Mutant promoting homologous pairing of heavy and light chains of multispecific antibody

Abstract

The present invention relates to a multispecific antibody, such as a bispecific antibody, comprising a mutated amino acid pair capable of promoting homologous pairing of a heavy chain and a light chain of the multispecific antibody.

Description

Mutants that promote homologous pairing of heavy and light chains of multispecific antibodies

[0001] The present invention relates to a multispecific antibody, such as a bispecific antibody, comprising a mutated amino acid pair that can promote cognate pairing of the heavy chain and the light chain of the multispecific antibody.

[0002] Background of the invention:

[0003] Antibodies dominate the current biotherapeutic market. Bispecific antibodies (bsAbs) are single molecules that simultaneously bind to two different antigens or two distinct epitopes on the same antigen; they differ from naturally occurring monospecific immunoglobulin G (IgG) antibodies (mAbs). Due to their additional targeting capabilities, bsAbs often offer improved clinical benefits in treating complex diseases, such as cancer and immune disorders, that involve multiple cell surface receptors or ligands. Numerous efforts have been made to engineer mAbs into bsAbs, resulting in over 60 different bsAb formats. Many bsAbs can be engineered by linking antibody fragments, such as single-chain variable fragments (scFv), antigen-binding fragments (Fab), and heavy (VH) and light (VL) chain variable domains. However, these novel formats, unlike conventional IgG structures, often exhibit inferior physicochemical properties, such as low solubility and aggregation, difficulties in large-scale production, poor thermal stability, poor pharmacokinetics, and potential immunogenicity. The significant production challenges of bispecific antibodies in terms of quantity, quality, and stability have hindered their wider clinical application and acceptance.

[0004] Heterodimeric Fc technology has been developed to facilitate the production of bispecific antibodies. For example, Carter et al. used the "Kih" (knobs-into-holes) model to modify some of the amino acids in the antibody heavy chain, achieving relatively successful bispecific antibodies (Ridgway, Presta et al. 1996; Carter 2001). They created a "knob" (e.g., T366Y) in the CH3 region of the first Fc heavy chain by mutating an amino acid with a small side chain to one with a large side chain. They also created a "hole" (e.g., Y407T) by mutating certain amino acids in the CH3 region of the second heavy chain to amino acids with small side chains. This exploits spatial conformational complementarity to achieve heterodimer formation (WO9627011). Subsequently, the proportion of heterodimers was further increased through random mutagenesis-phage display and other methods (WO1998050431), and disulfide bonds were introduced in the CH3 region to increase the heterodimerization. However, the ability to hinder the formation of homodimers was still insufficient, and the overall heterodimer ratio was about 70-80%.

[0005] Alternatively, chain exchange engineered domain (SEED) heterologous design represents another steric mutagenesis-based design strategy, utilizing interchangeable amino acid sequences between the CH3 domains of IgG and IgA (AG SEED CH3 and GA SEED CH3) to form complementary structures. IgG and IgA CH3 derivatives produce complementary sequences, allowing the two complementary heavy chains to assemble into heterodimers, thus avoiding the potential for homodimerization due to lack of complementarity (Muda, M et al. Therapeutic assessment of SEED: A new engineered antibody platform designed to generate mono- and bispecific antibodies. Protein Eng. Des. Sel. PEDS 2011, 24, 447–454.).

[0006] In addition to the earlier proposed steric mutations, electrostatic interactions have also been widely used to promote heavy chain heterodimer formation. This approach involves mutating individual amino acids in the CH3 domain of one heavy chain to positively charged Lys, while replacing individual amino acids in the CH3 domain of the other heavy chain with negatively charged Asp or Glu. The charged amino acids then readily form heterodimers through electrostatic attraction. Gunasekaran et al. first introduced charged amino acids to form Fc heterodimers in a bispecific antibody (Gunasekaran, K. et al. Enhancing antibody Fc heterodimer formation through electrostatic steering elements: applications to bispecific molecules and monovalent IgG. J. Biol. Chem. 2010, 285, 19637–19646).

[0007] CN104011221B provides a bispecific antibody with modified polypeptide chains containing disulfide bond remodeling mutations in both the variable and constant regions. However, these mutations are insufficient to achieve over 90% correct pairing of bispecific antibodies expressed in a single cell line. The amino acid pairs selected for disulfide bond remodeling in this patent are suboptimal, resulting in a portion of molecules failing to form disulfide bonds.

[0008] CN109475627A provides a method for preparing an antibody mixture, but it involves the preparation of a mixture of two monospecific antibodies, and charge mutations are mainly introduced in the constant region, which requires the introduction of a large number of mutations to achieve optimized pairing effects.

[0009] Therefore, there is still a need in the art for new bispecific antibody platforms that only require the introduction of fewer mutations so that the bispecific antibodies obtained therefrom have lower mismatches during expression while maintaining unchanged affinity and expression levels. At the same time, the immunogenicity of the obtained bispecific antibodies is reduced because the bispecific antibodies have fewer mutations, and the expression and recombination of antibodies of the bispecific antibody platform can be carried out in a single cell line.

[0010] Summary of the Invention

[0011] The present invention provides a novel multispecific antibody (e.g., novel bispecific antibody) platform, which introduces charge mutations in the variable region of the bispecific antibody and simultaneously introduces disulfide bond remodeling mutations in the CH1 / CL region, thereby obtaining a multispecific antibody.

[0012] (1) Fewer mismatches and a higher proportion of correctly paired molecules;

[0013] (2) has an immunogenicity risk comparable to that of natural IgG;

[0014] (3) the affinity is similar to or remains unchanged compared to the antibody before the mutation was introduced;

[0015] (4) having a higher expression level, for example, an expression level comparable to or higher than that of the antibody before the introduction of the mutation or a monospecific antibody;

[0016] (5) It can be expressed in one cell line, avoiding expression in different cell lines and in vitro recombination.

[0017] At the same time, the mutation positions and amino acids introduced into the multispecific antibody platform of the present invention are relatively conservative, and therefore are applicable to a variety of multispecific antibodies (bispecific antibodies).

[0018] Description of the accompanying drawings:

[0019] FIG1 shows an exemplary bispecific antibody structure (monovalent 1+1 configuration) and mutations that may be included therein.

[0020] Figure 2 shows the correct assembled molecule ratios of the mutation combinations shown in Table 7 analyzed by LC-MS, wherein A: heavy chain of antibody 1; a: light chain of antibody 1; B: heavy chain of antibody 2; b: light chain of antibody 2; AaBb: correctly matched bispecific antibodies;

[0021] Figure 2A: LC-MS results of combination 1 in Table 7;

[0022] Figure 2B: LC-MS results of combination 2 in Table 7

[0023] Figure 2C: LC-MS results of combination 3 in Table 7.

[0024] Figure 3 shows the proportion of correctly assembled molecules analyzed by LC-MS after the introduction of disulfide bond remodeling and charge mutations into the different antibody pairs shown in Table 8, where A: heavy chain of antibody 1; a: light chain of antibody 1; B: heavy chain of antibody 2; b: light chain of antibody 2; AaBb: correctly paired bispecific antibodies.

[0025] Figure 3A: Table 8 LC-MS results of Her2 / PD-1 combination.

[0026] Figure 3B: Table 8 LC-MS results of Her2 / CD47 combination.

[0027] Figure 3C: Table 8 LC-MS results of Her2 / cMET combination.

[0028] Figure 4 shows the proportion of correctly assembled molecules analyzed by LC-MS after different charge mutations were introduced into different antibody pairs shown in Table 9 in combination with the same disulfide bond remodeling mutations, where A: heavy chain of antibody 1; a: light chain of antibody 1; B: heavy chain of antibody 2; b: light chain of antibody 2; AaBb: correctly paired bispecific antibodies.

[0029] Figure 4A: LC-MS results of combination 1 in Table 9.

[0030] Figure 4B: LC-MS results of combination 2 in Table 9.

[0031] Figure 4C: LC-MS results of combination 3 in Table 9.

[0032] Figure 4D: LC-MS results of combination 4 in Table 9.

[0033] Figure 4E: LC-MS results of combination 5 in Table 9.

[0034] Figure 5 shows the configurations of three bispecific antibodies, wherein Figure 5A shows a bispecific antibody with a 2+1 N-terminal configuration; Figure 5B shows a bispecific antibody with a 2+1 C-terminal configuration; and Figure 5C shows a bispecific antibody with a 2+2 C-terminal configuration.

[0035] Figure 6 shows the multivalent configuration molecules constructed using the present invention as shown in Table 11, and the proportion of correctly assembled molecules was analyzed by LC-MS, wherein HC1: antibody 1 heavy chain; LC1: antibody 1 light chain; HC2: antibody 2 heavy chain; LC2: antibody 2 light chain; Intact: correctly paired multivalent bispecific antibody.

[0036] Figure 6A: Table 11 LC-MS results of GPRC5D / CD3-1.

[0037] Figure 6B: Table 11 LC-MS results of GPRC5D / CD3-2.

[0038] Figure 6C: Table 11 LC-MS results of GPRC5D / CD3-3.

[0039] Figure 6D: Table 11 LC-MS results of GPRC5D / CD3-4.

[0040] Figure 6E: Table 11 LC-MS results of GPRC5D / CD3-5.

[0041] Figure 6F: Table 11 LC-MS results of GPRC5D / CD3-6.

[0042] Figures 7A and 7B show examples of charge and disulfide bond remodeling mutations introduced into the BCMA / GPRC5D / CD3 triple antibody.

[0043] Figure 8 shows the exemplary antibody-mediated killing effect of PBMC on H929 cells, wherein Figure 8A shows the exemplary antibody-mediated killing effect of PBMC on BCMA-positive H929 cells, and Figure 8B shows the exemplary antibody-mediated killing effect of PBMC on GPRC5D-positive H929 cells.

[0044] Figures 9A-C show the results of non-reducing polyacrylamide gel electrophoresis of purified monoclonal antibodies with different mutants; Figures 9D-M show the results of denaturing non-reducing capillary electrophoresis (nrCE-SDS) of antibodies with disulfide bonds.

[0045] definition:

[0046] It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, as these may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only, and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0047] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0048] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0049] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0050] When "first" and "second" are mentioned herein, it is only to distinguish the two domains or two chains, but does not indicate the positions of the two domains in any way.

[0051] As used herein, the terms "comprising" or "including" are intended to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the stated elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0052] As used herein, amino acid positions throughout the variable regions of heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) and are referred to herein as “Kabat numbers.”

[0053] As used herein, when referring to amino acid positions in antibody domains other than the variable region (e.g., constant region, e.g., Fc region), the positions are numbered according to the EU numbering system described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and are referred to herein as "EU numbering." When position numbers and / or amino acid residues are assigned to a specific antibody isotype, it is intended to apply to the corresponding positions and / or amino acid residues of any other antibody isotype, as known to those skilled in the art.

[0054] General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0055] The term "amino acid substitution" or "amino acid mutation" refers to the replacement of at least one amino acid residue in a predetermined parent amino acid sequence with a different "substituted" amino acid residue. The replacement residue or residues can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and are selected from the group consisting of: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine ​​(Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). The definition of amino acid substitution herein also encompasses replacement with one or more non-naturally occurring amino acid residues. "Non-natural amino acid residues" refer to residues that are capable of covalently binding to adjacent amino acid residues in a polypeptide chain, in addition to those naturally occurring amino acid residues listed above. Examples of non-natural amino acid residues include norleucine, ornithine, norvaline, homoserine, Aib and other amino acid residue analogs.

[0056] The term "CH1 region" refers to the portion of an antibody heavy chain polypeptide extending from EU position 118 to EU position 220 (EU numbering system). In one embodiment, the CH1 domain comprises the amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 116). In one embodiment, internal disulfide bonds are removed by mutating cysteine ​​residues in CH1 to non-cysteine ​​residues, or new disulfide bonds are remodeled by mutating non-cysteine ​​residues to cysteine ​​residues.

[0057] The term "CH2 region" refers to the portion of an antibody heavy chain polypeptide extending from EU position 231 to EU position 340 (EU numbering system). In one embodiment, the CH2 domain comprises the amino acid sequence of APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 117).

