation T307P / V309Q or a fragment thereof, and a second heavy chain having wild-type amino acid residues at positions 307 and 309 or a fragment thereof, comprising: providing a first parental antibody comprising a first heavy chain having the mutation T307P / V309Q or a fragment thereof and a first light chain; providing a second parental antibody comprising a second heavy chain having wild-type amino acid residues at positions 307 and 309 or a fragment thereof and a second light chain; contacting the first parental antibody and the second parental antibody in a sample; incubating the sample; and purifying the multispecific antibody using protein A ligand affinity chromatography.

[0232] The present invention also provides a method for producing an isolated multispecific antibody comprising a first heavy chain having the mutation T307P / L309Q / Q311R or a fragment thereof, and a second heavy chain having wild-type amino acid residues at positions 307, 309 and 311 or a fragment thereof, comprising: providing a first parental antibody comprising a first heavy chain having the mutation T307P / L309Q / Q311R or a fragment thereof and a first light chain; providing a second parental antibody comprising a second heavy chain having wild-type amino acid residues at positions 307, 309 and 311 or a fragment thereof and a second light chain; contacting the first parental antibody and the second parental antibody in a sample; incubating the sample; and purifying the multispecific antibody using protein A ligand affinity chromatography.

[0233] The present invention also provides a method for producing an isolated multispecific antibody comprising a first heavy chain having the mutations T307P / V309Q / Q311R or a fragment thereof, and a second heavy chain having wild-type amino acid residues at positions 307, 309 and 311 or a fragment thereof, wherein: providing a first parental antibody comprising a first heavy chain having the mutations T307P / V309Q / Q311R or a fragment thereof and a first light chain; providing a second parental antibody comprising a second heavy chain having wild-type amino acid residues at positions 307, 309 and 311 or a fragment thereof and a second light chain; contacting the first parental antibody and the second parental antibody in a sample; incubating the sample; and purifying the multispecific antibody using protein A ligand affinity chromatography. Also provided is a method comprising:

[0234] The VH and VL regions of the multispecific antibody can be derived from the existing VH / VL regions of antibodies specific for a desired antigen, or from the VH / VL domains of newly generated parental antibodies.

[0235] The parental antibodies can be newly generated using a variety of techniques. For example, the parental antibodies can be generated using the hybridoma method of Kohler and Milstein, Nature 256:495, 1975. In the hybridoma method, a mouse or other host animal, such as a hamster, rat or monkey, is immunized with an antigen and then, using standard methods, spleen cells from the immunized animal are fused with myeloma cells to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1986). Colonies arising from a single immortalized hybridoma cell are screened for the production of antibodies having desired properties such as binding specificity, cross-reactivity or lack thereof, and antigen affinity.

[0236] Transgenic mice having a human immunoglobulin (Ig) locus in their genome can be used to generate parental antibodies against a desired antigen, for example, as described in WO 90 / 04036, US Patent No. 6150584, WO 99 / 45962, WO 02 / 066630, WO 02 / 43478, Lonberg et al., Nature 368:856-9, 1994; Green et al., Nature Genet 7:13-21, 1994; Green & Jakobovits, J. Exp. Med. 188:483-95, 1998; Lonberg and Huszar, Int Rev Immunol 13:65-93, 1995; Bruggemann et al., Eur J Immunol 21:1323-1326, 1991; Fishwild et al., Nat Biotechnol 14:845-851, 1996; Mendez et al., Nat Genet 15:146-156, 1997; Green, J Immunol Methods 231:11-23, 1999; Yang et al., Cancer Res 59:1236-1243, 1999; Bruggemann and Taussig, Curr Opin Biotechnol. 8:455-458, 1997; WO 02 / 043478). The endogenous immunoglobulin locus in such mice may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus can be inserted into the mouse genome using homologous or non-homologous recombination, transchromosome, or minigene.Companies such as Regeneron (http: / / _www_regeneron_com), Harbour Antibodies (http: / / _www_harbourantibodies_com), Open Monoclonal Technology, Inc. (OMT) (http: / / _www_omtinc_net), KyMab (http: / / _www_kymab_com), Trianni (http: / / _www.trianni_com), and Ablexis (http: / / _www_ablexis_com) may be working to use the above technologies to provide human antibodies targeting selected antigens.

[0237] The parental antibody can also be selected from a phage display library, where the phage is modified to express human immunoglobulins or their parts such as, for example, Fab, single-chain antibody (scFv), or unpaired or paired antibody variable regions. The parental antibody can be isolated, for example, from a phage display library that expresses the heavy and light chain variable regions of the antibody as a fusion protein with the bacteriophage pIX coat protein described in Shi et al., J Mol Biol 397:385-96, 2010 and International Publication No. 09 / 085462. The library can be screened for phage binding to the desired antigen, the resulting positive clones can be further characterized, and the Fab can be isolated from the clone lysate and expressed as a full-length IgG. Such phage display methods for isolating human antibodies are described, for example, in U.S. Patent Nos. 5,223,409, 5,403,484, 5,571,698, 5,427,908, 5,580,717, 5,969,108, 6,172,197, 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081.

[0238] The isolated VH / VL regions can be cloned using standard cloning methods as part of any Ig isotype or an antibody constant domain such as the CH2-CH3 region. Fc mutations can be introduced into the parental antibody using standard methods.

[0239] In some embodiments, the first parental antibody and the second parental antibody are provided as purified antibodies.

[0240] In some embodiments, the first parental antibody and the second parental antibody are provided in cell culture medium recovered from cells expressing the first parental antibody and the second parental antibody.

[0241] In some embodiments, the first parental antibody and the second parental antibody are co-expressed intracellularly.

[0242] It has been demonstrated herein that the production of the multispecific antibodies of the invention can occur when the parental antibodies are provided in a crude extract as unpurified antibodies. If it is possible to purify the multispecific antibodies from the crude extract, only one purification step is required, thus reducing the cost of downstream processing.

[0243] An incubation step is performed when the parental antibodies are brought into contact with each other.

[0244] In some embodiments, the incubation is performed at a temperature of about 20°C to about 37°C.

[0245] In some embodiments, the incubation is performed at a temperature of about 25°C to about 37°C.

[0246] In some embodiments, the incubation is performed at a temperature of about 25°C to about 37°C for about 90 minutes to about 6 hours.

[0247] In some embodiments, a reducing agent is added during the incubation step.

[0248] In some embodiments, the reducing agent is 2-mercaptoethylamine (2-MEA).

[0249] In some embodiments, the reducing agent is dithiothreitol (DTT).

[0250] In some embodiments, the reducing agent is dithioerythritol (DTE).

[0251] In some embodiments, the reducing agent is glutathione.

[0252] In some embodiments, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0253] In some embodiments, the reducing agent is L-cysteine.

[0254] In some embodiments, the reducing agent is β-mercaptoethanol.

[0255] In some embodiments, the reducing agent is present at a concentration of about 10 mM to about 100 mM.

[0256] In some embodiments, 2-MEA is present at a concentration of about 10 mM to about 100 mM.

[0257] In some embodiments, 2-MEA is present at a concentration of about 25 mM to about 75 mM.

[0258] For example, at a temperature of at least 20 °C, in the presence of at least 25 mM of 2-MEA or in the presence of at least 0.5 mM of dithiothreitol, at pH 5-8, such as pH 7.0 or pH 7.4, an incubation of at least 90 minutes can be used.

[0259] In some embodiments, a pH gradient is used in protein A ligand chromatography.

[0260] In some embodiments, the pH gradient is from about pH 7.0 to about pH 3.0.

[0261] In some embodiments, the pH gradient is from about pH 4.6 to about pH 3.4.

[0262] In some embodiments, the multimeric antibody elutes at from about pH 4.4 to about pH 4.1.

[0263] In some embodiments, the pH gradient is a step gradient of pH 4.6, pH 4.1, and pH 3.4.

[0264] In some embodiments, Protein A ligand chromatography uses a citrate buffer.

[0265] In some embodiments, Protein A ligand chromatography uses a 50 mM citrate buffer.

[0266] In some embodiments, Protein A ligand chromatography uses an acetate buffer.

[0267] In some embodiments, Protein A ligand chromatography uses a 40 mM acetate buffer.

[0268] Protein A chromatography can be performed using an mAbSelect Sure column (GE Healthcare) or in batch mode. The culture supernatant is loaded directly onto the column without additional treatment according to the manufacturer's column specifications. A buffer containing 50 mM citrate at pH 4.7, pH 4.2, or pH 3.4 is used, and a pH step gradient is used to elute the antibody. The eluted fractions are collected and concentrated to a concentration of greater than 1 mg / mL prior to analysis. The purity of the isolated multimeric antibody can be evaluated using hydrophobic interaction chromatography (HIC).

[0269] Composition of the substance: The multimeric protein of the present invention The mutations identified herein can be used to isolate any multimeric protein from its parent protein, as long as the multimeric protein has at least two polypeptide chains each having an asymmetric CH2-CH3 region with the Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R mutation.

[0270] The present invention also provides a multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region having the mutation Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R, the second polypeptide comprises a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, and the residue numbering follows the EU index.

[0271] The present invention also provides a multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region having the mutation Q311R, the second polypeptide comprises a second CH2-CH3 region having a wild-type amino acid residue at position 311, and the residue numbering follows the EU index.

[0272] The present invention also provides a multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region having the mutation Q311K, the second polypeptide comprises a second CH2-CH3 region having a wild-type amino acid residue at position 311, and the residue numbering follows the EU index.

[0273] The present invention also provides a multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region having the mutations T307P / L309Q, the second polypeptide comprises a second CH2-CH3 region having wild-type amino acid residues at positions 307 and 309, and the residue numbering follows the EU index.

[0274] The present invention also provides a multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region having the mutations T307P / V309Q, the second polypeptide comprises a second CH2-CH3 region having wild-type amino acid residues at positions 307 and 309, and the residue numbering follows the EU index.

[0275] The present invention also provides a multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region having the mutations T307P / L309Q / Q311R, the second polypeptide comprises a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309, and 311, and the residue numbering follows the EU index.

[0276] The present invention also provides a multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region having the mutations T307P / V309Q / Q311R, the second polypeptide comprises a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309, and 311, and the residue numbering follows the EU index.

[0277] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region are of the IgG1 isotype.

[0278] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region are of the IgG2 isotype.

[0279] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region are of the IgG4 isotype.

[0280] In some embodiments, the first CH2-CH3 region has a reduced binding to Protein A ligand compared to the second CH2-CH3 region.

[0281] In some embodiments, the Protein A ligand comprises Protein A of Staphylococcus aureus.

[0282] In some embodiments, the Protein A ligand comprises a Z domain.

[0283] In some embodiments, the Protein A ligand comprises a Y domain.

[0284] In some embodiments, the Z domain comprises the amino acid sequence of SEQ ID NO: 1.