[0058] The term "CH3 region" refers to the portion of an antibody heavy chain polypeptide extending from EU position 341 to EU position 447. In one embodiment, the CH3 domain comprises the amino acid sequence of GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 118).

[0059] The term "hinge region" refers to the portion of an antibody heavy chain polypeptide that connects the CH1 and CH2 regions in a wild-type antibody heavy chain, e.g., the IgG1 hinge region, e.g., the sequence from D221 to P230 according to EU numbering. Hinge regions of other IgG subclasses can be identified by aligning the hinge region cysteine ​​residues with those of the IgG1 subclass sequence.

[0060] The hinge region is typically a dimer composed of two polypeptides having the same amino acid sequence. In one embodiment, the hinge region comprises the amino acid sequence DKTHTCPXCP (SEQ ID NO: 119), wherein X is S or P. In one embodiment, the hinge region comprises the amino acid sequence HTCPXCP (SEQ ID NO: 120), wherein X is S or P. In one embodiment, the hinge region comprises the amino acid sequence CPXCP (SEQ ID NO: 121), wherein X is S or P. In one embodiment, the internal disulfide bond in the hinge region is removed by mutating the cysteine ​​residues in the hinge region to non-cysteine ​​residues.

[0061] The multispecific antibodies of the present invention may comprise a linker. As used herein, the term "linker" refers to any molecule that enables the direct connection of the different parts of a multispecific antibody. Examples of linkers for establishing covalent linkages between different parts of a multispecific antibody include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes or copolymers of polyethylene glycol and polypropylene glycol. In some embodiments, the term "peptide linker" according to the present invention refers to an amino acid sequence, wherein the sequence connects the amino acid sequences of the various parts of the multispecific antibody together. Preferably, the peptide linker has a length that is sufficient to connect the two entities in a manner that allows them to maintain their conformations relative to each other so as not to interfere with the desired activity. The peptide linker may or may not primarily include the following amino acid residues: Gly, Ser, Ala or Thr. Useful linkers include glycine-serine polymers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, (GGGS)n, and (GGGGS)nG, where n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10). Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.

[0062] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, the amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) in the Fc region may or may not exist. Two Fc regions can achieve dimerization to form a dimeric Fc, and two different Fc heterodimerizations form heterodimeric Fc. In this article, the terms "Fc region", "Fc portion" and "dimeric Fc (e.g., heterodimeric Fc)" do not include the heavy chain variable region VH and light chain variable region VL of an immunoglobulin and heavy chain constant region CH1 and light chain constant region CL, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases. In one embodiment, a human IgG heavy chain Fc region extends from Asp221, or from Cys226, or from Asp231, to the carboxyl-terminus of the heavy chain.

[0063] In one embodiment, the human IgG1 Fc region polypeptide (including the hinge region) comprises the following amino acid sequence:

[0064] The human IgG4 Fc region polypeptide (including the hinge region) comprises the following amino acid sequence:

[0065] The antibodies produced in the methods as reported herein comprise an Fc region, and in one embodiment, the Fc region is derived from a human Fc region. In one embodiment, the Fc region comprises the entirety of a human constant region. The antibody Fc region is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region is of the human IgG4 subclass. In one embodiment, the Fc region is of the human IgG1 subclass.

[0066] Herein, a "heterodimeric Fc scaffold" refers to a scaffold comprising two different Fc regions or formed by dimerization of two different Fc regions, which can be connected at its N-terminus or C-terminus to an antigen-binding domain (e.g., an antibody heavy chain and / or light chain variable region or an antibody antigen-binding fragment that can bind to a target molecule, or a soluble portion of a ligand or receptor that can bind to a target molecule) to form a multispecific antibody such as a bispecific antibody.

[0067] Amino acid mutations are represented by (original amino acid, amino acid position, mutated amino acid). For example, when the mutation site is located in the V region, "A43D" means that the alanine amino acid at Kabat position 43 is replaced by aspartic acid (D); when the mutation site is located in the C region, "Q124C" means that the glutamine at EU position 124 is replaced by cysteine ​​(C). When referring to a combination of mutations, the combined mutations are connected by a plus sign (+). "A43D+Q124C" means that the mutations A43D and Q124C are included at the same time. When a specific position can have multiple mutation possibilities, this is represented in this article by the symbol " / ". For example, the mutation "K370T / S" means that the residue K at position 370 can be replaced by a T or S residue.

[0068] When referring to an amino acid position in the present invention, unless otherwise specified, it refers to the amino acid position numbered according to the IgG1 heavy chain or kappa light chain, that is, it covers the amino acid position numbered according to the IgG1 heavy chain or kappa light chain, as well as the amino acid position corresponding to the amino acid position in other heavy chains or light chains. For example, when referring to C220, it covers amino acid position 220 in the IgG1 heavy chain according to EU numbering, as well as the corresponding amino acid in other heavy chains, such as amino acid position 131 in IgG2, IgG3 or IgG4.

[0069] It should be noted that when describing a mutation, the original amino acid at a specific position can be the amino acid being described, or it can be another amino acid at the corresponding position. For example, when describing a mutation, it is noted that the alanine at position 43 of the variable region is mutated to aspartic acid, and also when the corresponding position 43 is not alanine, the amino acid at the corresponding position 43 is mutated to aspartic acid, as long as the original amino acid corresponds to position 43 of the variable region. The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parent cell in terms of nucleic acid content, but may contain mutations. Such mutant progeny are included herein, and the mutant progeny have the same function or biological activity as screened or selected for in the initially transformed cells.

[0070] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0071] The term "binding molecule" refers to any molecule capable of specifically binding to a target, such as an antibody or antigen-binding fragment or fusion protein thereof.

[0072] The term "target" refers to the object to which a binding molecule is directed. A target can be an antigen, or it can be a ligand or a receptor. The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. The skilled person will understand that any macromolecule, including essentially all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the part of an antigen that specifically interacts with an antibody molecule. In some embodiments, the antigen is a tumor-associated antigen (i.e., an antigen related to the occurrence and progression of a tumor) or a T cell adaptor. When the binding molecule of the present invention relates to a target binding region derived from an antibody, "target" and "antigen" can be used interchangeably.

[0073] As used herein, the term "target binding region" refers to the portion of a binding molecule, such as a multispecific binding molecule or a bispecific binding molecule, that binds a specific target or antigen. The target binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such a target binding region may or may not have a tertiary structure independent of the rest of the binding molecule and may or may not bind to its target as a separate entity. The target binding region can also be a receptor or a ligand, or a domain of a receptor that is capable of binding a ligand. In the case of multispecific antibodies or bispecific antibodies, the "target binding region" is also referred to as an "antigen binding region."

[0074] As used herein, the term "antigen binding region" refers to any portion of an antibody or antigen binding fragment thereof, such as a multispecific antibody or bispecific antibody, that binds a specific target or antigen. The antigen binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such an antigen binding region may or may not have a tertiary structure independent of the remainder of the multispecific antibody or bispecific antibody and may or may not bind to its antigen / epitope as a separate entity. Where the binding molecules of the present invention relate to a target binding region derived from an antibody, "target binding region" and "antigen binding region" can be used interchangeably.

[0075] The term "multispecific binding molecule" refers to a multispecific binding molecule that is at least bispecific, e.g., a bispecific binding molecule, i.e., the molecule comprises at least a first target binding region and a second target binding region, wherein the first target binding region binds to one target or antigen and the second target binding region binds to another antigen or target. The multispecific binding molecules according to the present invention also encompass multispecific molecules comprising multiple target binding regions / binding sites, such as trispecific binding molecules. In some embodiments, the multispecific binding molecules of the present invention are multispecific antibodies. In some embodiments, the bispecific binding molecules of the present invention are bispecific antibodies. In some embodiments, the multispecific binding molecules of the present invention are fusion proteins, which may, for example, comprise a target binding region from an antibody and a target binding region from a receptor or ligand.

[0076] As used herein, the term "monospecific" refers to a polypeptide / protein molecule having one or more target binding sites, each of which binds to the same site or structure of the same target or the same epitope of the same antigen.

[0077] As used herein, the term "multispecific" binding molecule, such as an antibody, refers to a molecule having at least two target binding sites, each of which binds to a different site / structure of the same target or to a different target. A multispecific binding molecule is a binding molecule that has binding specificity for at least two different targets or sites / structures. In one embodiment, provided herein are bispecific binding molecules that have binding specificity for a first target and a second target.

[0078] As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of different antigens. A multispecific antibody is an antibody that has binding specificity for at least two different antigenic epitopes. In one embodiment, provided herein is a bispecific antibody that has binding specificity for a first antigen and a second antigen.

[0079] When referring to a "first antigen-binding region" in a multispecific antibody or bispecific antibody, it refers to the binding region that binds to the first antigen, and is not intended to limit the number of such antigen-binding regions contained in the antibody. For example, a multispecific antibody or bispecific antibody may contain one or more first antigen-binding regions. For example, a bispecific antibody contains a first antigen-binding region and a second antigen-binding region, but may contain one or more first antigen-binding regions and one or more second antigen-binding regions.

[0080] When referring to a "first target binding region" in a binding molecule, it refers to the binding region that binds to the first target, and is not intended to limit the number of target binding regions contained in the binding molecule. For example, a multispecific binding molecule or a bispecific binding molecule can contain one or more first target binding regions. For example, a bispecific binding molecule comprising a first target binding region and a second target binding region can contain one or more first target binding regions and one or more second target binding regions.

[0081] When referring to a "first antigen-binding region" in an antibody, it refers to the binding region that binds to the first antigen, and is not intended to limit the number of such antigen-binding regions contained in the antibody. For example, a multispecific antibody or a bispecific antibody may contain one or more first antigen-binding regions. For example, a bispecific antibody contains a first antigen-binding region and a second antigen-binding region, but may contain one or more first antigen-binding regions and one or more second antigen-binding regions.

[0082] When referring to "a target or antigen binding region is derived from an antibody", it means that the binding domain constituting the target / antigen binding region is or is derived from the binding domain of the antibody that specifically binds to the antigen, for example, the specific antigen-binding fragment of the antigen binding region, such as Fab, is or is derived from the corresponding fragment, such as Fab, of the antibody, or the heavy chain variable region and / or light chain variable region of the antigen binding region is or is derived from the heavy chain variable region and / or light chain variable region of the antibody, or one, two, three, four, five or six CDRs of the antigen binding region are CDRs of the antibody.

[0083] The term "derived from" means that the fragment in the antigen binding region is substantially identical to the fragment of the antibody from which it is derived, but has a mutation, such as a substitution, deletion or addition, at one or more sites. In a specific embodiment, the mutation is not in the CDR of the antibody.

[0084] The terms "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules with the structure of natural immunoglobulin molecules. In the case of conventional four-chain IgG antibodies, the full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy chain antibodies having only heavy chains and lacking light chains, the full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds. For conventional four-chain IgG antibodies, the full-length antibody heavy chain is generally composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains CH1, CH2 and CH3. The full-length antibody light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region are composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0085] The term "antigen-binding fragment" of an antibody is a molecule that is different from a full-length antibody and that contains a portion of the full-length antibody, but is capable of binding to the antigen of the full-length antibody or competing with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) for antigen binding. Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabodies, single-domain antibodies (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. In addition, digesting a full-length antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, which is a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab fragment with a hinge region. An Fv fragment consists of the VL and VH domains of a single arm of an antibody. The two domains of the Fv fragment, VL and VH, can be encoded by separate genes, but can also be produced by recombinant methods using a synthetic linker peptide to link the two domains as a single protein chain in which the VL region and VH region are paired to form a single-chain Fv (scFv).

[0086] "Fab fragment" or "Fab" are used interchangeably herein to refer to an immunoglobulin fragment composed of two polypeptide chains comprising an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CH1, a light chain variable domain VL, and a light chain constant domain CL, wherein one polypeptide chain comprises, from N-terminus to C-terminus, VH and a constant region selected from CH1 and CL, and the other polypeptide chain comprises, from N-terminus to C-terminus, VL and another constant region selected from CL and CH1, wherein the VH domain and the VL domain pair to form an antigen binding site. Herein, the Fab polypeptide chain comprising the heavy chain constant region CH1 is also referred to as the "Fab heavy chain"; accordingly, the Fab polypeptide chain comprising the light chain constant region CL is also referred to as the "Fab light chain."

[0087] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics The database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention).

[0088] Calculation of sequence identity between sequences is performed as follows.

[0089] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0090] Detailed Description of the Invention

[0091] I. Binding Molecules

[0092] The present invention relates to a binding molecule comprising a first target binding region and a second target binding region, and optionally one or more other target binding regions, wherein one or two or more of the target binding regions comprise one or more mutations that are conducive to preventing mispairing and / or promoting heterodimerization. In some embodiments, the binding molecule further comprises one or more mutations that promote purification of heterodimers. In some embodiments, the mutations comprised in the binding molecule of the present invention are selected from disulfide bond remodeling mutations, charge mutations and / or other mutations that are conducive to preventing mispairing and / or promoting heterodimerization. In some embodiments, the first target binding region and the second target binding region bind to the same target or site / structure on the target. In some embodiments, the first target binding region and the second target binding region bind to different targets or sites / structures on the target.

[0093] In some embodiments, the binding molecule is a fusion protein, which may, for example, comprise a target binding region from an antibody and a target binding region from a receptor or ligand. In some embodiments, illustrative examples of the ligand or receptor are granule-associated cytokines or their receptors, such as tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), or transforming growth factor (TGF, such as TGF-β), or their receptors, and fragments thereof.

[0094] In some embodiments, the binding molecule is an antibody. In some embodiments, the present invention relates to an antibody comprising a mutation, such as a monospecific antibody or a multispecific antibody, such as a bispecific antibody, which comprises a first antigen-binding region and a second antigen-binding region, and optionally one or more other antigen-binding regions, wherein one or two or more of the antigen-binding regions comprise one or more mutations that are conducive to preventing mispairing and / or promoting heterodimerization. In some embodiments, the antibody further comprises one or more mutations that promote heterodimer purification. In some embodiments, the mutations included in the antibodies of the present invention are selected from disulfide bond remodeling mutations, charge mutations and / or other mutations that are conducive to preventing mispairing and / or promoting heterodimerization. In some embodiments, the first antigen-binding region and the second antigen-binding region bind to the same antigen or epitope. In some embodiments, the first antigen-binding region and the second antigen-binding region bind to different antigens or epitopes.

[0095] In some embodiments, the target binding region or antigen binding region comprises a heavy chain variable region and a light chain variable region. In some embodiments, the target binding region or antigen binding region comprises a light chain constant region CL. In some embodiments, the target binding region or antigen binding region comprises a heavy chain constant region CH1.

[0096] In some embodiments, the CH1 region is a human IgG CH1, e.g., human IgG1 CH1, human IgG2 CH1, human IgG3 CH1, or human IgG4 CH1. In one embodiment, the CH1 region comprises the amino acid sequence of SEQ ID NO: 116, or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or 100% identity thereto, or consists thereof.

[0097] In some embodiments, the CL region is a human kappa light chain CL region or a human lambda light chain CL region. In some embodiments, the CL region comprises or consists of the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55, or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or 100% identity thereto.

[0098] In the present disclosure, the definition of the antigen binding region also applies to the target binding region.

[0099] I-1 disulfide bond remodeling mutation

[0100] Disulfide bond remodeling mutation is a strategy that can effectively force the pairing of homologous light and heavy chains to avoid mispairing between the light and heavy chains of different target binding regions of binding molecules or multispecific antibodies. The disulfide bonds connecting the light and heavy chains in one or more target binding regions are remodeled to change the position of the disulfide bonds, so that the mispaired light and heavy chains cannot effectively form disulfide bonds.

[0101] Therefore, in one embodiment, in a binding molecule of the invention, such as an antibody, at least one target binding region or antigen binding region comprises a CH1-CL, and the disulfide bonds at the CH1-CL interface are remodeled, for example, a disulfide bond remodeling mutation is present at the CH1-CL1 interface. In one embodiment, in a binding molecule of the invention, such as an antibody, two or more target binding regions comprise a disulfide bond remodeling mutation at the CH1-CL1 interface.

[0102] The "disulfide bond remodeling mutation" described herein refers to a mutation at the position of a pair of amino acids on the interface, wherein the non-cysteine ​​at the position of the amino acid pair is replaced by cysteine, for example, the replacement of two non-cysteine ​​at the position of the amino acid pair by cysteine ​​allows the formation of interchain disulfide bonds.

[0103] In some embodiments, the disulfide bond remodeling mutation of the present invention is a mutation at the position of an amino acid pair on the CH1-CL interface of an antigen binding region (e.g., an antigen binding region comprising a Fab), wherein the non-cysteine ​​at the position of the amino acid pair is substituted with cysteine. In some embodiments, the two non-cysteine ​​residues at the position of the amino acid pair are substituted with cysteine ​​residues to allow the formation of an interchain disulfide bond.

[0104] In some embodiments, the mutation is such that the distance between the alpha carbon atoms at the CH1-CL interface is less than More preferably less than In some embodiments, the amino acids in the non-cysteine ​​amino acid pair are all mutated to cysteine. In some embodiments, the amino acid side chains in the amino acid pair point toward each other. In some embodiments, the distance between the α carbon atoms at the CH1-CL interface is less than More preferably less than The amino acids in the non-cysteine ​​amino acid pair are both mutated to cysteine, and the side chains of the amino acids in the non-cysteine ​​amino acid pair point toward each other.

[0105] In some embodiments, the heavy chain CH1 (e.g., IgG1, IgG2, IgG3, or IgG4) in the antigen binding region has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 126, 128, 134, 136, 139, 168, 170, 171, or 173 (EU numbering). In some embodiments, the light chain CL in the antigen binding region has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 114, 116, 118, 124, 160, 162, or 164 (EU numbering).

[0106] In some embodiments, in the CH1-CL of the antigen binding region, (e.g., IgG1, IgG2, IgG3, or IgG4) the heavy chain CH1 has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 126 (EU numbering), and the light chain CL has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 124 (EU numbering).

[0107] In some embodiments, in the CH1-CL of the antigen binding region, (e.g., IgG1, IgG2, IgG3, or IgG4) the heavy chain CH1 has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 168 (EU numbering), and the light chain CL has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 164 (EU numbering).

[0108] In some embodiments, in the CH1-CL of the antigen binding region, (e.g., IgG1, IgG2, IgG3, or IgG4) the heavy chain CH1 has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 170 (EU numbering), and the light chain CL has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 162 or 164 (EU numbering).

[0109] In some embodiments, in the CH1-CL of the antigen binding region, (e.g., IgG1, IgG2, IgG3, or IgG4) the heavy chain CH1 has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 173 (EU numbering), and the light chain CL has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 160 or 162 (EU numbering).

[0110] In some embodiments, in the CH1-CL of the antigen binding region, (e.g., IgG1, IgG2, IgG3, or IgG4) the heavy chain CH1 has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 128 (EU numbering), and the light chain CL has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 118 (EU numbering).

[0111] In some embodiments, in the CH1-CL of the antigen binding region, (e.g., IgG1, IgG2, IgG3, or IgG4) the heavy chain CH1 has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 134 (EU numbering), and the light chain CL has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 116 (EU numbering).

[0112] In some embodiments, in the CH1-CL of the antigen binding region, (e.g., IgG1, IgG2, IgG3, or IgG4) the heavy chain CH1 has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 136 (EU numbering), and the light chain CL has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 114 (EU numbering).

[0113] In some embodiments, in the CH1-CL of the antigen binding region, (e.g., IgG1, IgG2, IgG3, or IgG4) the heavy chain CH1 has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 171 (EU numbering), and the light chain CL has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 162 (EU numbering).

[0114] In some embodiments, in the CH1-CL of the antigen binding region, (e.g., IgG1, IgG2, IgG3, or IgG4) the heavy chain CH1 has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 139 (EU numbering), and the light chain CL has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 116 (EU numbering).

[0115] Non-cysteine ​​amino acids include naturally occurring and / or non-classical amino acids in some aspects. Naturally occurring non-cysteine ​​amino acids include glycine, alanine, valine, leucine, isoleucine, proline, serine, threonine, methionine, histidine, lysine, arginine, glutamic acid, aspartic acid, glutamine, asparagine, phenylalanine, tyrosine and tryptophan. Non-classical amino acids can sometimes be incorporated via a cell expression system (e.g., a prokaryotic and / or eukaryotic cell expression system). Examples of non-classical amino acids include ornithine, diaminobutyric acid, norleucine, pyridine alanine (pyrylalanine), thienyl alanine, naphthylalanine and phenylglycine.

[0116] In some embodiments, the disulfide bond remodeling mutation comprises F126C in CH1 and Q124C in CL in the antigen binding region (EU numbering).

[0117] In some embodiments, the disulfide bond remodeling mutation comprises H168C in CH1 and T164C in CL in the antigen binding region (EU numbering).

[0118] In some embodiments, the disulfide bond remodeling mutation comprises F170C in CH1 and T164C in CL in the antigen binding region (EU numbering).

[0119] In some embodiments, the disulfide bond remodeling mutation comprises F170C in CH1 and S162C in CL in the antigen binding region (EU numbering).

[0120] In some embodiments, the disulfide bond remodeling mutation comprises V173C in CH1 and S162C in CL in the antigen binding region (EU numbering).

[0121] In some embodiments, the disulfide bond remodeling mutation comprises V173C in CH1 and Q160C in CL in the antigen binding region (EU numbering).

[0122] In some embodiments, the disulfide bond remodeling mutation comprises L128C in CH1 and F118C in CL in the antigen binding region (EU numbering).

[0123] In some embodiments, the disulfide bond remodeling mutation comprises S134C in CH1 and F116C in CL in the antigen binding region (EU numbering).

[0124] In some embodiments, the disulfide bond remodeling mutation comprises S136C in CH1 and S114C in CL in the antigen binding region (EU numbering).

[0125] In some embodiments, the disulfide bond remodeling mutation comprises P171C in CH1 and S162C in CL in the antigen binding region (EU numbering).

[0126] In some embodiments, the disulfide bond remodeling mutation comprises T139C in CH1 and F116C in CL in the antigen binding region (EU numbering).

[0127] Therefore, in one embodiment of the present invention, the present invention relates to a binding molecule, such as a multispecific antibody, comprising a first antigen-binding region and a second antigen-binding region and optionally other antigen-binding regions, wherein the first antigen-binding region comprises a first CH1 and a first CL,

[0128] (i) the first CH1 comprises an F126C mutation, and the first CL comprises a Q124C mutation (EU numbering);

[0129] (ii) the first CH1 comprises an H168C mutation, and the first CL comprises a T164C mutation (EU numbering);

[0130] (iii) the first CH1 comprises an F170C mutation, and the first CL comprises a T164C mutation (EU numbering);

[0131] (iv) the first CH1 comprises an F170C mutation, and the first CL comprises an S162C mutation (EU numbering);

[0132] (v) the first CH1 comprises a V173C mutation, and the first CL comprises an S162C mutation (EU numbering);

[0133] (vi) the first CH1 comprises a V173C mutation, and the first CL comprises a Q160C mutation (EU numbering);

[0134] (vii) the first CH1 comprises an L128C mutation, and the first CL comprises an F118C mutation (EU numbering);

[0135] (viii) the first CH1 comprises an S134C mutation, and the first CL comprises an F116C mutation (EU numbering);

[0136] (ix) the first CH1 comprises an S136C mutation, and the first CL comprises an S114C mutation (EU numbering);

[0137] (x) the first CH1 comprises a P171C mutation, and the first CL comprises an S162C mutation (EU numbering); or

[0138] (xi) the first CH1 comprises a T139C mutation, and the first CL comprises a F116C mutation (EU numbering).

[0139] In some embodiments, the CH1 is derived from IgG1, IgG2, IgG3, or IgG4, preferably from IgG1 or IgG4. In some embodiments, the CL is derived from a kappa light chain or a lambda light chain.