[0285] In some embodiments, the Protein A ligand has the amino acid sequence of SEQ ID NO: 99, 100, or 101.

[0286] In some embodiments, the multimeric protein further has asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region.

[0287] In some embodiments, the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region are F405L and K409R, respectively, wild type and F405L / R409K, respectively, T366W and T366S / L368A / Y407V, respectively, T366Y / F405A and T394W / Y407T, respectively, T366W / F405W and T394S / Y407A, respectively, F405W / Y407A and T366W / T394S, respectively, L351Y / F405A / Y407V and T394W, respectively, T366I / K392M / T394W and F405A / Y407V, respectively, T366L / K392M / T394W and F405A / Y407V, respectively, L351Y / Y407A and T366A / K409F, respectively, L351Y / Y407A and T366V / K409F, respectively, Y407A and T366A / K409F, respectively, D399K / E356K and K409D / K392D, respectively, or D399K / E356K / E357K and K409D / K392D / K370, respectively.

[0288] In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region are SEQ ID NO: 2 and 22, respectively, SEQ ID NO: 3 and 22, respectively, SEQ ID NO: 4 and 22, respectively, SEQ ID NO: 5 and 22, respectively, SEQ ID NO: 6 and 23, respectively, SEQ ID NO: 7 and 23, respectively, SEQ ID NO: 8 and 23, respectively, SEQ ID NO: 9 and 23, respectively, SEQ ID NO: 10 and 24, respectively, SEQ ID NO: 11 and 24, respectively, SEQ ID NO: 12 and 24, respectively, SEQ ID NO: 13 and 24, respectively, SEQ ID NO: 14 and 25, respectively, SEQ ID NO: 15 and 25, respectively, SEQ ID NO: 16 and 25, respectively, Each has SEQ ID NOs: 17 and 25, Each has SEQ ID NOs: 18 and 26, Each has SEQ ID NOs: 19 and 26, Each has SEQ ID NOs: 20 and 26, Each has SEQ ID NOs: 21 and 26, Each has SEQ ID NOs: 52 and 54, Each has SEQ ID NOs: 52 and 55, Each has SEQ ID NOs: 53 and 54, Each has SEQ ID NOs: 53 and 55, Each has SEQ ID NOs: 56 and 54, or Each has an amino acid sequence of SEQ ID NOs: 56 and 55.

[0289] In some embodiments, the first CH2-CH3 region and / or the second CH2-CH3 region is conjugated to a heterologous protein.

[0290] In some embodiments, the heterologous protein is a peptide.

[0291] In some embodiments, the heterologous protein is an extracellular domain of a receptor.

[0292] In some embodiments, the heterologous protein is an extracellular domain of a ligand.

[0293] In some embodiments, the heterologous protein is a secreted protein.

[0294] In some embodiments, the heterologous protein is a scFV.

[0295] In some embodiments, the heterologous protein is a heavy chain variable region (VH).

[0296] In some embodiments, the heterologous protein is a light chain variable region (VL).

[0297] In some embodiments, the heterologous protein is a fibronectin type III domain.

[0298] In some embodiments, the heterologous protein is a finomer.

[0299] In some embodiments, the heterologous protein is attached to the N-terminus of the first CH2-CH3 region and / or the second CH2-CH3 region, optionally via a linker.

[0300] In some embodiments, the heterologous protein is attached to the C-terminus of the first CH2-CH3 region and / or the second CH2-CH3 region, optionally via a linker.

[0301] In some embodiments, the linker has the amino acid sequence of SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 92, 93, 94, 95, 96, 97 or 98.

[0302] In some embodiments, the multimeric protein is an antibody.

[0303] In some embodiments, the antibody is multispecific.

[0304] In some embodiments, the antibody is bispecific.

[0305] In some embodiments, the antibody is monospecific.

[0306] In some embodiments, the multimeric protein comprises two polypeptide chains.

[0307] In some embodiments, the multimeric protein comprises three polypeptide chains.

[0308] In some embodiments, the multimeric protein comprises four polypeptide chains.

[0309] Table 5 shows exemplary formats of multimeric proteins encompassed by the present invention. In this format, the peptide (P) may be an extracellular domain of a receptor, an extracellular domain of a ligand, a secreted protein, a scFv, a Fab, a heavy chain variable region (VH), a light chain variable region (VL), a fibronectin type III domain, or a finomer. In this format, the linker (L) may optionally be absent. Exemplary linkers are shown in Table 6. The asterisk ( * ) in the table indicates that, as described herein, the two CH2-CH3 domains have asymmetric mutations.

[0310] The multimeric proteins of the present invention can be further modified as described herein for multispecific antibodies using standard methods. The multimeric proteins of the present invention can be produced using standard cloning methods.

[0311] [Table 5]

[0312] [Table 6]

[0313] Polynucleotides, Vectors, and Host Cells The present invention also provides an isolated polynucleotide encoding any one of the CH2-CH3 region, an antibody heavy chain, an antibody light chain, or a polypeptide of the multimeric protein of the present invention.

[0314] The present invention also provides an isolated polynucleotide that comprises a polynucleotide encoding a first CH2-CH3 region having the mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R, or A polynucleotide encoding a first CH2-CH3 region having mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R, and a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, or Also provided are isolated polynucleotides comprising the polynucleotide sequences of SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 87, 88 or 91.

[0315] The polynucleotide sequences of the present invention may be operably linked to one or more regulatory elements such as a promoter or enhancer that enables the expression of the nucleotide sequence in a desired host cell. The polynucleotide may be cDNA.

[0316] The present invention also provides a vector comprising the polynucleotide of the present invention. Such a vector may be a plasmid vector, a viral vector, a baculovirus expression vector, a transposon-based vector, or any other vector suitable for introducing the synthetic polynucleotide of the present invention into a given organism or genetic background by any means. The polynucleotide of the present invention may be operably linked to control sequences within an expression vector (s) that ensure the expression of the CH2-CH3 region encoded by the polynucleotide. Such control sequences include signal sequences, promoters (e.g., native or heterologous promoters), enhancer elements, and transcription termination sequences, and are selected to be compatible with the host cell selected to express the antibody. After the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the protein encoded by the incorporated polynucleotide.

[0317] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 27 and 47.

[0318] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 28 and 47.

[0319] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 29 and 47.

[0320] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 30 and 47.

[0321] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 31 and 48.

[0322] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 32 and 48.

[0323] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 33 and 48.

[0324] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 34 and 48.

[0325] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 35 and 49.

[0326] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 36 and 49.

[0327] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 37 and 49.

[0328] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 38 and 49.

[0329] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 39 and 50.

[0330] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 40 and 50.

[0331] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 41 and 50.

[0332] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 42 and 50.

[0333] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 43 and 51.

[0334] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 44 and 51.

[0335] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 45 and 51.

[0336] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 46 and 51.

[0337] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 87 and 89.

[0338] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 87 and 90.

[0339] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 88 and 89.

[0340] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 88 and 90.

[0341] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 92 and 89.

[0342] In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 92 and 90.

[0343] Table 7 shows the cDNA sequences in the representative CH2-CH3 region.

[0344]

Table 7-1

[0345]

Table 7-2

[0346]

Table 7-3

[0347]

Table 7-4

[0348]

Table 7-5

[0349]

Table 7-6

[0350]

Table 7-7

[0351]

Table 7-8

[0352]

Table 7-9

[0353]

Table 7-10

[0354] Suitable expression vectors are typically replicable in the host organism either as episomes or as an integral part of the host chromosomal DNA. Usually, the expression vector contains a selectable marker such as ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance to enable the detection of cells transformed with the desired DNA sequence.

[0355] Suitable promoter and enhancer elements are known in the art. For expression in eukaryotic cells, typical promoters include the light chain and / or heavy chain immunoglobulin gene promoters and enhancer elements, the cytomegalovirus immediate early promoter, the herpes simplex virus thymidine kinase promoter, the early and late SV40 promoters, the promoters present in the terminal repeat sequences derived from retroviruses, the mouse metallothionein-I promoter, and tissue-specific promoters known in various fields. The selection of appropriate vectors and promoters is within the skill of the art.

[0356] Many suitable vectors and promoters are known, and many of them are commercially available for constructing recombinant constructs. Representative vectors include those for expression in bacteria such as pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia (Uppsala, Sweden)), and eukaryotic vectors such as pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0357] The present invention also provides a host cell comprising one or more vectors of the present invention. A "host cell" refers to a cell into which a vector has been introduced. It is understood that the term "host cell" is intended to refer not only to a particular target cell, but also to the progeny of such a cell, as well as stable cell lines generated from a particular target cell. Although such progeny may not be identical to the parental cell due to either mutation or environmental influences that may result in certain modifications in subsequent generations, they are still included within the scope of the term "host cell" as used herein. Such host cells may be eukaryotic cells, prokaryotic cells, plant cells, or archaeal cells. Examples of prokaryotic host cells include Escherichia coli, bacilli such as Bacillus subtilis, and other Enterobacteriaceae such as species of the genus Salmonella, Serratia, and various species of the genus Pseudomonas. Other microorganisms such as yeast are also useful for expression. Examples of suitable yeast host cells are Saccharomyces (e.g., S. cerevisiae) and Pichia. Representative eukaryotic cells may be derived from mammals, insects, birds, or other animals. Mammalian eukaryotic cells include immortalized cell lines (e.g., hybridomas) or myeloma cell lines (e.g., SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) mouse cell lines). An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell lines include those derived from Chinese hamster ovary (CHO) cells such as CHO-K1SV (Lonza Biologics (Walkersville, MD)), CHO-K1 (ATCC CRL-61), or DG44.

[0358] The present invention also provides a method for producing an isolated multispecific antibody of the present invention, which comprises culturing the host cell of the present invention under conditions under which the multispecific antibody is expressed, and purifying the multispecific antibody using protein A affinity chromatography.

[0359] Pharmaceutical compositions, administration, and methods of treatment The present invention also provides a pharmaceutical composition comprising the multispecific antibody or multimeric protein of the present invention and a pharmaceutically acceptable carrier. For therapeutic use, the multispecific antibody or multimeric protein of the present invention can be prepared as a pharmaceutical composition containing an effective amount of the multispecific antibody or multimeric protein of the present invention as an active ingredient in a pharmaceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient, or vehicle administered together with the active compound. Such vehicles may be water and oils of petroleum, animal, vegetable, or synthetic origin, for example, liquids such as peanut oil, soybean oil, mineral oil, sesame oil, etc. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterilized and generally contain no particulate matter. They can be sterilized by conventional well-known sterilization techniques (e.g., filtration). This composition can contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusters and buffers, stabilizers, thickeners, lubricants, and coloring agents, etc. The concentration of the multispecific antibody or multimeric protein of the present invention in such pharmaceutical formulations can vary widely, i.e., from less than about 0.5% by weight, usually at least about 1% by weight, up to 15 or 20% by weight at most, and is also selected mainly based on the required dose, fluid volume, viscosity, etc. according to the particular administration method chosen. Suitable vehicles and formulations containing other human proteins, such as human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp691 - 1092, see particularly pp. 958 - 989.