[0140] In some embodiments, the disulfide bond remodeling mutation also includes replacing natural cysteine ​​with non-cysteine. In some embodiments, the interchain cysteine ​​in the heavy chain constant region-light chain constant region interface in one or two or more antigen-binding regions is replaced by non-cysteine. In some embodiments, the CH1 in the antigen-binding region has a cysteine ​​amino acid replaced by a non-cysteine ​​amino acid at position 220 (EU numbering). In some embodiments, the IgG1 heavy chain CH1 in the antigen-binding region has a cysteine ​​amino acid replaced by a non-cysteine ​​amino acid at position 220 (EU numbering), or the IgG2, IgG3 or IgG4 heavy chain CH1 in the antigen-binding region has a cysteine ​​replaced by a non-cysteine ​​at position 131 (EU numbering). In some embodiments, the light chain CL in the antigen-binding region has a cysteine ​​amino acid replaced by a non-cysteine ​​amino acid at position 214 (EU numbering). In some embodiments, in the antigen-binding region, the IgG1 heavy chain CH1 has a cysteine ​​amino acid to a non-cysteine ​​amino acid substitution at position 220 (EU numbering), and the light chain CL has a cysteine ​​amino acid to a non-cysteine ​​amino acid substitution at position 214 (EU numbering), or the IgG2, IgG3 or IgG4 heavy chain CH1 region has a cysteine ​​amino acid to a non-cysteine ​​amino acid substitution at position 131 (EU numbering), and the light chain CL has a cysteine ​​amino acid to a non-cysteine ​​amino acid substitution at position 214 (EU numbering). Exemplary cysteine ​​to non-cysteine ​​mutations are also described in, for example, CN104011221B. In some embodiments, the cysteine ​​is substituted for serine, alanine or valine, for example, serine.

[0141] In some embodiments, the disulfide bond remodeling mutation comprises C220S / A / V in CH1 in the antigen binding region (IgG1 subtype, or C131S / A / V at the corresponding position in IgG2, IgG3 or IgG4) and C214S / A / V in CL (EU numbering).

[0142] Therefore, in one embodiment of the present invention, the present invention relates to a bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region comprises CH1 and CL,

[0143] (i) the first CH1 comprises an F126C mutation, and the first CL comprises a Q124C mutation (EU numbering);

[0144] (ii) the first CH1 comprises an H168C mutation, and the first CL comprises a T164C mutation (EU numbering);

[0145] (iii) the first CH1 comprises an F170C mutation, and the first CL comprises a T164C mutation (EU numbering);

[0146] (iv) the first CH1 comprises an F170C mutation, and the first CL comprises an S162C mutation (EU numbering);

[0147] (v) the first CH1 comprises a V173C mutation, and the first CL comprises an S162C mutation (EU numbering);

[0148] (vi) the first CH1 comprises a V173C mutation, and the first CL comprises a Q160C mutation (EU numbering);

[0149] (vii) the first CH1 comprises an L128C mutation, and the first CL comprises an F118C mutation (EU numbering);

[0150] (viii) the first CH1 comprises an S134C mutation, and the first CL comprises an F116C mutation (EU numbering);

[0151] (ix) the first CH1 comprises an S136C mutation, and the first CL comprises an S114C mutation (EU numbering);

[0152] (x) the first CH1 comprises a P171C mutation, and the first CL comprises an S162C mutation (EU numbering); or

[0153] (xi) the first CH1 comprises a T139C mutation, and the first CL comprises a F116C mutation (EU numbering)

[0154] The CH1 further comprises a C220S / A / V mutation (or the corresponding C131S / A / V), and the CL further comprises a C214S / A / V mutation (EU numbering);

[0155] Preferably, the CH1 further comprises C220S (EU numbering), and the CL further comprises C214S (EU numbering).

[0156] In some embodiments, the first antigen binding region comprises CH1 and CL, wherein CH1 comprises T139C and C220S mutations (or the corresponding C131S), and wherein CL comprises F116C and C214S mutations (EU numbering).

[0157] In some embodiments, the first antigen binding region comprises CH1 and CL, wherein CH1 comprises F126C and C220S mutations (or the corresponding C131S), and wherein CL comprises Q124C and C214S mutations (EU numbering).

[0158] In some embodiments, the first antigen binding region comprises CH1 and CL, wherein CH1 comprises H168C and C220S mutations (or the corresponding C131S), and wherein CL comprises T164C and C214S mutations (EU numbering).

[0159] In some embodiments, the first antigen binding region comprises CH1 and CL, wherein CH1 comprises F170C and C220S mutations (or the corresponding C131S), and wherein CL comprises T164C and C214S mutations (EU numbering).

[0160] In some embodiments, the first antigen binding region comprises CH1 and CL, wherein CH1 comprises F170C and C220S mutations (or the corresponding C131S), and wherein CL comprises S162C and C214S mutations (EU numbering).

[0161] In some embodiments, the first antigen binding region comprises CH1 and CL, wherein CH1 comprises V173C and C220S mutations (or the corresponding C131S), and wherein CL comprises S162C and C214S mutations (EU numbering).

[0162] In some embodiments, the first antigen binding region comprises CH1 and CL, wherein CH1 comprises V173C and C220S mutations (or the corresponding C131S), and wherein CL comprises Q160C and C214S mutations (EU numbering).

[0163] In some embodiments, the first antigen binding region comprises CH1 and CL, wherein CH1 comprises L128C and C220S mutations (or the corresponding C131S), and wherein CL comprises F118C and C214S mutations (EU numbering).

[0164] In some embodiments, the first antigen binding region comprises CH1 and CL, wherein CH1 comprises S134C and C220S mutations (or the corresponding C131S), and wherein CL comprises F116C and C214S mutations (EU numbering).

[0165] In some embodiments, the first antigen binding region comprises CH1 and CL, wherein CH1 comprises S136C and C220S mutations (or the corresponding C131S), and wherein CL comprises S114C and C214S mutations (EU numbering).

[0166] In some embodiments, the first antigen binding region comprises CH1 and CL, wherein CH1 comprises P171C and C220S mutations (or the corresponding C131S), and wherein CL comprises S162C and C214S mutations (EU numbering).

[0167] In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1 and comprises F126C. In some embodiments, CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 129 and comprises F126C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 129.

[0168] In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 132, and comprises H168C. ​​In some embodiments, CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 132. In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 133, and comprises H168C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 133.

[0169] In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 134 and comprises F170C. In some embodiments, CH1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 134. In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 135 and comprises F170C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135.

[0170] In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 136 and comprises V173C. In some embodiments, CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 136. In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 137 and comprises V173C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 137.

[0171] In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 138 and comprises L128C. In some embodiments, CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 138. In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 139 and comprises L128C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 139.

[0172] In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 140 and comprises S134C. In some embodiments, CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 140. In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 141 and comprises S134C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 141.

[0173] In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 142, and comprises S136C. In some embodiments, CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 142. In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 143, and comprises S136C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 143.

[0174] In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 144 and comprises P171C. In some embodiments, CH1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 144. In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 145 and comprises P171C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 145.

[0175] In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 146, and comprises T139C. In some embodiments, CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 146. In some embodiments, CH1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 147, and comprises T139C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 147.

[0176] In some embodiments, CL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2 and comprises Q124C. In some embodiments, CL comprises or consists of the amino acid sequence of SEQ ID NO: 2. In some embodiments, CL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 128 and comprises Q124C and C124S. In some embodiments, CL comprises or consists of the amino acid sequence of SEQ ID NO: 128.

[0177] In some embodiments, CL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 148 and comprises T164C. In some embodiments, CL comprises or consists of the amino acid sequence of SEQ ID NO: 148. In some embodiments, CL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 149 and comprises T164C and C124S. In some embodiments, CL comprises or consists of the amino acid sequence of SEQ ID NO: 149.

[0178] In some embodiments, CL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 150 and comprises S162C. In some embodiments, CL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 150. In some embodiments, CL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 151 and comprises S162C and C124S. In some embodiments, CL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 151.

[0179] In some embodiments, CL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 152 and comprises Q160C. In some embodiments, CL comprises or consists of the amino acid sequence of SEQ ID NO: 152. In some embodiments, CL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 35 and comprises Q160C and C124S. In some embodiments, CL comprises or consists of the amino acid sequence of SEQ ID NO: 35.

[0180] In some embodiments, CL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 36 and comprises F118C. In some embodiments, CL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, CL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 37 and comprises F118C and C124S. In some embodiments, CL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37.

[0181] In some embodiments, CL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 38 and comprises F116C. In some embodiments, CL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, CL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 98 and comprises F116C and C124S. In some embodiments, CL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 98.

[0182] In some embodiments, CL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 99 and comprises S114C. In some embodiments, CL comprises or consists of the amino acid sequence of SEQ ID NO: 99. In some embodiments, CL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 100 and comprises S114C and C124S. In some embodiments, CL comprises or consists of the amino acid sequence of SEQ ID NO: 100.

[0183] I-2 charge mutation

[0184] Charge mutation is a strategy that can effectively force the pairing of homologous light and heavy chains and avoid mispairing between the light and heavy chains in the two target binding regions. Amino acid pairs are searched on the contact surface of the heavy and light chains and mutated into amino acids with opposite charges, using the principle of opposite charges attracting each other to promote homologous pairing.

[0185] In one embodiment, the binding molecules of the present invention, such as antibodies (e.g., multispecific antibodies, such as bispecific antibodies), comprise charge mutations. In some embodiments, the charge mutations are mutations of amino acid pairs on the contact surface of the heavy chain and light chain of at least one antigen-binding region, wherein the two amino acids in the amino acid pair are mutated to opposite charges. In some embodiments, the amino acid pair has one or more of the following characteristics:

[0186] a. The amino acid pair is a conserved amino acid pair located at the interface between the heavy chain variable region and the light chain variable region of the antigen binding region;

[0187] b. The distance between the α carbon atoms of the amino acid pair is less than and / or

[0188] c. The side chains of the amino acid pair are oriented towards each other.

[0189] In some embodiments, the charge mutation comprises mutating two amino acids to amino acids with opposite charges at amino acid pairs selected from the following positions: position 39 of the heavy chain variable region and position 38 of the light chain variable region, position 45 of the heavy chain variable region and position 87 of the light chain variable region, position 45 of the heavy chain variable region and position 44 of the light chain variable region, position 91 of the heavy chain variable region and position 43 of the light chain variable region, or position 91 of the heavy chain variable region and position 44 of the light chain variable region (Kabat numbering).

[0190] In some embodiments, the charge mutation comprises mutating two amino acids into amino acids with opposite charges at amino acid pairs selected from the following positions: Q39 of the heavy chain variable region and Q38 of the light chain variable region, L45 of the heavy chain variable region and Y87 of the light chain variable region, L45 of the heavy chain variable region and P44 of the light chain variable region, Y91 of the heavy chain variable region and A43 of the light chain variable region, or Y91 of the heavy chain variable region and P44 of the light chain variable region (kabat numbering).

[0191] In some embodiments, charge mutations are respectively included at the positions of one or more amino acid pairs in the antigen binding regions that bind to different antigens. In some embodiments, the amino acid positions that include charge mutations in the antigen binding regions that bind to different antigens can be the same or different. When the amino acid positions that include charge mutations are the same, the mutated amino acids in the amino acid pairs in the antigen binding regions that bind to different antigens are different. For example, in some embodiments, charge mutations are included at the positions of one or more identical amino acid pairs in the first antigen binding region and the second antigen binding region, but the charge mutations on the amino acid pairs in one antigen binding region are different from the charge mutations on the amino acid pairs in the other antigen binding region, for example, at the same position, the charge of the mutated amino acid in the first antigen binding region is opposite to the charge of the mutated amino acid in the second antigen binding region. For example, in the first antigen-binding region, the heavy chain amino acid at position X1 is mutated to a positively charged amino acid, and the light chain amino acid at position X2 is mutated to a negatively charged amino acid, while in the second antigen-binding region, the heavy chain amino acid at position X1 is mutated to a negatively charged amino acid, and the light chain amino acid at position X2 is mutated to a positively charged amino acid, wherein the positively charged amino acid in the first antigen-binding region and the positively charged amino acid in the second antigen-binding region may be the same or different, for example, the same; and the negatively charged amino acid in the first antigen-binding region and the negatively charged amino acid in the second antigen-binding region may be the same or different, for example, the same.

[0192] In some embodiments, the amino acids in the heavy chain variable region of the amino acid pairs are substituted by positively charged amino acids, and the amino acids in the light chain variable region are substituted by negatively charged amino acids. In some embodiments, the amino acids in the heavy chain variable region of the amino acid pairs are substituted by negatively charged amino acids, and the amino acids in the light chain variable region are substituted by positively charged amino acids.

[0193] In some embodiments, in the first antigen-binding region, the amino acid at position 39 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 38 of the light chain variable region is substituted with a negatively charged amino acid; and / or in the second antigen-binding region, the amino acid at position 39 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 38 of the light chain variable region is substituted with a positively charged amino acid.

[0194] In some embodiments, in the first antigen binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 87 of the light chain variable region is substituted with a negatively charged amino acid; and / or in the second antigen binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 87 of the light chain variable region is substituted with a positively charged amino acid.

[0195] In some embodiments, in the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a negatively charged amino acid; and / or in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a positively charged amino acid.

[0196] In some embodiments, in the first antigen binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 43 of the light chain variable region is substituted with a negatively charged amino acid; and / or in the second antigen binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 43 of the light chain variable region is substituted with a positively charged amino acid.

[0197] In some embodiments, in the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a negatively charged amino acid; and / or in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a positively charged amino acid.

[0198] In some embodiments, the positively charged amino acid is selected from K, R, and H, and / or the negatively charged amino acid is selected from E or D. In some embodiments, the positively charged amino acid is selected from K and R, and / or the negatively charged amino acid is selected from E or D. In some embodiments, the positively charged amino acid is K, and / or the negatively charged amino acid is D.

[0199] In some embodiments, in one antigen binding region, the amino acid at position 39 of the heavy chain variable region is substituted with K, and the amino acid at position 38 of the light chain variable region is substituted with D. In some embodiments, in one antigen binding region, the amino acid at position 39 of the heavy chain variable region is substituted with D, and the amino acid at position 38 of the light chain variable region is substituted with K.

[0200] In some embodiments, in one antigen binding region, the amino acid at position 45 of the heavy chain variable region is substituted with K, and the amino acid at position 87 of the light chain variable region is substituted with D. In some embodiments, in one antigen binding region, the amino acid at position 45 of the heavy chain variable region is substituted with D, and the amino acid at position 87 of the light chain variable region is substituted with K.

[0201] In some embodiments, in one antigen binding region, the amino acid at position 45 of the heavy chain variable region is substituted with K, and the amino acid at position 44 of the light chain variable region is substituted with D. In some embodiments, in one antigen binding region, the amino acid at position 45 of the heavy chain variable region is substituted with D, and the amino acid at position 44 of the light chain variable region is substituted with K.

[0202] In some embodiments, in one antigen binding region, the amino acid at position 91 of the heavy chain variable region is substituted with K, and the amino acid at position 43 of the light chain variable region is substituted with D. In some embodiments, in one antigen binding region, the amino acid at position 91 of the heavy chain variable region is substituted with D, and the amino acid at position 43 of the light chain variable region is substituted with K.

[0203] In some embodiments, in one antigen binding region, the amino acid at position 91 of the heavy chain variable region is substituted with K, and the amino acid at position 44 of the light chain variable region is substituted with D. In some embodiments, in one antigen binding region, the amino acid at position 91 of the heavy chain variable region is substituted with D, and the amino acid at position 44 of the light chain variable region is substituted with K.

[0204] In some embodiments, a binding molecule of the invention, e.g., an antibody, such as a multispecific antibody, e.g., a bispecific antibody, comprises a first antigen binding region and a second antigen binding region comprising a substitution selected from the group consisting of:

[0205] (i) In the first antigen-binding region, the amino acid at position 39 of the heavy chain variable region is substituted with K, and the amino acid at position 38 of the light chain variable region is substituted with D. Simultaneously, in the second antigen-binding region, the amino acid at position 39 of the heavy chain variable region is substituted with D, and the amino acid at position 38 of the light chain variable region is substituted with K;

[0206] (ii) In the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with K, and the amino acid at position 87 of the light chain variable region is substituted with D. Simultaneously, in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with D, and the amino acid at position 87 of the light chain variable region is substituted with K;

[0207] (iii) In the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with K, and the amino acid at position 44 of the light chain variable region is substituted with D. Simultaneously, in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with D, and the amino acid at position 44 of the light chain variable region is substituted with K.

[0208] (iv) In the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with K, and the amino acid at position 43 of the light chain variable region is substituted with D. Simultaneously, in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with D, and the amino acid at position 43 of the light chain variable region is substituted with K.

[0209] (v). In the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with K, and the amino acid at position 44 of the light chain variable region is substituted with D. Simultaneously, in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with D, and the amino acid at position 44 of the light chain variable region is substituted with K.

[0210] I-3 combined mutations

[0211] In some embodiments, a binding molecule of the invention, such as an antibody, such as a multispecific antibody, such as a bispecific antibody, comprises a first target binding region (antigen binding region) and a second target binding region (antigen binding region) comprising a disulfide bond remodeling mutation, and a charge mutation.

[0212] In some embodiments, the present invention relates to an antibody, such as a multispecific antibody, such as a bispecific antibody, comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region comprises a heavy chain variable region, a heavy chain constant region CH1, a light chain variable region and a light chain constant region CL, and the second antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL.

[0213] In some embodiments, the first antigen binding region comprises disulfide bond remodeling mutations at the CH1-CL interface and charge mutations in VH and VL.

[0214] In some embodiments, the first antigen-binding region comprises a disulfide bond remodeling mutation at the CH1-CL interface and charge mutations on VH and VL; the second antigen-binding region comprises charge mutations on VH and VL.

[0215] In some embodiments, the first antigen-binding region comprises a combination of mutations selected from the following:

[0216] In some embodiments, the second antigen-binding region comprises a combination of mutations selected from the following:

[0217] In some embodiments, the present invention relates to an antibody, such as a multispecific antibody, such as a bispecific antibody, comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region comprises a heavy chain variable region VH, a heavy chain constant region CH1, a light chain variable region VL, and a light chain constant region CL, and the second antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein the antibody comprises the following mutation combination:

[0218] In some embodiments, in the antibody, eg, a multispecific antibody (eg, a bispecific antibody), one or more or all antigen binding regions that bind the same antigen comprise the same mutation.

[0219] In some embodiments, in the antibody, eg, a multispecific antibody (eg, a bispecific antibody), one or more or all antigen binding regions that bind the same antigen comprise different mutations.

[0220] I-4 light chain constant region replacement

[0221] In some embodiments, the binding molecules of the present invention, such as antibodies, such as multispecific antibodies, comprise a first target binding region (antigen binding region) and a second target binding region (antigen binding region), wherein the light chain constant region comprised by one or more or all of the first target binding region (antigen binding region) is a Kappa light chain constant region, and the light chain constant region comprised by one or more or all of the second target binding region (antigen binding region) is a Lambda light chain constant region, or wherein the light chain constant region comprised by one or more or all of the first target binding region (antigen binding region) is a Lambda light chain constant region, and the light chain constant region comprised by one or more or all of the second target binding region (antigen binding region) is a Kappa light chain constant region.

[0222] I-5 Fc region mutations

[0223] In some embodiments, one or more target binding regions (antigen binding regions) of a binding molecule of the invention, such as an antibody, such as a multispecific antibody (e.g., a bispecific antibody), further comprise an Fc region, wherein the Fc regions comprised may be the same or different.

[0224] In some embodiments, the first and second Fc regions are different and are capable of dimerizing to form a heterodimeric Fc scaffold.

[0225] In this article, Fc district refers to the C-terminal region of the immunoglobulin heavy chain containing the constant region of at least a portion, and can include native sequence Fc district and variant Fc district.Native sequence F district covers naturally occurring various immunoglobulin Fc sequences, such as various Ig subtypes and the Fc district (Gestur Vidarsson et al., IgG subclasses and allotypes:from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520) of its allotype.In some embodiments, Fc district of the present invention comprises antibody CH2 and CH3.In some embodiments, antibody Fc district can also carry IgG hinge region or part IgG hinge region at N end, such as, IgG1 hinge region or part IgG1 hinge region, such as according to EU numbering, the sequence of D221 to P230.In described hinge region, can contain sudden change.

[0226] Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region is according to the EU numbering system, also known as the EU index, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0227] In some embodiments, the Fc region is a human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In one embodiment, the Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 122 or 123, or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or higher identity thereto.

[0228] As will be appreciated by those skilled in the art, to promote the formation of heterodimers of the multispecific antibodies of the present invention, the Fc regions comprised by the multispecific antibodies of the present invention may include mutations that facilitate heterodimerization of the first Fc region and the second Fc region. In one embodiment, mutations are introduced into the CH3 regions of both Fc regions.

[0229] Methods for promoting heterodimerization of Fc regions are known in the art. For example, the CH3 region of a first Fc region and the CH3 region of a second Fc region are engineered in a complementary manner such that each CH3 region (or a heavy chain comprising it) is no longer able to homodimerize with itself but is forced to heterodimerize with the other complementary engineered CH3 region (such that the CH3 regions of the first and second Fc regions heterodimerize and homodimers are not formed between the two first CH3 regions or the two second CH3 regions).

[0230] Preferably, corresponding Knob mutations and Hole mutations are introduced into the first and second Fc regions based on the Knob-in-Hole technique, as described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).

[0231] In a specific embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (knob mutation); and in the CH3 region of another Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (numbering according to the EU index).

[0232] In a further embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine residue at position 354 is replaced with a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine ​​residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine ​​residue); and in the CH3 region of another Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the EU index), optionally the tyrosine residue at position 349 is replaced with a cysteine ​​residue (Y349C) (numbering according to the EU index).

[0233] In a specific embodiment, one Fc region comprises the amino acid substitution T366W and the other Fc region comprises the amino acid substitutions T366S, L368A, and Y407V (numbering is according to the EU index).

[0234] In a specific embodiment, one Fc region comprises amino acid substitutions S354C and T366W, and the other Fc region comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering is according to the EU index).

[0235] Corresponding mutations can also be introduced into the first and second Fc regions based on the Innobody technology. For this technology, see, for example, PCT / CN2021 / 143141.

[0236] In a specific embodiment,

[0237] The first CH3 region comprises an S364R / K mutation (preferably S364R), and optionally one or more other mutations. In some embodiments, the second CH3 region comprises a K370S / T / A / V mutation (preferably K370S), and optionally one or more other mutations. In some embodiments, the first CH3 region comprises an S364R / K mutation, and the second CH3 region comprises a K370S / T / A / V mutation. In some embodiments, the first CH3 region comprises an S364R mutation, and the second CH3 region comprises a K370S mutation.

[0238] In some embodiments, the first CH3 region comprises S364R / K (preferably S364R) and D399K / R (preferably D399K) mutations. In some embodiments, the second CH3 region comprises K370S / T / A / V (preferably K370S) mutations and K409D / E (preferably K409D) mutations. In some embodiments, the first CH3 region comprises S364R / K+D399K / R, and the second CH3 region comprises K370S / T / A / V+Y349T / S / A / V. In some embodiments, the first CH3 region comprises S364R+D399K, and the second CH3 region comprises K370S+Y349T. In some embodiments, the first CH3 region further comprises E375N / Q (preferably E375N) and / or T350V / A (preferably T350V). In some embodiments, the second CH3 region further comprises K409D / E (preferably K409D), Q347D / E (preferably Q347D) and / or T350V / A (preferably T350V).

[0239] In some embodiments, the first CH3 region comprises S364R + D399K, and the second CH3 region comprises K370S + Y349T + K409D. In some embodiments, the first CH3 region further comprises E357N. In some embodiments, the second CH3 region further comprises Q347D. In some embodiments, the first CH3 region further comprises E357N, and the second CH3 region further comprises Q347D. In some embodiments, the first CH3 region and the second CH3 region further comprise T350V, or both comprise T350V.