[0360] The methods of administration in the therapeutic use of the multispecific antibodies or multimeric proteins of the present invention can be parenteral administration, for example, intradermal, intramuscular, intraperitoneal, intravenous, or subcutaneous, intralung; transmucosal (oral, intranasal, intravaginal, rectal) administration, using tablets, capsules, solutions, powders, gels, granular preparations, or contained in syringes, implantable devices, osmotic pumps, cartridges, micropumps, or any other suitable route for delivering the drug to the host, such as other means recognized by those skilled in the art and well-known in the art. Site-specific administration can be achieved, for example, by intra-articular, intratracheal, intra-abdominal, intra-articular capsule, intra-cartilage, intra-cavity, intracerebellar, intraventricular, intra-colon, endocervical, intra-gastric, intra-hepatic, intramyocardial, intra-bone, intra-pelvic, intra-pericardial, intraperitoneal, intra-pleural, intra-prostatic, intralung, intra-rectal, intra-renal, intra-retinal, intraspinal, intra-synovial sac, intrathoracic, intra-uterine, intra-vascular, intra-bladder, intra-lesion, intravaginal, rectal, intra-oral, sublingual, intranasal, or transdermal delivery.

[0361] The pharmaceutical composition can be supplied as a kit containing a container containing the pharmaceutical composition described herein. The pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder to be reconstituted before injection. Alternatively, such a kit can include a dry powder disperser, a liquid aerosol generator, or a nebulizer for administering the pharmaceutical composition. Such a kit can further include information written about the instructions and use of the pharmaceutical composition.

[0362] Multispecific antibodies and other multimeric proteins can be used to treat any condition of a human subject depending on their specificities.

[0363] Although the present invention has been described in general terms, embodiments of the present invention are further disclosed in the following examples and should not be construed as limiting the scope of the claims.

[0364] Example 1. Design of Fc mutations that can reduce the binding of Fc to Protein A The Z domains of FcRn and Protein A bind to Fc at the interface between the CH2 and CH3 domains and contact many of the same residues on Fc. Mouse IgG2a / b binds more weakly to the Z domain than human IgG1, but binds to FcRn in both cases. Therefore, positions not conserved in the CH2 domain of mouse IgG2a were identified at the Z domain binding interface of the CH2 domain of human IgG1. Figure 1A shows the alignment between the CH2 domain of human IgG1 and residues 305 and 315 of the CH2 domain of mouse IgG2a. Since the residues at positions 305, 307, 309, 314, and 315 differed between the human and mouse sequences, it was hypothesized that introducing the reverse mutations T307P and / or L309Q into human IgG1 would result in engineered IgG1 variants with reduced binding to Protein A without affecting the interaction with FcRn. Valine 305 of human IgG1 is located within a β-strand of the CH2 domain and does not interact with either Protein A or FcRn. Leucine 314 and asparagine 315 differ between human and mouse IgG, but these differences are conservative (e.g., L314 of human IgG is replaced by another hydrophobic residue L / M314 in mouse IgG, and N315 is replaced by another polar residue S315 in mouse IgG). Therefore, it was inferred that the changes at positions 307 and 309 have the greatest impact on the interaction of human IgG1 with Protein A and FcRn.

[0365] Analysis of the crystal structure of the complex of the Z domain and Fc (Z34C peptide, a bundle of two disulfide - linked helices derived from the Z domain, PDB ID: 1L6X) revealed that residue Q311 of IgG1 Fc interacts mainly via hydrophobic interactions with F9, L13, R23, N24, and I27 on the Z domain (residue numbering follows SEQ ID NO: 99) (Figure 1B). In contrast, residue Q311 of IgG1 Fc interacted with the mainly acidic surface of FcRn containing E115 and E116 of the α - subunit of FcRn (corresponding to residues E4 and E5 of SEQ ID NO: 103) (PDB ID: 4N0U). It was hypothesized that mutating residue Q311 of Fc would have different effects on the binding of the resulting mutant(s) to the Z domain and FcRn. Figure 1B shows Fc residues in contact with FcRn or the Z domain at a cutoff distance of 5 Å.

[0366] Example 2. Preparation of monospecific and bispecific antibodies used in the experiments Mutations T307A, Q311A, Q311K, Q311E, T307P / L309Q, or T307P / L309Q / Q311R were introduced by genetic manipulation into the heavy chains of both of the different monospecific antibodies using standard molecular biology techniques.

[0367] Bispecific mA was prepared by using common light - chain technology or by promoting Fab - arm exchange using Duobdy® technology or knob - in - hole technology. In common light - chain technology, anti - TNFα and anti - αVβ5 antibodies known to share a light chain were used. In knob - in - hole technology, either a knob (T366W mutation) or a hole (T366S, L368A, Y407V mutations) was introduced into the parental monospecific antibody. In Duobdy® technology, F405L or K409R mutations were introduced into the parental monospecific antibody.

[0368] The produced bispecific antibody carried a mutation(s) that could inhibit or reduce Protein A binding only in one of the heavy chains (T307A, Q311A, Q311K, Q311, T307P / L309Q, and T307P / L309Q / Q311R) (e.g., asymmetric mutations).

[0369] Ab was expressed in Expi293F cells (Invitrogen) according to the manufacturer's protocol using a molar ratio of light chain:heavy chain plasmid = 3:1. The co-transfection mixture was prepared using a molar ratio of light chain:heavy chain 1:heavy chain 2 plasmid = 3:0.5:0.5. The culture supernatant was collected by filtration after a 5-day expression period. The titer was estimated using surface plasmon resonance against an isotype control standard of known concentration. The parental Ab was purified by Protein A affinity chromatography using MabSelect SuRe resin (GE Healthcare) according to the manufacturer's protocol. Variants with altered binding to Protein A were purified by Protein G affinity chromatography (GE Healthcare) according to the manufacturer's protocol.

[0370] Table 8 shows the produced monospecific antibodies.

[0371] Table 9 shows the produced bispecific (bs) antibodies.

[0372]

Table 8

[0373]

Table 9

[0374] Amino acid sequences of the heavy and light chains of the produced antibodies: SEQ ID NO: 68 gp120-R HC QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIHWVRQAPGQRFEWMGWINPYNGNKEFSAKFQDRVTFTADTSANTAYMELRSLRSADTAVYYCARVGPYSWDDSPQDNYYMDVWGKGTTVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0375] Sequence number 69 gp120 - R LC EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQHKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDFTLTITRVEPEDFALYYCQVYGASSYTFGQGTKLERKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0376] Sequence number 70 RSV - L HC QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0377] Sequence number 71 RSV LC DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0378] Sequence number 72 RSV-L[Q311A]HC QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHADWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0379] Sequence number 73 RSV-L[Q311K]HC QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHKDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0380] Accession No. 74 RSV-L[Q311R]HC(Q311R / F405L) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHRDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0381] Accession No. 75 RSV-L[Q311H]HC(Q311H / F405L) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHHDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0382] Sequence number 76 RSV-L[TL]HC (T307P / L309Q / F405L) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLPVQHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0383] Accession No. 77 RSV-L[TLQ]HC(T307P / L309Q / Q311R / F405L) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLPVQHRDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0384] Accession No. 78 RSV-L[I253D]HC(I253D / F405L) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMDSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0385] Sequence number 79 aVb5 HC QVQLVESGGGVVQPGRSRRLSCAASGFTFSRYTMHWVRQAPGKGLEWVAVISFDGSNKYYVGSVKGRFTISRDNSENTLYLQVNILRAEDTAVYYCAREARGSYAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0386] SEQ ID NO: 80 TNF and anti-αVβ5 LC EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0387] SEQ ID NO: 81 TNF HC EVQLVESGGGVVQPGGSLSLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDGSNKKYADSVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0388] SEQ ID NO: 82 TNF-[Q311R] HC EVQLVESGGGVVQPGGSLSLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDGSNKKYADSVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHRDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0389] Sequence number 83 TNF-[TLQ]HC (T307P / L309Q / Q311R): EVQLVESGGGVVQPGGSLSLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDGSNKKYADSVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLPVQHRDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0390] Sequence number 84 TNF-knob [Q311R] HC (Q311R / T366W) EVQLVESGGGVVQPGGSLSLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDGSNKKYADSVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHRDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0391] Accession number 85 TNF-knob [TLQ]HC (T307P / L309Q / Q311R / T366W) EVQLVESGGGVVQPGGSLSLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDGSNKKYADSVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLPVQHRDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0392] Accession number 86 aVb5-hole HC (T366S / L368A / Y407V) QVQLVESGGGVVQPGRSRRLSCAASGFTFSRYTMHWVRQAPGKGLEWVAVISFDGSNKYYVGSVKGRFTISRDNSENTLYLQVNILRAEDTAVYYCAREARGSYAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0393] Example 3. Effects of T307, L309 and / or Q311 mutations on binding to the Z domain and FcRn Binding of monospecific IgG1 variants to the Z domain Monospecific anti-RSV antibodies having Fc mutations as shown in Table 8 were used in the experiment.

[0394] RSV-L eluted from Protein A resin at pH 4.09. The T307A mutation did not affect the binding to Protein A (data not shown), whereas the T307P / L309Q mutation (mAb RSV-L[TL]) resulted in a moderate decrease in the binding to Protein A and this mAb was eluted at pH 4.48. A further attenuating effect on the binding to Protein A was obtained by the symmetric Q311K or Q311R mutations, but not by the Q311A mutation. The introduction of the triple mutation T307P / L309Q / Q311R (mAb RSV-L[TLQ]) further impaired the interaction with Protein A as shown by the increase in the elution pH (pH 4.70). Table 10 shows the values of the elution pH of the prepared IgG1 variants.

[0395] These results indicate that each of the Q311K, Q311R, T307P / L309Q, and T307P / L309Q / Q311R symmetric mutations can reduce the binding of the mutant IgG1 to Protein A, enabling the purification and separation of bispecific antibodies with asymmetric mutations generated from the parental mutant IgG1 based on the differential elution of Protein A.