[0240] Thus, in some embodiments, the first CH3 region comprises S364R+D399K, and the second CH3 region comprises K370S+Y349T+K409D+Q347D. In some embodiments, the first CH3 region comprises S364R+D399K+E357N, and the second CH3 region comprises K370S+Y349T+K409D+Q347D. In some embodiments, the first CH3 region comprises S364R+D399K+E357N+T350V, and the second CH3 region comprises K370S+Y349T+K409D+Q347D+T350V.

[0241] In some embodiments, the first CH3 region comprises K409E / D (preferably K409E). In some embodiments, the second CH3 region comprises D399K / R (preferably D399K) or K370T / S / A / V (preferably K370T). In some embodiments, the first CH3 region comprises K409E / D (preferably K409E), and the second CH3 region comprises D399K / R (preferably D399K). In some embodiments, the first CH3 region further comprises T411R / K (preferably T411R). In some embodiments, the second CH3 region further comprises K370T / S / A / V (preferably K370T). In some embodiments, the CH3 region comprises K409E / D+T411R / K, and the second CH3 region comprises D399K / R+K370T / S / A / V. In some embodiments, the CH3 region comprises K409E+T411R, and the second CH3 region comprises D399K+K370T.

[0242] In some specific embodiments, the first and second CH3 regions have the following mutation combinations:

[0243] In one embodiment, the CH3 of one Fc region comprises S364R and D399K mutations, and the CH3 mutations of the other Fc region comprise Y349T, K370S, and K409D mutations.

[0244] Thus, in a specific embodiment, a binding molecule of the invention, such as an antibody, such as a multispecific antibody, comprises two Fc regions that heterodimerize, wherein

[0245] a) one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises T366S, L368A and Y407V, or

[0246] b) one Fc-region polypeptide comprises the mutations S354C and T366W and the other Fc-region polypeptide comprises the mutations Y349C, T366S, L368A and Y407V, or

[0247] c) one Fc-region polypeptide comprises the mutations S364R and D399K, and the other Fc-region polypeptide comprises the mutations Y349T, K370S and K409D.

[0248] Thus, in a specific embodiment, a binding molecule of the invention, such as an antibody, such as a multispecific antibody, comprises two Fc regions that heterodimerize, wherein

[0249] a) one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 20 or 131, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 101 or 130

[0250] b) one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 125, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 124; or

[0251] c) one Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 126, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 127.

[0252] Thus, in a specific embodiment, a binding molecule of the invention, such as an antibody, such as a multispecific antibody, comprises two Fc regions that heterodimerize, wherein

[0253] a) one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 20 or 131, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 101 or 130; or

[0254] b) one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 125, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 124; or

[0255] c) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 126, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 127.

[0256] Thus, in a specific embodiment, a binding molecule of the invention, such as an antibody, such as a multispecific antibody, comprises two Fc regions that heterodimerize, wherein

[0257] a) one Fc-region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 20 or 131 and comprises the mutation T366W, and the other Fc-region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 101 or 130 and comprises the mutations T366S, L368A and Y407V; or

[0258] b) one Fc-region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 125 and comprises the mutations S354C and T366W, and the other Fc-region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 124 and comprises the mutations Y349C, T366S, L368A, and Y407V; or

[0259] c) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence of amino acids set forth in SEQ ID NO: 126 and comprises the mutations S364R and D399K, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence of amino acids set forth in SEQ ID NO: 127 and comprises the mutations Y349T, K370S, and K409D.

[0260] In some embodiments, the Fc region further comprises other mutations that facilitate purification of the heterodimer.

[0261] The Fc region of the binding molecules of the invention, such as antibodies, can also be mutated to obtain desired properties. Mutations in the Fc region are known in the art.

[0262] In one embodiment, the Fc region is modified with respect to the properties of the effector function of the Fc region (e.g., the complement activation function of the Fc region). In one embodiment, the effector function has been reduced or eliminated relative to a wild-type Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from the group consisting of: using an Fc isotype that naturally has reduced or eliminated effector function, and Fc region modification.

[0263] In a preferred embodiment, the Fc region has reduced effector function mediated by the Fc region, such as reduced or abolished ADCC or ADCP or CDC effector function, eg, comprises a mutation that achieves the above function.

[0264] As will be appreciated by those skilled in the art, depending on the intended use of the binding molecules of the present invention, such as antibody molecules, the binding molecules of the present invention, such as antibody molecules, may also include modifications in the Fc domain that alter the binding affinity for one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region comprises a mutation that reduces binding to the Fcγ receptor. For example, in some embodiments, the Fc region used in the present invention has an L234A / L235A mutation that reduces binding to the Fcγ receptor. In another preferred embodiment, the Fc fragment may have a mutation that results in increased serum half-life, such as a mutation that improves binding of the Fc fragment to FcRn.

[0265] I-6. Bispecific Antibodies or Multispecific Antibodies

[0266] In some embodiments, the antibodies of the invention are bispecific antibodies.

[0267] The term "bispecific antibody" refers to an antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds to one antigen or epitope and the second antigen-binding region binds to another antigen or another epitope. Therefore, the bispecific antibody according to the present invention comprises specificity for two different antigens, or for two different epitopes of one antigen. Bispecific antibody formats include IgG-like antibodies (Fan et al. (2015) Journal of Hematology & Oncology. 8: 130). The most common IgG-like antibody type comprises two Fab regions and two Fc regions, and the heavy chain and light chain of each Fab can be derived from a separate monoclonal antibody.

[0268] The bispecific antibodies of the present invention can be prepared using bispecific antibody forms or technologies known in the art.Specific exemplary bispecific forms that can be used in the context of the present invention are shown in, for example, Labrijn, et al.Bispecific antibodies:a mechanistic review of the pipeline.Nature Reviews Drug Discovery, 2019, 18 (8): 1-24. In some embodiments, the bispecific antibodies of the present invention have the following patterns: bispecific antibodies (Fig. 5A) of 2+1N-terminal configurations, bispecific antibodies (Fig. 5B) of 2+1C-terminal configurations, and bispecific antibodies (Fig. 5C) of 2+2C-terminal configurations. In some embodiments, the bispecific antibodies of the present invention also include 2+2N configurations or tandem Fab configurations.

[0269] In some embodiments, the bispecific antibody of the present invention comprises a first antigen-binding region that specifically binds to a first antigen and a second antigen-binding region that specifically binds to a second antigen, wherein the first antigen-binding region comprises a first Fab, and the second antigen-binding region comprises a second Fab, wherein the first Fab is connected to the N-terminus of the first Fc region at the C-terminus of its CH1 (via or without a linker, such as a hinge region), and the second Fab is connected to the N-terminus of the second Fc region at the C-terminus of its CH1 (via or without a linker, such as a hinge region). In a specific embodiment, the bispecific antibody is an IgG-like antibody having a configuration as shown in Figure 1. In a specific embodiment, the first Fab comprises a charge mutation and a disulfide bond remodeling mutation, the second Fab comprises a charge mutation, and optionally, the first Fc region and the second Fc region comprise an Innobody mutation or a Knob-into-hole mutation. Preferably, the charge mutation contained on the first Fab is at the same position as the charge mutation contained on the second Fab, but the charge of the mutated amino acid at the same position is opposite. In one embodiment, the bispecific antibody comprises:

[0270] Heavy chain 1: comprises or consists of the following from N-terminus to C-terminus: a first Fab heavy chain variable region-a heavy chain constant region CH1-a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region);

[0271] Light chain 1: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the first Fab - the light chain constant region;

[0272] Heavy chain 2: comprises or consists of the following from N-terminus to C-terminus: a second Fab heavy chain variable region - a heavy chain constant region CH1 - a second Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region);

[0273] Light chain 2: From N-terminus to C-terminus, it comprises the following: the light chain variable region of the second Fab - the light chain constant region.

[0274] In some embodiments, the bispecific antibody of the present invention comprises a first antigen-binding region that specifically binds to a first antigen and two second antigen-binding regions that specifically bind to a second antigen, wherein the first antigen-binding region comprises a first Fab, the second antigen-binding region comprises a second Fab, wherein one of the second Fabs is connected to the N-terminus of the first Fc region at the C-terminus of its CH1 (via or without a joint, such as a hinge region), one first Fab is connected to the N-terminus of the second Fc region at the C-terminus of its CH1 (via or without a joint, such as a hinge region), and another second Fab is connected to the N-terminus of the heavy chain variable region of the first Fab at the C-terminus of its CH1 (via or without a joint). In a specific embodiment, the bispecific antibody is a 2+1 N-terminal configuration having a configuration as shown in Figure 5A. In a specific embodiment, one or two second Fabs comprise charge mutations according to the present invention, and the first Fab comprises charge mutations and disulfide bond remodeling mutations according to the present invention, and optionally, the first Fc region and the second Fc region introduce mutations based on Innobody or Knob-into-hole. Preferably, the two second Fabs comprise the same charge mutations. Preferably, the charge mutation contained in the second Fab is at the same position as the charge mutation contained in the first Fab, but the charge of the mutated amino acid at the same position is opposite. In one embodiment, the bispecific antibody comprises:

[0275] Heavy chain 1: from N-terminus to C-terminus, comprises or consists of: a second Fab heavy chain variable region-heavy chain constant region CH1-a first Fab heavy chain variable region-heavy chain constant region CH1-a second Fc region, wherein the first Fab heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region), and the second Fab is connected at the C-terminus of its CH1 to the N-terminus of the heavy chain variable region of the first Fab (via or without a linker);

[0276] Light chain 1: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the first Fab - the light chain constant region;

[0277] Heavy chain 2: comprises or consists of the following from N-terminus to C-terminus: a second Fab heavy chain variable region - a heavy chain constant region CH1 - a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region);

[0278] Light chain 2: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the second Fab - the light chain constant region,

[0279] The bispecific antibody comprises two light chains 2.

[0280] In some embodiments, the bispecific antibody of the present invention comprises a first antigen-binding region that specifically binds to a first antigen and two second antigen-binding regions that specifically bind to a second antigen, wherein the first antigen-binding region comprises a first Fab and the second antigen-binding region comprises a second Fab, wherein the two second Fabs are connected to the N-terminus of the first Fc region and the N-terminus of the second Fc region at the C-terminus of their CH1 (via or without a linker, such as a hinge region), and the first Fab is connected to the C-terminus of the second Fc region at the N-terminus of the heavy chain variable region (via or without a linker). In a specific embodiment, the bispecific antibody is a 2+1 C-terminal configuration having a configuration as shown in Figure 5B. In a specific embodiment, one or two second Fabs comprise a charge mutation according to the present invention, and the first Fab comprises a charge mutation and a disulfide bond remodeling mutation according to the present invention, and optionally, the first Fc region and the second Fc region comprise an Innobody mutation or a Knob-into-hole mutation. Preferably, the two second Fabs comprise the same charge mutation. Preferably, the charge mutation contained on the first Fab is in the same position as the charge mutation contained on the second Fab, but the charge of the mutated amino acid at the same position is opposite. In one embodiment, the bispecific antibody comprises

[0281] Heavy chain 1: comprises or consists of the following from N-terminus to C-terminus: a second Fab heavy chain variable region-a heavy chain constant region CH1-a second Fc region-a first Fab heavy chain variable region-a heavy chain constant region CH1, wherein the second Fab is linked to the N-terminus of the second Fc region at the C-terminus of its CH1 (via or without a linker, such as a hinge region), and the first Fab is linked to the C-terminus of the second Fc region at the N-terminus of the heavy chain variable region (via or without a linker);

[0282] Light chain 1: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the first Fab - the light chain constant region;

[0283] Heavy chain 2: comprises or consists of the following from N-terminus to C-terminus: a second Fab heavy chain variable region - a heavy chain constant region CH1 - a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region);

[0284] Light chain 2: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the second Fab - the light chain constant region,

[0285] The bispecific antibody comprises two light chains 2.

[0286] In some embodiments, the bispecific antibody of the present invention comprises two first antigen-binding regions that specifically bind to the first antigen and two second antigen-binding regions that specifically bind to the second antigen, wherein the first antigen-binding region comprises a first Fab, and the second antigen-binding region comprises a second Fab, wherein the two second Fabs are connected to the N-termini of two Fc regions at the C-terminus of their CH1 (via or without a joint, such as a hinge region) (the Fc regions may be the same or different), and the two first Fabs are connected to the C-termini of two Fc regions at the N-terminus of the heavy chain variable region (via or without a joint). In a specific embodiment, the bispecific antibody is a 2+2 C-terminal configuration having a configuration as shown in Figure 5C. In a specific embodiment, one or two second Fabs comprise charge mutations according to the present invention, and one or two first Fabs comprise charge mutations and disulfide bond remodeling mutations according to the present invention. In a specific embodiment, two second Fabs comprise the same charge mutations according to the present invention, and / or two first Fabs comprise the same charge mutations and disulfide bond remodeling mutations according to the present invention. Preferably, the charge mutation contained in the first Fab is at the same position as the charge mutation contained in the second Fab, but the charge of the mutated amino acid at the same position is opposite. In one embodiment, the bispecific antibody comprises

[0287] Heavy chain: from N-terminus to C-terminus, comprising or consisting of: a second Fab heavy chain variable region-heavy chain constant region CH1-Fc region-a first Fab heavy chain variable region-heavy chain constant region CH1, wherein the second Fab is linked to the N-terminus of the Fc region at the C-terminus of its CH1 (via or without a linker, such as a hinge region), and the first Fab is linked to the C-terminus of the Fc region at the N-terminus of the heavy chain variable region (via or without a linker);

[0288] Light chain 1: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the first Fab - the light chain constant region;

[0289] Light chain 2: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the second Fab - the light chain constant region,

[0290] The bispecific antibody comprises two heavy chains.

[0291] In some embodiments, the antibodies of the invention are multispecific antibodies. In some embodiments, the multispecific antibodies of the invention are trispecific antibodies.

[0292] In some embodiments, the trispecific antibodies of the present invention comprise a first antigen-binding region that specifically binds to a first antigen, a second antigen-binding region that specifically binds to a second antigen, and a third antigen-binding region that specifically binds to a third antigen, wherein the first antigen-binding region comprises a first Fab, the second antigen-binding region comprises a second Fab, wherein the first Fab is connected to the N-terminus of the first Fc region at the C-terminus of its CH1 (via or without a linker, such as a hinge region), the second Fab is connected to the N-terminus of the second Fc region at the C-terminus of its CH1 (via or without a linker, such as a hinge region), and the third antigen-binding region comprises an scFv (e.g., VH-VL, or VL-VH), which is connected to the C-terminus of the second Fc region at the N-terminus or C-terminus of its VH or VL (e.g., the scFv is VL-VH, such as the N-terminus of VL) via or without a linker. In a specific embodiment, the configuration of the trispecific antibody is a 2+1 C-terminal configuration (Figure 7A or B). In a specific embodiment, the first Fab comprises a charge mutation and a disulfide bond remodeling mutation, the second Fab comprises a charge mutation, and optionally, the first Fc region and the second Fc region comprise an Innobody mutation or a Knob-into-hole mutation. Preferably, the charge mutation contained on the first Fab is at the same position as the charge mutation contained on the second Fab, but the charge of the mutated amino acid at the same position is opposite. In one embodiment, the multispecific antibody comprises

[0293] Heavy chain 1: comprises or consists of the following from N-terminus to C-terminus: a first Fab heavy chain variable region-a heavy chain constant region CH1-a first Fc region, wherein the C-terminus of the first Fab is connected to the N-terminus of the first Fc region (via or without a linker, such as a hinge region);

[0294] Light chain 1: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the first Fab - the light chain constant region;

[0295] Heavy chain 2: comprises or consists of the following from N-terminus to C-terminus: a second Fab heavy chain variable region-a heavy chain constant region CH1-a second Fc region-scFv, wherein the heavy chain constant region CH1 is linked at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region), and the scFv is linked at the N-terminus or C-terminus of its VH or VL (e.g., the scFv is VL-VH, e.g., the N-terminus of VL) to the C-terminus of the second Fc region via or without a linker;

[0296] Light chain 2: comprises or consists of the following from N-terminus to C-terminus: the light chain variable region of the second Fab - the light chain constant region.

[0297] I-7. Antigen and Antigen Binding Region

[0298] Binding molecules of the invention, such as antibodies, eg, multispecific antibodies, eg, bispecific antibodies, can bind to any target, such as an antigen, or a ligand or receptor suitable for an antibody.

[0299] In some embodiments, the antigen is a tumor-associated antigen (TA). In some embodiments, the tumor-associated antigen is an immune checkpoint molecule.

[0300] In some embodiments, the target or antigen is selected from FAP, CEA, p95HER2, BCMA, EpCAM, MSLN, MCSP, HER-1, HER-2, HER-3, CD19, CD20, CD22, CD33, CD38, CD52Flt3, EpCAM, IGF-1R, FOLR1, Trop-2, CA-12-5, HLA-DR, MUC-1 (mucin), GD2, A33-antigen, PSMA, PSCA, transferrin-receptor, TNC (tenascin), CA-IX, CD3, B7H3, EGFR, Hel, cMET, Axl, GPRC5D, PD-1, PD-L1, CD47, immune activation molecules such as 4-1BB, CD40, OX40, etc.

[0301] In some embodiments, the target or antigen is, for example, one or more targets / antigens selected from the group consisting of CD3, BCMA, CD20, Her2, Her3, B7H3, EGFR, Hel, cMET, Axl, GPRC5D, immune checkpoint molecules such as PD-1, PD-L1, CD47, , 4-1BB, CD40, OX40, or different epitopes of the same antigen.

[0302] In some embodiments, the bispecific antibodies of the invention bind to two different epitopes of one antigen or bind to two antigens.

[0303] In some specific embodiments, the bispecific antibodies of the invention bind to two antigens selected from the following:

[0304] CD3 / BCMA, CD3 / CD20, CD3 / Her2, Her2 / Her3, B7H3 / EGFR, PD-L1 / CD47, PD-L1 / CD40, PD-L1 / 4-1BB, EGFR / Her3, Her2 / Hel, Her2 / PD-1, Her2 / PD-L1, Her2 / CD47, Her2 / cMet, CD3 / GPRC5D.

[0305] In some specific embodiments, the trispecific antibodies of the invention bind BCMA, GPRC5D, and CD3.

[0306] The target binding region or antigen binding region of the binding molecule of the present invention, such as an antibody, may be derived from an antibody that binds to the above-mentioned antigen.

[0307] Antigen binding region that specifically binds to Her2

[0308] In some embodiments, the antigen binding region is derived from an antibody that specifically binds Her2, such as trastuzumab.

[0309] In some embodiments, the antigen binding region comprises 1, 2, 3, 4, 5, or 6 CDRs of an antibody known to specifically bind Her2, such as trastuzumab.

[0310] In some embodiments, the antigen binding region comprises one, two, and three heavy chain variable region CDRs, namely, HCDR1, HCDR2, and HCDR3, of antibodies known to specifically bind to Her2, such as trastuzumab.

[0311] In some embodiments, the antigen binding region comprises 1, 2, and 3 light chain variable region CDRs, ie, LCDR1, LCDR2, and LCDR3, of antibodies known to specifically bind Her2, such as trastuzumab.

[0312] In some embodiments, the antigen binding region comprises three heavy chain variable region CDRs and three light chain variable region CDRs of an antibody known to specifically bind Her2, such as trastuzumab.

[0313] In some embodiments, the antigen binding region comprises the heavy chain variable region and the light chain variable region of a known antibody that specifically binds to Her2, such as trastuzumab, and the mutations described herein.

[0314] In some embodiments, the antigen binding region comprises a Fab of a known antibody that specifically binds to Her2, such as trastuzumab, and a mutation described herein.

[0315] Specific binding to the antigen-binding region of PD-1

[0316] In some embodiments, the antigen binding region is derived from an antibody that specifically binds to PD-1, such as the PD-1 antibodies disclosed in WO2017025016, such as Sintilimab monoclonal antibody.

[0317] In some embodiments, the antigen binding region comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to PD-1, such as the PD-1 antibodies disclosed in WO2017025016, such as Sintilimab monoclonal antibody.

[0318] In some embodiments, the antigen binding region comprises 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of a known antibody that specifically binds to PD-1, such as the PD-1 antibody disclosed in WO2017025016, such as Sintilimab monoclonal antibody.

[0319] In some embodiments, the antigen binding region comprises 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of a known antibody that specifically binds to PD-1, such as the PD-1 antibody disclosed in WO2017025016, such as Sintilimab monoclonal antibody.

[0320] In some embodiments, the antigen binding region comprises three heavy chain variable region CDRs and three light chain variable region CDRs of a known antibody that specifically binds to PD-1, such as the PD-1 antibody disclosed in WO2017025016, such as Sintilimab monoclonal antibody.

[0321] In some embodiments, the antigen binding region comprises the heavy chain variable region and light chain variable region of a known antibody that specifically binds to PD-1, such as the PD-1 antibody disclosed in WO2017025016, such as Sintilimab monoclonal antibody, and the mutations described herein.

[0322] In some embodiments, the antigen binding region comprises a known antibody that specifically binds to PD-1, such as the PD-1 antibody disclosed in WO2017025016, such as the Fab of Sintilimab monoclonal antibody, and the mutations described herein.

[0323] Antigen binding region that specifically binds to CD47

[0324] In some embodiments, the antigen binding region is derived from an antibody that specifically binds to CD47, such as an antibody that specifically binds to CD47 disclosed in WO2019042285A1, such as ADI26630 disclosed therein.

[0325] In some embodiments, the antigen binding region comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to CD47, such as a CD47 antibody disclosed in WO2019042285A1, such as ADI26630 disclosed therein.

[0326] In some embodiments, the antigen binding region comprises a known antibody that specifically binds to CD47, such as the CD47 antibody disclosed in WO2019042285A1, such as 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of ADI26630 disclosed therein.

[0327] In some embodiments, the antigen binding region comprises a known antibody that specifically binds to CD47, such as the CD47 antibody disclosed in WO2019042285A1, such as 1, 2, and 3 light chain variable region CDRs of ADI26630 disclosed therein, i.e., LCDR1, LCDR2, and LCDR3.

[0328] In some embodiments, the antigen binding region comprises a known antibody that specifically binds to CD47, such as the CD47 antibody disclosed in WO2019042285A1, such as the three heavy chain variable region CDRs and three light chain variable region CDRs of ADI26630 disclosed therein.

[0329] In some embodiments, the antigen binding region comprises a known antibody that specifically binds to CD47, such as the CD47 antibodies disclosed in WO2019042285A1, such as the heavy chain variable region and light chain variable region of ADI26630 disclosed therein, and the mutations described herein.

[0330] In some embodiments, the antigen binding region comprises a known antibody that specifically binds to CD47, such as an antibody that specifically binds to CD47 disclosed in WO2019042285A1, such as the Fab of ADI26630 disclosed therein, and the mutations described herein.

[0331] Antigen binding region that specifically binds to cMet

[0332] In some embodiments, the antigen binding region is derived from an antibody that specifically binds cMet, such as Onartuzumab.

[0333] In some embodiments, the antigen binding region comprises 1, 2, 3, 4, 5, or 6 CDRs of an antibody known to specifically bind cMet, such as Onartuzumab.

[0334] In some embodiments, the antigen binding region comprises 1, 2, and 3 heavy chain variable region CDRs, ie, HCDR1, HCDR2, and HCDR3, of an antibody known to specifically bind cMet, such as Onartuzumab.

[0335] In some embodiments, the antigen binding region comprises 1, 2, and 3 light chain variable region CDRs, ie, LCDR1, LCDR2, and LCDR3, of an antibody known to specifically bind cMet, such as Onartuzumab.

[0336] In some embodiments, the antigen binding region comprises three heavy chain variable region CDRs and three light chain variable region CDRs of an antibody known to specifically bind cMet, such as Onartuzumab.

[0337] In some embodiments, the antigen binding region comprises the heavy chain variable region and the light chain variable region of a known antibody that specifically binds to cMet, such as Onartuzumab, and a mutation described herein.

[0338] Some antigen-binding regions comprise known antibodies that specifically bind to cMet, such as Fab of Onartuzumab, and the mutations described herein.