[0396]

Table 10

[0397] Binding of IgG1 variants to FcRn In none of the single-site mutants (Q311R, Q311A, Q311K, and Q311H) introduced at position 311 was the interaction with FcRn of the monospecific antibody inhibited. The Q311R mutation improved the binding ability to FcRn to some extent, suggesting that this mutation may extend the serum half-life. RSV-L[TLQ] bound to FcRn with a similar affinity as compared to RSV-L. Bispecific IgG1 antibodies with asymmetric F405L Q311R (bsRSV-L[Q311R]) or F405L / T307P / L309Q / Q311R mutations (bsRSV-L[TLQ]) also bound to FcRn with an equivalent affinity as compared to wild-type IgG1. Figure 2A shows the dose-response curves of competitive binding of IgG1 variants with Q311R, Q311A, Q311K, or Q311H mutations to FcRn. Figure 2B shows the dose-response curves of competitive binding of IgG1 variants with either symmetric (e.g., RSV-L, RSV-L[Q311R], RSV-L[TLQ] as monospecific mAbs) or asymmetric (e.g., bsRSV-L[Q311R], bsRSV-L[TLQ] as bispecific mAbs) Q311R or T307P / L309Q / Q311R mutations to FcRn. The I253D mutation is known to inhibit the interaction with FcRn and was used as a negative control.

[0398] The mutations of Q311R, Q311A, Q311K or Q311H did not inhibit the interaction with FcRn. The mutation of Q311R enhanced the interaction with FcRn. The introduction of the T307P / L309Q / Q311R mutation into one or both heavy chains did not inhibit the interaction with FcRn. These results suggest that bispecific antibodies with asymmetric Q311R or T307P / L309Q / Q311R mutations can be isolated and purified from their parental monospecific antibodies by differential protein A purification. Furthermore, these antibodies may have a longer half-life in serum compared to wild-type IgG1.

[0399] Method The T307A, Q311A, Q311K, Q311R, Q311H, T307P / L309Q and T307P / L309Q / Q311R mutations were introduced by genetic engineering into monospecific parental anti-RSV antibodies or anti-gp120 antibodies. To generate bispecific anti-RSV / gp120 antibodies using Fab-arm exchange, the parental antibodies were further engineered to have an F405L mutation (anti-RSV mAb) or a K409R mutation (anti-gp120 mAb). The extent to which these mutations modulate the binding to the Z domain and FcRn was evaluated.

[0400] The Z domain used in the experiment has the amino acid sequence of SEQ ID NO: 1.

[0401] Binding to Protein A For each parental mAb with mutations in both arms, 1 mg was loaded onto a 1 mL mAbSelect sure column (GE Healthcare) and eluted at 1 mL / min using a 30 mL gradient from 1×PBS (pH 7.2) to 50 mM citrate (pH 3.5). The absorbance at 280 nm and the pH were monitored. The elution pH for the fractionation experiment was determined using the pH value at the peak maximum.

[0402] Binding to FcRn The binding to FcRn was evaluated in vitro using the AlphaScreen assay. In these assays, biotinylated IgG was bound to donor beads coated with streptavidin, and His-tagged FcRn was bound to acceptor beads coated with Ni. A luminescence signal was generated by the binding between the two proteins. When unlabeled wild-type or mutant IgG was used to compete for the binding, the signal decreased in a dose-dependent manner. The mAb was biotinylated using the SureLINK Chromophoric Biotin Labeling kit (KPL Inc.) according to the manufacturer's protocol. His-tagged FcRn was purchased from Sino Biological. The assay was performed in 1×PBS adjusted to pH 6.0 supplemented with 0.05% (w / v) bovine serum albumin (BSA) and 0.01% (w / v) Tween-20. 1 μg / mL of biotinylated wild-type IgG1 was bound to donor beads conjugated with streptavidin, and 0.2 μg / mL of His-tagged FcRn was bound to acceptor beads conjugated with nickel. The competing Ab was prepared at 0.4 mg / mL and serially diluted 3-fold at each point. Luminescence between 520 and 620 nm was recorded using an EnVision plate reader (Perkin Elmer). The data were analyzed using Prism 6.01 software (GraphPad Software, Inc.) and fitting with a four-parameter competition model as described above (Vafa et al., Methods 65:114-126, 2014).

[0403] Example 4. The T307P, L309Q, and Q311R mutations do not affect Fcγ receptor (FcγR) binding or antibody stability Mutations at the CH2-CH3 interface have been reported to change the structure of Fc, enhance the dynamics of Fc, reduce the thermal stability, and change the interaction with Fcγ receptors (Majumdar et al., MAbs 7:84-95, 2015). To investigate whether the Q311R or T307P / L309Q / Q311R mutations have a similar effect on the structure of Fc, antibodies with these mutations were evaluated for their ability to bind to Fcγ receptors and their thermal stability.

[0404] None of the symmetric or asymmetric Q311R or T307P / L309Q / Q311R mutations in either the monospecific or bispecific antibodies affected the ability of the mutant IgG1 to interact with Fcγ receptors in vitro. Fcγ receptors bind to the interface of C H 2-C H 3 instead of the interface of C H 2-hinge, so this result was as expected to some extent. These results also suggested that the introduced mutations did not affect the overall structure of Fc. Figures 3A, 3B, 3C, and 3D show the dose-response curves of the competitive binding of the selected antibodies to FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa, respectively. The graph shows the maximum signal (%) plotted against the concentration of the competing antibody.

[0405] Comparison of the T m values showed that the Q311R or T307P / L309Q / Q311R mutations did not affect the thermal stability of the mAb. Table 11 shows the parameters of differential scanning calorimetry (Tm and enthalpy values) of the tested antibodies. Combining these results suggests that the effects of the Q311R and T307P / L309Q / Q311R mutations are limited to the interaction between protein A and FcRn.

[0406]

Table 11

[0407] Method Using the α screen assay, the binding of IgG1 variants to FcγR was evaluated using the protocol described in Example 2 with some modifications. The soluble extracellular domain of FcγR with a C-terminal His tag was purchased from R&D Systems. The assay was performed in 1×PBS (pH 7.2) supplemented with 0.05% (w / v) bovine serum albumin (BSA) and 0.01% (w / v) Tween-20. 1 μg / mL of biotinylated wild-type IgG1 was bound to donor beads conjugated with streptavidin, and His-tagged FcγR was bound to acceptor beads conjugated with nickel. For FcγRI, a biotinylated IgG1-L234A / L235A variant that bound to the receptor weaker than wild-type IgG1 was used to increase the signal window. The concentrations of FcγR used were 200 ng / mL (FcγRI and FcγRIIIa), 10 ng / mL (FcγRIIa), or 14 ng / mL (FcγRIIb). The competing Ab was prepared at 0.4 mg / mL and serially diluted 3-fold at each point.

[0408] Differential scanning calorimetry (DSC) was used to determine the T m and enthalpy of antibody unfolding. Samples were diluted to 1 mg / mL in 1×PBS, pH 7.2. After equilibrating the samples at 25 °C for 15 minutes, the temperature was raised from 25 to 95 °C at a rate of 1 °C / min. Data were analyzed using Origin software.

[0409] Example 5. Separation of bispecific antibodies from parental monospecific mAbs after in vitro Fab arm exchange of purified antibodies by elution from protein A resin The introduction of asymmetric Q311R or T307P / L309Q / Q311R mutations into bispecific antibodies facilitated the purification of bispecific antibodies from parental monospecific mAbs.

[0410] The 1:1:1 mixture of the parental antibodies RSV-L[TLQ] and gp120-R and the bispecific bsRSV-L[TLQ] produced after in vitro Fab arm exchange was purified by differential protein A affinity chromatography, and the elution peaks were pooled and analyzed by HIC.

[0411] Figure 4A shows that both the parental mAb and the bispecific mAb could be separated using an HIC chromatograph and the developed conditions. Figure 4B shows the HIC chromatograph of an equimolar mixture of antibodies injected into a protein A column. Figure 4C shows the elution profile of the antibody mixture from the protein A resin that produced three different elution peaks at pH 4.7, pH 4.2, and pH 3.4, consistent with the presence of the two parental antibodies and the bispecific antibody. Figure 4D shows the HIC analysis of the elution peak of protein A. Analysis of the elution peaks by HIC showed that the eluate at high pH (pH 4.8) mainly contained the parental RSV-L[TLQ] mAb, while the eluate at pH 3.4 mainly contained the gp120-R parental mAb. The eluate at intermediate pH (pH 4.2) contained approximately 94% pure bispecific bsRSV-L[TLQ] mAb. Table 12 shows the elution purity of bsRSV-L[TLQ] obtained from differential protein A purification.

[0412]

Table 12

[0413] Method RSV-L[TLQ] and gp120-R were used as parental antibodies, and bsRSV-L[TLQ] mAb was used as the bispecific in the experiment.

[0414] RSV-L[TLQ] was purified using a protein G affinity chromatograph and dialyzed into 1×PBS. gp120-R was purified by protein A affinity chromatography and dialyzed into 1×PBS. Then, Fab-arm exchange was performed with 1 mg / mL of the two parental mAbs. Briefly, 5 mg of each parental antibody was mixed in a buffer containing 1×PBS and 75 mM 2-mercaptoethylamine, incubated at 31 °C for 5 h, and then dialyzed extensively against 1×PBS. The resulting material containing >95% BsAb was then mixed with the two purified parental mAbs in a 1:1:1 molar ratio, and this mixture was used in a differential protein A purification experiment.

[0415] Differential protein A purification was performed using a 1 mL mAb Select Sure column (GE Healthcare). The mixture was eluted in three steps using a buffer containing 50 mM citric acid (pH 4.7, pH 4.2, or pH 3.4). The eluted fractions were collected and concentrated to a concentration of >1 mg / mL before analysis.

[0416] The elution peaks obtained from differential protein A purification were analyzed by hydrophobic interaction chromatography (HIC) using a butyl NPR column (Tosoh Biosciences). Approximately 30 μg of each sample was injected onto the column and eluted using a 0–100% gradient of a buffer containing 100 mM sodium phosphate (pH 6.0), 1.5 M (NH4)2SO4, or 100 mM sodium phosphate (pH 6.0).

[0417] Example 6. Separation of bispecific antibodies from parental monospecific mAbs after in vitro Fab-arm exchange in the crude supernatant by elution from protein A resin Purification of bispecific antibodies generated from parental antibodies was facilitated by introducing asymmetric Q311R or T307P / L309Q / Q311R into the bispecific antibodies made from the cross materials in the supernatant.

[0418] In the DuoBody® technology for generating bispecific antibodies, it is necessary to purify the parental mAbs individually before performing Fab-arm exchange. However, the cFAE reaction often has residual amounts of bivalent parental mAbs, which may require additional downstream purification steps. Therefore, the purification of bispecific antibodies can be simplified by using differential protein A chromatography with a pH gradient. As another way to reduce the number of purification steps, the Fab-arm exchange protocol may be performed using culture supernatants. In this method, the titers of the parental mAbs are accurately determined so that the parental mAbs are mixed at a 1:1 molar ratio. By controlling the Fab-arm exchange using culture supernatants, the number of protein A purification steps is reduced by one, eliminating the need for two rounds of parental antibody purification and characterization, saving time, and thus reducing the cost of generating bispecific antibodies.

[0419] bsRSV-L[TLQ] and bsFSV-L[Q311R] were generated using Fab-arm exchange in cell culture supernatants containing equimolar amounts of parental antibodies RSV-L[TLQ] and gp120-R, and the resulting samples were applied to a protein A affinity column.

[0420] Figure 5A shows the protein A chromatogram of a sample of bsRSV-L[TLQ] with Fab-arm exchange in the supernatant, showing three different peaks eluting at pH 4.7, 4.2, and 3.4.

[0421] Figure 5B shows the HIC analysis of the eluate at pH 4.7 from a protein A affinity column of a sample from bsRSV-L[TLQ] with Fab-arm exchange in the supernatant.

[0422] Figure 5C shows the HIC analysis of the eluate at pH 4.2 from a protein A affinity column of a sample from bsRSV-L[TLQ] with Fab-arm exchange in the supernatant.

[0423] Figure 5D shows the HIC analysis of the eluate at pH 3.4 from a protein A affinity column of a sample from bsRSV-L[TLQ] with Fab-arm exchange in the supernatant.

[0424] Figure 6A shows a Protein A chromatogram of a sample of bsRSV-L[Q311R] with Fab arm exchange in the supernatant, showing three different peaks eluting at pH 4.7, 4.2, and 3.4.

[0425] Figure 6B shows a HIC analysis of the eluate at pH 4.7 from a Protein A affinity column of a sample from bsRSV-L[Q311R] with Fab arm exchange in the supernatant.

[0426] Figure 6C shows a HIC analysis of the eluate at pH 4.2 from a Protein A affinity column of a sample from bsRSV-L[Q311R] with Fab arm exchange in the supernatant.

[0427] Figure 6D shows a HIC analysis of the eluate at pH 3.4 from a Protein A affinity column of a sample from bsRSV-L[Q311R] with Fab arm exchange in the supernatant.

[0428] In the supernatant Fab arm exchange for preparing bsRSV-L[TLQ], the remaining parental RSV-L[TLQ] was removed by eluting at pH 4.7 (Figure 5B), and the remaining parental gp120-R was removed by eluting at pH 3.4 (Figure 5D). At pH 4.2, only bsRSV-L[TLQ] eluted (Figure 5C). Some bsRSV-L[TLQ] eluted at pH 3.4 (Figure 5D) and pH 4.7 (Figure 5B), leading to a decrease in the final yield of purified bsRSV-L[TLQ]. Table 13 shows the purity of the eluate by differential Protein A purification of bsRSV-L[TLQ] prepared by supernatant Fab arm exchange. bsRSV-L[TLQ] was isolated with a purity of over 95%.

[0429]

Table 13

[0430] In the supernatant for the production of bsRSV-L[Q311R] by Fab-arm exchange, the remaining parental RSV-L[Q311R] was removed by elution at pH 4.6 (Figure 6B), and the remaining parental gp120-R was removed by elution at pH 3.4 (Figure 6D). Due to the stronger binding of the single mutation to Protein A than that of the triple mutant T307P / L309Q / Q311R, a slightly more acidic pH was required for the efficient elution of the parental RSV-L[Q311R]. At pH 4.2, only the BsAb was eluted (Figure 6C). Some bsRSV-L[Q311R] was eluted at pH 3.4 (Figure 6D) and pH 4.6 (Figure 6B), leading to a decrease in the final yield of the purified BsAb.

[0431] Table 14 shows the purity of the eluate by differential Protein A purification of bsRSV-L[Q311R] prepared by Fab-arm exchange in the supernatant. bsRSV-L[Q311R] was purified with a purity of over 95%.

[0432]

Table 14

[0433] In conclusion, this experiment demonstrated the usefulness of the Q311R and T307P / L309Q / Q311R mutations for the efficient separation of bispecific antibodies prepared by Fab-arm exchange in the supernatant.

[0434] Method The parental mAbs RSV-L[Q311R] or RSV-L[TLQ] and gp120-R were expressed in Expi293 cells, and the antibody titers were determined (Octet, ForteBio). To generate the bispecific antibodies bsRSV-L[Q311R] and bsRSV-L[TLQ], equal milligram amounts of the culture supernatants containing RSV-L[Q311R] and gp120-R, or RSV-L[TLQ] and gp120-R were combined, 2-mercaptoethylamine was added to a final concentration of 75 mM, and the mixture was then incubated at 31 °C for 5 h and dialyzed extensively against 1×DPBS (pH 7.4) to perform a Fab-arm exchange reaction at a final protein concentration of 0.2 mg / mL (Labrijn Aran F, Meesters Joyce I et al. 2014). After dialysis, the protein was applied to a 1 mL mAbSelect Sure column (GE) and eluted using a pH step gradient.

[0435] Prior to purification, optimal elution conditions were determined by separating a control mixture containing 1 mg equivalent of purified parental mAb and bispecific antibody on a 1 mL mAbSelect Sure column (GE). The purified protein mix containing RSV-L[Q311R], gp120-R, and bsRSV-L[Q311R] showed optimal separation of the parental mAb from the BsAb by eluting over 30 column volumes (CV) with 50 mM citrate (pH 4.6), then over 30 CV with 50 mM citrate (pH 4.2), and then over 20 CV with 50 mM citrate (pH 3.4). The purified protein mix containing RSV-L[TLQ], gp120-R, and bsRSV-L[TLQ] showed optimal separation of the parental mAb from the bispecific mAb by eluting over 30 CV with 50 mM citrate (pH 4.7), then over 30 CV with 50 mM citrate (pH 4.2), and then over 20 CV with 50 mM citrate (pH 3.4).

[0436] Therefore, bsRSV-L[Q311R] prepared from the parental mAb cross-linked in the supernatant was eluted in subsequent experiments with 50 mM citrate (pH 4.6) over 30 CV, then with 50 mM citrate (pH 4.2) over 30 CV, and then with 50 mM citrate (pH 3.4) over 20 CV. bsRSV-L[TLQ] prepared from the parental mAb cross-linked in the supernatant was eluted in subsequent experiments with 50 mM citrate (pH 4.7) over 30 CV, then with 50 mM citrate (pH 4.2) over 30 CV, and then with 50 mM citrate (pH 3.4) over 20 CV. The optimal elution conditions for each pair of bispecific antibodies were used in subsequent experiments.

[0437] Hydrophobic interaction chromatography was used to evaluate the separation efficiency. The elution fractions from each pH step were pooled, neutralized with Tris (pH 7.5), and concentrated for analysis. Samples were prepared at equal protein concentrations, diluted 1:2 in binding buffer (0.1 M NaHPO4 (pH 6.5), 1.5 M (NH4)2SO4), and applied to a 4.6 mm × 10 cm TSKgel Butyl-NPR column (Tosoh Bioscience, LLC) equilibrated with 0.1 M NaHPO4 (pH 6.5), 1.5 M (NH4)2SO4, and eluted at 0.5 mL / min using a gradient to 0.1 M NaHPO4 (pH 6.5) over 25 minutes.

[0438] Example 7. Separation of bispecific antibodies from parental monospecific mAbs after in vitro Fab arm exchange starting from co-transfected materials The usefulness of utilizing the Q311R or T307P / L309Q / Q311R mutations was evaluated for the purification of bispecific antibodies prepared using the common light chain technology instead of Fab arm exchange.

[0439] The prepared bispecific antibodies bsTNF-[TLQ] and bsTNF-[Q311R] were isolated to a purity of over 95% using the three-step pH elution method described in the above examples. bsTNF-[TLQ] and bsTNF-[Q311R] eluted at pH 4.2. Furthermore, the parental TNF-[TLQ] and TNF-[Q311R] efficiently eluted at pH 4.7, and no mAb was detected in other eluents. The purity of bsTNF[TLQ] and bsTNF[Q311R] isolated from the eluate at pH 4.2 was high, but the yields of these bispecific antibodies were slightly lower compared to the bispecific antibodies prepared using Fab arm exchange because the expression levels of the two parental mAbs were significantly different when co-transfected (about 300 mg / L for the parental TNF-[TLQ] versus about 35 mg / L for aVb5). Despite a difference of about 10-fold in the expression levels of the parental mA, the introduction of the T307P / L309Q / Q311R mutation promoted the isolation of bsTNF-[TLQ], which accounted for only about 10% of the total antibody population in the initial sample, to a purity of over 95%. Table 15 shows the purity of the eluate obtained from the secondary protein A purification of bsTNF-[TLQ] prepared using the common light chain technology. Table 16 shows the purity of the eluate obtained from the secondary protein A purification of bsTNF-[Q311R] prepared using the common light chain technology.

[0440] Figure 7A shows the protein A chromatogram of a sample of bsTNF-[TLQ] prepared using the common light chain technology, showing three different peaks eluting at pH 4.7, 4.2, and 3.4.

[0441] Figure 7B shows the HIC analysis of the eluate at pH 4.7 of the protein A affinity column of a sample of bsTNF-[TLQ] prepared using the common light chain technology.

[0442] Figure 7C shows the HIC analysis of the eluate at pH 4.2 of the protein A affinity column of a sample of bsTNF-[TLQ] prepared using the common light chain technology.

[0443] Figure 7D shows the HIC analysis of the eluate at pH 3.4 of a protein A affinity column of a bsTNF-[TLQ] sample prepared using the common light chain technology.

[0444] A similar chromatogram was obtained from a sample of bsTNF-[Q311R] prepared using the common light chain technology.

[0445] [Table 15]

[0446] [Table 16]

[0447] Method The parental antibodies TNF-[Q311R], TNF-[TLQ] and aVb5 (see Table 8) were used in the experiments. The parental anti-TNF antibody and anti-αVβV antibody share a common light chain, and thus, by using these mAbs in the experiments, the mAb species that could be caused by light chain mispairing were minimized.

[0448] Co-transfection of TNF-[Q311R] and aVb5, or TNF-[TLQ] and aVb5 was performed in Expi293 cells according to the manufacturer's protocol using a molar ratio of plasmid of heavy chain of TNF-[Q311R] or TNF-[TLQ]:heavy chain of aVb5:light chain of 0.5:0.5:3.0. To determine the approximate relative expression levels, separate transfections of the parental mAbs were also performed using a molar ratio of plasmid of heavy chain:light chain of 1.0:3.0, and the titers were determined using Octet. Approximately 50 mL of each supernatant was applied to a 1 mL mAbSelect Sure column and eluted using a three-step pH gradient of 50 mM citrate at pH 4.7 (or 4.6), 4.2, and 3.4. The fractions were collected, concentrated, buffer-exchanged into 1×PBS, and then subjected to HIC analysis.

[0449] Example 8. The Q311R or T307P / L309Q / Q311R mutations do not affect the serum half-life of the antibody The PK properties of the selected antibodies were examined using Tg32 hemizygous mice. In these experiments, RSV-L had a half-life of approximately 7 days. The homodimeric parental Abs (antibodies RSV-L[Q311R] and RSV-L[TLQ]) having either the Q311R or T307P / L309Q / Q311R mutation both had half-lives at least as long as that of the wild-type mAb (approximately 7 days and 9 days, respectively). These mutations had little effect on the serum half-life even when introduced asymmetrically into the bispecific antibody. bsRSV-L[Q311R] and bsRSV-L[TLQ] had serum half-lives of 11 / 1 ± 3.6 days and 4.8 ± 2.0 days, respectively. The serum half-life of RSV-L was 7.0 ± 3.9 days, the half-life of RSV-L[TLQ] was 9.0 ± 4.0 days, and the serum half-life of RSV-L[Q311R] was 6.7 ± 3.4 days. The I253D mutant Ab did not bind to FcRn and was used as a control in this experiment.

[0450] Figure 8 shows the results of the pharmacokinetic analysis of the selected mutants. These results are consistent with the in vitro FcRn binding analysis (Example 3). These experimental results indicate that the Q311R or T307P / L309Q / Q311R mutations introduced asymmetrically into bispecific antibodies produced using a wide range of techniques retain a normal serum half-life and give rise to antibodies that allow differential protein A affinity purification of the bispecific antibody from the contaminating parental monospecific antibodies.

[0451] Method Tg32 hemizygous mice (Jackson Laboratories stock number 014565) were used for the pharmacokinetic (PK) studies of the antibodies. These mice are transgenic for the human α-microglobulin subunit of FcRn and thus useful for predicting serum half-life in humans (Petkova et al., Int Immunol 18:1759-1769, 2006). Mice were intravenously injected via the tail vein with a dose of 2 mg / kg of the test Ab into four animals per group. Blood samples were taken at 1 h, 1 d, 3 d, 7 d, 14 d, and 21 d. At the indicated times, serial blood collections were performed from the retro-orbital venous plexus of mice under CO2 anesthesia, and the final blood was collected by cardiac puncture. After 30 min at room temperature, the blood samples were centrifuged at 3,000×g for 15 min, and serum for analysis was collected.

[0452] An electrochemiluminescence immunoassay was used to detect the test Ab in mouse serum. A streptavidin gold multiarray 96-well plate (Meso Scale Discovery) was coated overnight with 50 μL / well of 3 μg / mL biotin-F(ab’)2 fragment g anti-h IgG (Fc fragment specific) (Jackson Immunoresearch catalog number 109-066-008) in Starting Block (Thermo) and then washed with Tris-buffered saline containing Tween 20 (TBST). Serum samples were diluted in 5% CD-1 mouse serum in Starting Block (1:20 and then serially diluted 2-fold), incubated on the plate for 2 h, and then washed. Ru ++ labeled anti-h IgG F(ab’)2 (prepared from Jackson 109-006-097) was added, incubated on the plate for 1.5 h, and then washed. Read Buffer containing surfactant was added at 200 μL / well, and the plate was read on an MSD Sector Imager 6000 plate reader. The serum concentration of the IgG2b Ab was determined from the standard curve using the 4-parameter non-linear regression program of Prism 6.01 software.

[0453] Using the one-phase exponential decay model fitted by non-linear regression of natural logarithm concentration against time with Prism version 6.01 software, the calculated value of the terminal half-life (t 1 / 2 ) in the elimination phase (β-phase) of the PK test was determined. The least-squares non-linear decay model was weighted by the reciprocal of the fitted concentration. The formula t 1 / 2 = ln2 / β (where β is the slope of the straight line fitted by regression analysis using the least-squares method starting after the first administration) was used to determine the calculated value of the half-life in the elimination phase (β-phase). The terminal half-life value of Ab was determined by taking the average of the t 1 / 2 values calculated for each animal within the test group. The inventions described in the original claims of the present application are listed below. [Invention 1] An isolated multispecific antibody comprising a first CH2-CH3 region having mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R, or T307P / V309Q / Q311R, and a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309, and 311, with residue numbering according to the EU index. [Invention 2] The isolated multispecific antibody according to Invention 1, wherein the antibody is of the IgG1, IgG2, or IgG4 isotype. [Invention 3] The isolated multispecific antibody according to Invention 1 or 2, wherein the binding of the first CH2-CH3 region to the protein A ligand is reduced compared to the second CH2-CH3 region. [Invention 4] The isolated multispecific antibody according to Invention 3, wherein the protein A ligand comprises protein A, Z domain, or Y domain of Staphylococcus aureus. [Invention 5] The isolated multispecific antibody according to Invention 4, wherein the Z domain has the amino acid sequence of SEQ ID NO: 1. [Invention 6] The isolated multispecific antibody according to any one of Inventions 1 to 5, further comprising asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region. [Invention 7] The asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are a) F405L and K409R, respectively, b) wild-type and F405L / R409K, respectively, c) T366W and T366S / L368A / Y407V, respectively, d) T366Y / F405A and T394W / Y407T, respectively, e) T366W / F405W and T394S / Y407A, respectively, f) F405W / Y407A and T366W / T394S, respectively, g) L351Y / F405A / Y407V and T394W, respectively, h) respectively, T366I / K392M / T394W and F405A / Y407V, i) respectively, T366L / K392M / T394W and F405A / Y407V, j) respectively, L351Y / Y407A and T366A / K409F, k) respectively, L351Y / Y407A and T366V / K409F, l) respectively, Y407A and T366A / K409F, m) respectively, D399K / E356K and K409D / K392D, or n) respectively, D399K / E356K / E357K and K409D / K392D / K370 and is the isolated multispecific antibody according to Invention 6. [Invention 8] The first CH2-CH3 region and the second CH2-CH3 region are a) respectively, SEQ ID NO: 2 and 22, b) respectively, SEQ ID NO: 3 and 22, c) respectively, SEQ ID NO: 4 and 22, d) respectively, SEQ ID NO: 5 and 22, e) respectively, SEQ ID NO: 6 and 23, f) respectively, SEQ ID NO: 7 and 23, g) respectively, SEQ ID NO: 8 and 23, h) respectively, SEQ ID NO: 9 and 23, i) respectively, SEQ ID NO: 10 and 24, j) respectively, SEQ ID NO: 11 and 24, k) respectively, SEQ ID NO: 12 and 24, l) respectively, SEQ ID NO: 13 and 24, m) respectively, SEQ ID NO: 14 and 25, n) respectively, SEQ ID NO: 15 and 25, o) respectively, SEQ ID NO: 16 and 25, p) respectively, SEQ ID NO: 17 and 25, q) respectively, SEQ ID NO: 18 and 26, r) respectively, SEQ ID NO: 19 and 26, s) respectively, SEQ ID NO: 20 and 26, t) respectively, SEQ ID NO: 21 and 26, u) respectively, SEQ ID NO: 52 and 54, v) respectively, SEQ ID NO: 52 and 55, w) respectively, SEQ ID NO: 53 and 54, x) respectively, SEQ ID NO: 53 and 55, y) respectively, SEQ ID NO: 56 and 54, or z) respectively, SEQ ID NO: 56 and 55 and has the amino acid sequences of any one of Inventions 1 to 7. [Invention 9] The isolated multispecific antibody according to any one of Inventions 1 to 8, further comprising at least one mutation that modulates the binding of the antibody to FcγR or FcRn. [Invention 10] The at least one mutation that modulates the binding of said antibody to FcγR or FcRn is L234A, F234A, V234A, L235A, G237A, P238S, H268A, V309L, A330A, P331S, L234A / L235A, F234A / L235A, V234A / L235A, V234A / G237A / P238S / H268A / V309L / A330S / P331S, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, G236A / S239D / I332E, S267E, S267E / L328F, S267E / I332E or M252Y / S254T / T256E, an isolated multispecific antibody according to invention 9. [Invention 11] An isolated multispecific antibody according to any one of inventions 1 to 10, comprising a first light chain and a second light chain. [Invention 12] The isolated multispecific antibody according to invention 11, wherein the first light chain and the second light chain have the same amino acid sequence. [Invention 13] The isolated multispecific antibody according to any one of inventions 1 to 12, wherein the isolated multispecific antibody binds to two or more antigens. [Invention 14] The two antigens are any two of PD1, CD27, CD28, NKP46, ICOS, GITR, OX40, CTLA4, LAG3, TIM3, KIRa, CD73, CD39, IDO, BTLA, VISTA, TIGIT, CD96, CD30, HVEM, DNAM-1, LFA, tumor antigen, EGFR, cMet, FGFR, ROR1, CD123, IL1RAP, FGFR, mesothelin, CD3, T cell receptor, CD32b, CD32a, CD16a, CD16b, NKG2D, NKP46, CD28, CD47, DLL, CD8, CD89, HLA, B cell receptor, or CD137, an isolated multispecific antibody according to invention 13. [Invention 15] The isolated multispecific antibody according to any one of inventions 1 to 14, which is a bispecific antibody. [Invention 16] A pharmaceutical composition comprising the isolated multispecific antibody according to any one of inventions 1 to 15. [Invention 17] a) a polynucleotide encoding the first CH2-CH3 region having mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R, b) a polynucleotide encoding the first CH2-CH3 region having mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R and the second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, c) an isolated polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 87, 88 or 91. [Invention 18] a) the isolated polynucleotide encoding the first CH2-CH3 region having mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R, b) the isolated polynucleotide having a polynucleotide sequence of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 87, 88 or 91, c) the isolated polynucleotide comprising a polynucleotide encoding the first CH2-CH3 region having mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R and the second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, d) i) SEQ ID NO: 27 and 47, respectively, ii) SEQ ID NO: 28 and 47, respectively, iii) SEQ ID NO: 29 and 47, respectively, iv) SEQ ID NO: 30 and 47, respectively, v) SEQ ID NO: 31 and 48, respectively, vi) SEQ ID NO: 32 and 48, respectively, vii) SEQ ID NO: 33 and 48, respectively, viii) SEQ ID NO: 34 and 48, respectively, ix) SEQ ID NO: 35 and 49, respectively, x) SEQ ID NO: 36 and 49, respectively, xi) SEQ ID NO: 37 and 49, respectively, xii) SEQ ID NOs: 38 and 49, respectively, xiii) SEQ ID NOs: 39 and 50, respectively, xiv) SEQ ID NOs: 40 and 50, respectively, xv) SEQ ID NOs: 41 and 50, respectively, xvi) SEQ ID NOs: 42 and 50, respectively, xvii) SEQ ID NOs: 43 and 51, respectively, xviii) SEQ ID NOs: 44 and 51, respectively, xix) SEQ ID NOs: 45 and 51, respectively, xx) SEQ ID NOs: 46 and 51, respectively, xxi) SEQ ID NOs: 87 and 89, respectively, xxii) SEQ ID NOs: 87 and 90, respectively, xxiii) SEQ ID NOs: 88 and 89, respectively, xxiv) SEQ ID NOs: 88 and 90, respectively, xxv) SEQ ID NOs: 92 and 89, respectively, or xxvi) SEQ ID NOs: 92 and 90, respectively, the isolated polynucleotide having the same, and a vector comprising the same. [Invention 19] A host cell comprising the vector according to Invention 18. [Invention 20] The host cell according to Invention 19, wherein the host cell is a hybridoma, myeloma, SP2 / 0, NS0, U266, CHO, CHO-K1SV, CHO-K1, DG44 or Hek293. [Invention 21] A method for producing the isolated multispecific antibody according to Invention 1, comprising: a) culturing the host cell according to Invention 19 under conditions under which the multispecific antibody is expressed; and b) purifying the multispecific antibody using protein A ligand affinity chromatography. [Invention 22] A method for producing an isolated multispecific antibody comprising a first heavy chain or a fragment thereof having a mutation Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R and a second heavy chain or a fragment thereof having wild-type amino acid residues at positions 307, 309 and 311, comprising: a) providing a first parental antibody comprising the first heavy chain or a fragment thereof having the mutation Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R and a first light chain; b) providing a second parental antibody comprising the second heavy chain or a fragment thereof having wild-type amino acid residues at positions 307, 309 and 311 and a second light chain; and c) contacting the first parental antibody and the second parental antibody in a sample. d) incubating the sample; and e) purifying the multispecific antibody using protein A ligand affinity chromatography, a method comprising: [Inventive Step 23] The method according to Inventive Step 22, wherein the isolated multispecific antibody further has asymmetric stabilizing mutations in the first heavy chain or a fragment thereof and the second heavy chain or a fragment thereof. [Inventive Step 24] The asymmetric stabilizing mutations in the first heavy chain or a fragment thereof and the second heavy chain or a fragment thereof, or in the second heavy chain or a fragment thereof and the first heavy chain or a fragment thereof are a) F405L and K409R, respectively; b) wild type and F405L / R409K, respectively; c) T366W and T366S / L368A / Y407V, respectively; d) T366Y / F405A and T394W / Y407T, respectively; e) T366W / F405W and T394S / Y407A, respectively; f) F405W / Y407A and T366W / T394S, respectively; g) L351Y / F405A / Y407V and T394W, respectively; h) T366I / K392M / T394W and F405A / Y407V, respectively; i) T366L / K392M / T394W and F405A / Y407V, respectively; j) L351Y / Y407A and T366A / K409F, respectively; k) L351Y / Y407A and T366V / K409F, respectively; l) Y407A and T366A / K409F, respectively; m) D399K / E356K and K409D / K392D, respectively, or n) D399K / E356K / E357K and K409D / K392D / K370, respectively, the method according to Inventive Step 23. [Inventive Step 25] [Inventive Step 26] The method according to any one of Inventive Steps 22 to 24, wherein the isolated multispecific antibody is of the IgG1, IgG2 or IgG4 isotype. [Inventive Step 27] The method according to any one of Inventive Steps 22 to 25, wherein the first light chain and the second light chain have the same amino acid sequence. [Inventive Step 28] The method according to any one of Inventive Steps 22 to 25, wherein the first parent antibody and the second parent antibody are provided as purified antibodies. [Inventive Step 29] The method according to any one of Inventions 22 to 26, wherein the first parental antibody and the second parental antibody are provided in a cell culture medium recovered from cells expressing the first parental antibody and the second parental antibody. [Invention 29] The method according to Invention 22, wherein a reducing agent is added in step d). [Invention 30] The method according to Invention 29, wherein the reducing agent is 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, or β-mercaptoethanol. [Invention 31] The method according to Invention 30, wherein 2-MEA is present at a concentration of about 10 mM to about 100 mM. [Invention 32] The method according to Invention 31, wherein 2-MEA is present at a concentration of about 25 mM to about 75 mM. [Invention 33] The method according to Invention 22, wherein step d) is performed at a temperature of about 20°C to about 37°C for about 90 minutes to about 6 hours. [Invention 34] The method according to any one of Inventions 22 to 33, wherein protein A ligand affinity chromatography uses a pH gradient. [Invention 35] The method according to Invention 34, wherein the pH gradient is from about pH 7.0 to about pH 3.0. [Invention 36] The method according to Invention 34, wherein the pH gradient is from about pH 4.6 to about pH 3.4. [Invention 37] The method according to any one of Inventions 34 to 46, wherein the multimeric antibody elutes at about pH 4.4 to about pH 4.1. [Invention 38] The method according to any one of Inventions 34 to 37, wherein protein A ligand affinity chromatography uses a citrate buffer. [Invention 39] The method according to any one of Inventions 22 to 38, wherein the multispecific antibody is a bispecific antibody. [Invention 40] An isolated antibody comprising two heavy chains or fragments thereof having the same amino acid sequence and two light chains or fragments thereof, wherein the two heavy chains have the mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R, or T307P / V309Q / Q311R, and the residue numbering follows the EU index. [Invention 41] The isolated antibody according to Invention 40, wherein the two heavy chains or fragments thereof further have the mutations F405L, K409R, F405L / R409K, T366W, or T366S / L368A / Y407V. [Invention 42] The isolated antibody according to invention 40 or 41, wherein the antibody is of the IgG1, IgG2 or IgG4 isotype. [Invention 43] The isolated antibody according to any one of inventions 40 to 42, comprising the heavy chain CH2-CH3 region of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 52, 53 or 56. [Invention 44] a) Encoding an antibody heavy chain comprising the CH2-CH3 region of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 52, 53 or 56, or b) A polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 87, 88 or 91. [Invention 45] A vector comprising the polynucleotide according to invention 44. [Invention 46] A host cell comprising the vector according to invention 45. [Invention 47] A method for producing the isolated antibody according to invention 40, comprising culturing the host cell according to invention 46 under conditions under which the antibody is expressed. [Invention 48] A multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region having a mutation Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R, and the second polypeptide comprises a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, and the residue numbering follows the EU index. [Invention 49] The multimeric protein according to invention 48, wherein the first CH2-CH3 region and the second CH2-CH3 region are of the IgG1, IgG2 or IgG4 isotype. [Invention 50] The multimeric protein according to invention 48 or 49, wherein the binding of the first CH2-CH3 region to the protein A ligand is reduced as compared to the second CH2-CH3 region. [Invention 51] The multimeric protein according to invention 50, wherein the protein A ligand comprises protein A, Z domain or Y domain of Staphylococcus aureus. [Invention 52] The multimeric protein according to Invention 51, wherein the Z domain has the amino acid sequence of SEQ ID NO: 1. [Invention 53] The multimeric protein according to any one of Inventions 48 to 52, further having asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region. [Invention 54] The asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are a) F405L and K409R, respectively, b) wild type and F405L / R409K, respectively, c) T366W and T366S / L368A / Y407V, respectively, d) T366Y / F405A and T394W / Y407T, respectively, e) T366W / F405W and T394S / Y407A, respectively, f) F405W / Y407A and T366W / T394S, respectively, g) L351Y / F405A / Y407V and T394W, respectively, h) T366I / K392M / T394W and F405A / Y407V, respectively, i) T366L / K392M / T394W and F405A / Y407V, respectively, j) L351Y / Y407A and T366A / K409F, respectively, k) L351Y / Y407A and T366V / K409F, respectively, l) Y407A and T366A / K409F, respectively, m) D399K / E356K and K409D / K392D, respectively, or n) D399K / E356K / E357K and K409D / K392D / K370, respectively The multimeric protein according to Invention 53. [Invention 55] The first CH2-CH3 region and the second CH2-CH3 region are a) SEQ ID NO: 2 and SEQ ID NO: 22, respectively, b) SEQ ID NO: 3 and SEQ ID NO: 22, respectively, c) SEQ ID NO: 4 and SEQ ID NO: 22, respectively, d) SEQ ID NO: 5 and SEQ ID NO: 22, respectively, e) SEQ ID NO: 6 and SEQ ID NO: 23, respectively, f) SEQ ID NO: 7 and SEQ ID NO: 23, respectively, g) SEQ ID NO: 8 and SEQ ID NO: 23, respectively, h) SEQ ID NO: 9 and SEQ ID NO: 23, respectively, i) SEQ ID NO: 10 and SEQ ID NO: 24, respectively, j) SEQ ID NO: 11 and SEQ ID NO: 24, respectively, k) SEQ ID NO: 12 and SEQ ID NO: 24, respectively, l) SEQ ID NO: 13 and SEQ ID NO: 24, respectively, m) SEQ ID NO: 14 and SEQ ID NO: 25, respectively, n) SEQ ID NO: 15 and SEQ ID NO: 25, respectively, o) SEQ ID NO: 16 and SEQ ID NO: 25, respectively, p) SEQ ID NO: 17 and SEQ ID NO: 25, respectively, q) respectively, SEQ ID NO: 18 and 26, r) respectively, SEQ ID NO: 19 and 26, s) respectively, SEQ ID NO: 20 and 26, t) respectively, SEQ ID NO: 21 and 26, u) respectively, SEQ ID NO: 52 and 54, v) respectively, SEQ ID NO: 52 and 55, w) respectively, SEQ ID NO: 53 and 54, x) respectively, SEQ ID NO: 53 and 55, y) respectively, SEQ ID NO: 56 and 54, or z) respectively, SEQ ID NO: 56 and 55 A multimeric protein according to any one of Inventions 48 to 54, having the amino acid sequences. [Invention 56] The multimeric protein according to any one of Inventions 48 to 55, wherein the first CH2-CH3 region and / or the second CH2-CH3 region is bound to a heterologous protein. [Invention 57] The multimeric protein according to Invention 56, wherein the heterologous protein is a peptide, an extracellular domain of a receptor, an extracellular domain of a ligand, a secreted protein, an scFv, a Fab, a heavy chain variable region (VH), a light chain variable region (VL), a fibronectin type III domain and / or a finomer. [Invention 58] The multimeric protein according to Invention 57, wherein the heterologous protein is bound to the N-terminus or C-terminus of the first CH2-CH3 region and / or the second CH2-CH3 region, optionally via a linker. [Invention 59] The multimeric protein according to Invention 68, wherein the linker has the amino acid sequence of SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 92, 93, 94, 95, 96, 97 or 98. [Invention 60] The multimeric protein according to Invention 48, wherein the multimeric protein is an antibody. [Invention 61] The multimeric protein according to Invention 60, wherein the antibody is multispecific, bispecific or monospecific. [Invention 62] The multimeric protein according to any one of Inventions 48 to 61, comprising 2, 3 or 4 polypeptide chains. [Invention 63] A pharmaceutical composition comprising the multimeric protein according to any one of Inventions 48 to 62. [Invention 64] A method for producing an isolated multimeric protein comprising a first CH2-CH3 region having the mutations Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R and a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, a) providing a first parent protein comprising the first CH2-CH3 region having the mutation Q311R, Q311K, T307P / L309Q, T307P / V309Q, T307P / L309Q / Q311R or T307P / V309Q / Q311R; b) providing a second parent protein comprising the second CH2-CH3 region having the wild-type amino acid residues at positions 307, 309 and 311; c) contacting the first parent protein and the second parent protein in a sample; d) incubating the sample; e) purifying the multispecific protein using protein A ligand affinity chromatography; [Invention 65] The method according to invention 64, wherein the multimeric protein further has asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region. [Invention 66] The asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region, are a) F405L and K409R, respectively; b) wild-type and F405L / R409K, respectively; c) T366W and T366S / L368A / Y407V, respectively; d) T366Y / F405A and T394W / Y407T, respectively; e) T366W / F405W and T394S / Y407A, respectively; f) F405W / Y407A and T366W / T394S, respectively; g) L351Y / F405A / Y407V and T394W, respectively; h) T366I / K392M / T394W and F405A / Y407V, respectively; i) T366L / K392M / T394W and F405A / Y407V, respectively; j) L351Y / Y407A and T366A / K409F, respectively; k) L351Y / Y407A and T366V / K409F, respectively; l) Y407A and T366A / K409F, respectively; m) D399K / E356K and K409D / K392D, respectively, or n) D399K / E356K / E357K and K409D / K392D / K370, respectively The method according to invention 65. [Invention 67] The method according to any one of inventions 64 to 66, wherein the first CH2-CH3 region and the second CH2-CH3 region are of the IgG1, IgG2 or IgG4 isotype. [Invention 68] The first CH2-CH3 region and the second CH2-CH3 region are a) respectively, SEQ ID NO: 2 and 22, b) respectively, SEQ ID NO: 3 and 22, c) respectively, SEQ ID NO: 4 and 22, d) respectively, SEQ ID NO: 5 and 22, e) respectively, SEQ ID NO: 6 and 23, f) respectively, SEQ ID NO: 7 and 23, g) respectively, SEQ ID NO: 8 and 23, h) respectively, SEQ ID NO: 9 and 23, i) respectively, SEQ ID NO: 10 and 24, j) respectively, SEQ ID NO: 11 and 24, k) respectively, SEQ ID NO: 12 and 24, l) respectively, SEQ ID NO: 13 and 24, m) respectively, SEQ ID NO: 14 and 25, n) respectively, SEQ ID NO: 15 and 25, o) respectively, SEQ ID NO: 16 and 25, p) respectively, SEQ ID NO: 17 and 25, q) respectively, SEQ ID NO: 18 and 26, r) respectively, SEQ ID NO: 19 and 26, s) respectively, SEQ ID NO: 20 and 26, t) respectively, SEQ ID NO: 21 and 26, u) respectively, SEQ ID NO: 52 and 54, v) respectively, SEQ ID NO: 52 and 55, w) respectively, SEQ ID NO: 53 and 54, x) respectively, SEQ ID NO: 53 and 55, y) respectively, SEQ ID NO: 56 and 54, or z) respectively, SEQ ID NO: 56 and 55 and have the amino acid sequences of any one of inventions 64 to 67. [Invention 69] The method according to any one of inventions 64 to 68, wherein the first CH2-CH3 region and / or the second CH2-CH3 region is bound to a heterologous protein. [Invention 70] The method according to invention 69, wherein the heterologous protein is a peptide, an extracellular domain of a receptor, an extracellular domain of a ligand, a secreted protein, an scFv, a Fab, a heavy chain variable region (VH), a light chain variable region (VL), a fibronectin type III domain and / or a finomer. [Invention 71] The method according to invention 69 or 70, wherein the heterologous protein is bound to the N-terminus or C-terminus of the first CH2-CH3 region and / or the second CH2-CH3 region, optionally via a linker. [Invention 72] The method according to invention 71, wherein the linker has the amino acid sequence of SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 92, 93, 94, 95, 96, 97 or 98. [Invention 73] The method according to any one of inventions 64 to 72, wherein the first parent protein and the second parent protein are provided as purified proteins. [Invention 74] The method according to any one of inventions 64 to 72, wherein the first parent protein and the second parent protein are provided in a cell culture medium recovered from cells expressing the first parent protein and the second parent protein. [Invention 75] The method according to invention 64, wherein a reducing agent is added in step d). [Invention 76] The method according to invention 75, wherein the reducing agent is 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine or β-mercaptoethanol. [Invention 77] The method according to invention 76, wherein 2-MEA is present at a concentration of about 10 mM to about 100 mM. [Invention 78] The method according to invention 76, wherein 2-MEA is present at a concentration of about 25 mM to about 75 mM. [Invention 79] The method according to invention 64, wherein step d) is carried out at a temperature of about 25°C to about 37°C for about 90 minutes to about 6 hours.

Claims

**Claim 1** An isolated multispecific antibody, wherein the antibody is of the IgG1, IgG2 or IgG4 isotype, wherein when the antibody is of the IgG1 or IgG4 isotype, the antibody comprises a first CH2-CH3 region having the mutations Q311R, or T307P / L309Q / Q311R, and a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, wherein when the antibody is of the IgG2 isotype, the antibody comprises a first CH2-CH3 region having the mutations Q311R, or T307P / V309Q / Q311R, and a second CH2-CH3 region having wild-type amino acid residues at positions 307, 309 and 311, the isolated multispecific antibody, wherein residue numbering follows the EU index. **Claim 2** The isolated multispecific antibody according to claim 1, wherein binding of the first CH2-CH3 region to a protein A ligand is reduced compared to the second CH2-CH3 region, the protein A ligand comprising protein A, Z domain or Y domain of Staphylococcus aureus, and the Z domain having the amino acid sequence of SEQ ID NO:

1. **Claim 3** The antibody further comprises asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, and the asymmetric stabilizing mutations in the first CH2-CH3 region and the second CH2-CH3 region, or in the second CH2-CH3 region and the first CH2-CH3 region are i) F405L and K409R, respectively, ii) wild-type and F405L / R409K, respectively, iii) T366W and T366S / L368A / Y407V, respectively, iv) T366Y / F405A and T394W / Y407T, respectively, v) T366W / F405W and T394S / Y407A, respectively, vi) F405W / Y407A and T366W / T394S, respectively, vii) L351Y / F405A / Y407V and T394W, respectively, viii) T366I / K392M / T394W and F405A / Y407V, respectively, ix) T366L / K392M / T394W and F405A / Y407V, respectively, x) L351Y / Y407A and T366A / K409F, respectively, xi) L351Y / Y407A and T366V / K409F, respectively, xii) Y407A and T366A / K409F, respectively, xiii) D399K / E356K and K409D / K392D, respectively, or xiv) D399K / E356K / E357K and K409D / K392D / K370, respectively, the isolated multispecific antibody according to claim 1.

4. The first CH2-CH3 region and the second CH2-CH3 region are i) SEQ ID NO: 2 and SEQ ID NO: 22, respectively, ii) SEQ ID NO: 3 and SEQ ID NO: 22, respectively, iii) SEQ ID NO: 4 and SEQ ID NO: 22, respectively, iv) SEQ ID NO: 5 and SEQ ID NO: 22, respectively, v) SEQ ID NO: 6 and SEQ ID NO: 23, respectively, vi) SEQ ID NO: 7 and SEQ ID NO: 23, respectively, vii) SEQ ID NO: 8 and SEQ ID NO: 23, respectively, viii) SEQ ID NO: 9 and SEQ ID NO: 23, respectively, ix) SEQ ID NO: 10 and SEQ ID NO: 24, respectively, x) SEQ ID NO: 11 and SEQ ID NO: 24, respectively, xi) SEQ ID NO: 12 and SEQ ID NO: 24, respectively, xii) SEQ ID NO: 13 and SEQ ID NO: 24, respectively, xiii) SEQ ID NO: 14 and SEQ ID NO: 25, respectively, xiv) SEQ ID NO: 15 and SEQ ID NO: 25, respectively, xv) SEQ ID NO: 16 and SEQ ID NO: 25, respectively, xvi) SEQ ID NO: 17 and SEQ ID NO: 25, respectively, xvii) SEQ ID NO: 18 and SEQ ID NO: 26, respectively, xviii) SEQ ID NO: 19 and SEQ ID NO: 26, respectively, xix) SEQ ID NO: 20 and SEQ ID NO: 26, respectively, xx) SEQ ID NO: 21 and SEQ ID NO: 26, respectively, xxi) SEQ ID NO: 52 and SEQ ID NO: 54, respectively, xxii) SEQ ID NO: 52 and SEQ ID NO: 55, respectively, xxiii) SEQ ID NO: 53 and SEQ ID NO: 54, respectively, xxiv) SEQ ID NO: 53 and SEQ ID NO: 55, respectively, xxv) SEQ ID NO: 56 and SEQ ID NO: 54, respectively, or xxvi) the isolated multispecific antibody according to claim 1, having the amino acid sequences of SEQ ID NO: 56 and SEQ ID NO: 55, respectively. **Claim 5**: The isolated multispecific antibody further comprises at least one mutation that modulates the binding of the antibody to FcγR or FcRn, and the at least one mutation that modulates the binding of the antibody to FcγR or FcRn is L234A, F234A, V234A, L235A, G237A, P238S, H268A, V309L, A330A, P331S, L234A / L235A, F234A / L235A, V234A / L235A, V234A / G237A / P238S / H268A / V309L / A330S / P331S, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, G236A / S239D / I332E, S267E, S267E / L328F, S267E / I332E or M252Y / S254T / T256E. The isolated multispecific antibody according to claim 1. **Claim 6**: The antibody comprises a first light chain and a second light chain, and the first light chain and the second light chain have the same amino acid sequence. The isolated multispecific antibody according to any one of claims 1 to 5. **Claim 7**: The isolated multispecific antibody binds to two or more antigens, and the two antigens are any two of PD1, CD27, CD28, NKP46, ICOS, GITR, OX40, CTLA4, LAG3, TIM3, KIRa, CD73, CD39, IDO, BTLA, VISTA, TIGIT, CD96, CD30, HVEM, DNAM-1, LFA, tumor antigen, EGFR, cMet, FGFR, ROR1, CD123, IL1RAP, FGFR, mesothelin, CD3, T cell receptor, CD32b, CD32a, CD16a, CD16b, NKG2D, NKP46, CD28, CD47, DLL, CD8, CD89, HLA, B cell receptor, or CD137. The isolated multispecific antibody according to any one of claims 1 to 6. **Claim 8**: The antibody is a bispecific antibody. The isolated multispecific antibody according to any one of claims 1 to 7.

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