[0339] Antigen binding region that specifically binds to GPRC5D

[0340] In some embodiments, the antigen binding region is derived from an antibody that specifically binds to GPRC5D, such as an antibody that specifically binds to GPRC5D disclosed in PCT / CN2022 / 076832.

[0341] In some embodiments, the antibody that specifically binds to GPRC5D comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region as shown in SEQ ID NO: 87.

[0342] In some embodiments, the antibody that specifically binds to GPRC5D comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light chain variable region as shown in SEQ ID NO:83.

[0343] In some embodiments, the HCDR1 of the antibody that specifically binds to GPRC5D comprises or consists of the amino acid sequence of SEQ ID NO:88; the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:89, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:90, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:84, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:85, and / or the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:86.

[0344] In some embodiments, an antibody that specifically binds to GPRC5D comprises three complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, and three complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:88; HCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO:89, HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO:90, LCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:84, LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO:85, and LCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO:86.

[0345] In some aspects, an antibody that specifically binds to GPRC5D comprises a heavy chain variable region (VH), wherein the VH

[0346] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 87; or

[0347] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 87; or

[0348] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 87, preferably, the amino acid changes do not occur in the CDR regions.

[0349] In some aspects, an antibody that specifically binds to GPRC5D comprises a light chain variable region (VL), wherein the VL

[0350] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 83; or

[0351] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 83; or

[0352] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 83, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0353] In some embodiments, the antibody that specifically binds to GPRC5D comprises a VH and a VL, wherein the VH comprises or consists of the sequence shown in SEQ ID NO: 87; and / or the VL comprises or consists of the sequence shown in SEQ ID NO: 83.

[0354] In some embodiments, the antigen binding region comprises 1, 2, 3, 4, 5, or 6 CDRs of a known or described antibody that specifically binds to GPRC5D.

[0355] In some embodiments, the antigen binding region comprises one, two, and three heavy chain variable region CDRs, namely, HCDR1, HCDR2, and HCDR3, of a known or aforementioned antibody that specifically binds to GPRC5D.

[0356] In some embodiments, the antigen binding region comprises 1, 2, and 3 light chain variable region CDRs, ie, LCDR1, LCDR2, and LCDR3, of a known or described antibody that specifically binds to GPRC5D.

[0357] In some embodiments, the antigen binding region comprises three heavy chain variable region CDRs and three light chain variable region CDRs of a known or aforementioned antibody that specifically binds to GPRC5D.

[0358] In some embodiments, the antigen binding region comprises the heavy chain variable region and the light chain variable region of a known or aforementioned antibody that specifically binds to GPRC5D, and the mutations described herein.

[0359] Some antigen-binding regions comprise Fabs of known or aforementioned antibodies that specifically bind to GPRC5D, as well as the mutations described herein.

[0360] Antigen binding region that specifically binds to CD3

[0361] In some embodiments, the antigen binding region is derived from an antibody that specifically binds to CD3, such as the CD3 antibodies disclosed in WO2022068809, such as sp34.24 or sp34.87 therein.

[0362] In some embodiments, the antigen binding region comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to CD3, such as the CD3 antibodies disclosed in WO2022068809, such as sp34.24 or sp34.87 therein.

[0363] In some embodiments, the antigen binding region comprises a known antibody that specifically binds to CD3, such as the CD3 antibody disclosed in WO2022068809, such as 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of sp34.24 or sp34.87 therein.

[0364] In some embodiments, the antigen binding region comprises a known antibody that specifically binds to CD3, such as the CD3 antibody disclosed in WO2022068809, such as 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of sp34.24 or sp34.87 therein.

[0365] In some embodiments, the antigen binding region comprises a known antibody that specifically binds to CD3, such as the CD3 antibody disclosed in WO2022068809, such as three heavy chain variable region CDRs and three light chain variable region CDRs of sp34.24 or sp34.87 therein.

[0366] In some embodiments, the antigen binding region comprises a known antibody that specifically binds to CD3, such as the CD3 antibodies disclosed in WO2022068809, such as the heavy chain variable region and light chain variable region of sp34.24 or sp34.87 therein, and the mutations described herein.

[0367] Some antigen-binding regions include known antibodies that specifically bind to CD3, such as the CD3 antibodies disclosed in WO2022068809, such as the Fab of sp34.24 or sp34.87 therein, and the mutations described in the present invention.

[0368] Antigen binding region that specifically binds to BCMA

[0369] In some embodiments, the antigen binding region is derived from an antibody that specifically binds BCMA, such as an antibody that specifically binds BCMA disclosed in PCT / CN2022 / 076832.

[0370] In some embodiments, the antibody that specifically binds to BCMA comprises three complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region set forth in SEQ ID NO: 103.

[0371] In some embodiments, the antibody that specifically binds to BCMA comprises three complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are LCDR1, LCDR2, and LCDR3 having a light chain variable region as set forth in SEQ ID NO: 107.

[0372] In some embodiments, the HCDR1 of the antibody that specifically binds to BCMA comprises or consists of the amino acid sequence of SEQ ID NO: 104; the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 105, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 106, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 108, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 109, and / or the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 110.

[0373] In some embodiments, the antibody that specifically binds BCMA comprises three complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, and three complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 104; HCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 105, HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 106, LCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 108, LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 109, and LCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 110.

[0374] In some aspects, the antibody that specifically binds to BCMA comprises a heavy chain variable region (VH), wherein the VH

[0375] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 103; or

[0376] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 103; or

[0377] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 103, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0378] In some aspects, the antibody that specifically binds to BCMA comprises a light chain variable region (VL), wherein the VL

[0379] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 107; or

[0380] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 107; or

[0381] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 107, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0382] In some embodiments, the antibody that specifically binds to BCMA comprises a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 103; and / or the VL comprises or consists of the sequence set forth in SEQ ID NO: 107.

[0383] In some embodiments, the antigen binding region comprises 1, 2, 3, 4, 5, or 6 CDRs of a known or described antibody that specifically binds BCMA.

[0384] In some embodiments, the antigen binding region comprises one, two, and three heavy chain variable region CDRs, namely, HCDR1, HCDR2, and HCDR3, of a known or aforementioned antibody that specifically binds to BCMA.

[0385] In some embodiments, the antigen binding region comprises 1, 2, and 3 light chain variable region CDRs, namely LCDR1, LCDR2, and LCDR3, of a known or described antibody that specifically binds to BCMA.

[0386] In some embodiments, the antigen binding region comprises three heavy chain variable region CDRs and three light chain variable region CDRs of a known or described antibody that specifically binds to BCMA.

[0387] In some embodiments, the antigen binding region comprises the heavy chain variable region and light chain variable region of a known or above-described antibody that specifically binds to BCMA, and a mutation described herein.

[0388] Some antigen-binding regions comprise Fabs of known or aforementioned antibodies that specifically bind to BCMA, as well as the mutations described herein.

[0389] I.8. ​​Binding Fragments of Binding Molecules

[0390] The present invention also relates to fragments of the binding molecules described herein, such as antibodies, particularly fragments that specifically bind to the target to which the molecules bind, such as antigen-binding fragments.

[0391] In some embodiments, the antigen binding fragment is a Fab.

[0392] In some embodiments, the antigen binding fragment comprises one or more Fabs, wherein one or more Fabs comprises a mutation or combination of mutations described herein.

[0393] I.9. Exemplary Implementations

[0394] 1. An antibody or antigen-binding fragment thereof, comprising one or more first antigen-binding regions that specifically bind to a first antigen and one or more second antigen-binding regions that specifically bind to a second antigen, wherein at least one or more of the antigen-binding regions comprises a heavy chain CH1 and a light chain CL, and comprises a disulfide bond remodeling mutation at the CH1-CL interface, and / or at least one or more of the antigen-binding regions comprises a heavy chain variable region and a light chain variable region, and an amino acid pair at the interface between the heavy chain variable region and the light chain variable region comprises a charge mutation, wherein the charge mutation is a mutation in which two amino acids in the amino acid pair are mutated to amino acids with opposite charges;

[0395] wherein the first antigen and the second antigen may be the same or different;

[0396] Preferably, the disulfide bond remodeling mutations are only contained at the CH1-CL interface of one antigen binding region.

[0397] 2. The antibody or antigen-binding fragment thereof of embodiment 1, wherein the disulfide bond remodeling mutation comprises

[0398] (i) a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 126 of the heavy chain CH1 (EU numbering), and a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 124 of the light chain CL (EU numbering);

[0399] (ii) a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 168 of the heavy chain CH1 (EU numbering), and a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 164 of the light chain CL (EU numbering);

[0400] (iii) a non-cysteine ​​amino acid to a cysteine ​​amino acid substitution at position 170 of the heavy chain CH1 (EU numbering), and a non-cysteine ​​amino acid to a cysteine ​​amino acid substitution at position 162 or 164 of the light chain CL (EU numbering);

[0401] (iv) a non-cysteine ​​amino acid to a cysteine ​​amino acid substitution at position 173 of the heavy chain CH1 (EU numbering), and a non-cysteine ​​amino acid to a cysteine ​​amino acid substitution at position 160 or 162 of the light chain CL (EU numbering);

[0402] (v) a non-cysteine ​​amino acid to a cysteine ​​amino acid substitution at position 128 of the heavy chain CH1 (EU numbering), and a non-cysteine ​​amino acid to a cysteine ​​amino acid substitution at position 118 of the light chain CL (EU numbering);

[0403] (vi) a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 134 of the heavy chain CH1 (EU numbering), and a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 116 of the light chain CL (EU numbering);

[0404] (vii) a non-cysteine ​​amino acid to a cysteine ​​amino acid substitution at position 136 of the heavy chain CH1 (EU numbering), and a non-cysteine ​​amino acid to a cysteine ​​amino acid substitution at position 114 of the light chain CL (EU numbering);

[0405] (viii) a non-cysteine ​​amino acid to a cysteine ​​amino acid substitution at position 171 of the heavy chain CH1 (EU numbering), and a non-cysteine ​​amino acid to a cysteine ​​amino acid substitution at position 162 of the light chain CL (EU numbering);

[0406] (ix) a non-cysteine ​​amino acid to a cysteine ​​amino acid substitution at position 139 of the heavy chain CH1 (EU numbering), and a non-cysteine ​​amino acid to a cysteine ​​amino acid substitution at position 116 of the light chain CL (EU numbering).

[0407] 3. The antibody or antigen-binding fragment thereof of embodiment 2, wherein the disulfide bond remodeling mutation comprises

[0408] (i) contains F126C in CH1 and Q124C in CL (EU numbering);

[0409] (ii) contains H168C in CH1 and T164C in CL (EU numbering);

[0410] (iii) contains F170C in CH1 and T164C in CL (EU numbering);

[0411] (iv) contains F170C in CH1 and S162C in CL (EU numbering);

[0412] (v) contains V173C in CH1 and S162C in CL (EU numbering);

[0413] (vi) contains V173C in CH1 and Q160C in CL (EU numbering);

[0414] (vii) contains L128C in CH1 and F118C in CL (EU numbering);

[0415] (viii) contains S134C in CH1 and F116C in CL (EU numbering);

[0416] (ix) contains S136C in CH1 and S114C in CL (EU numbering);

[0417] (x) contains P171C in CH1 and S162C in CL (EU numbering);

[0418] (xi) Contains T139C in CH1 and F116C in CL (EU numbering).

[0419] 4. An antibody or antigen-binding fragment thereof, comprising one or more first antigen-binding regions that specifically bind to a first antigen and one or more second antigen-binding regions that specifically bind to a second antigen, wherein at least one of the antigen-binding regions comprises a heavy chain CH1 and a light chain CL, and comprises a disulfide bond remodeling mutation at the CH1-CL interface, wherein the disulfide bond remodeling mutation comprises T139C in CH1 and F116C in CL, wherein the first antigen-binding region and the second antigen-binding region may be the same or different.

[0420] 5. The antibody or antigen-binding fragment thereof according to any one of embodiments 2-4, wherein the disulfide bond remodeling mutation further comprises substitution of an interchain cysteine ​​with a non-cysteine ​​within the heavy chain constant region-light chain constant region interface.

[0421] 6. The antibody or antigen-binding fragment thereof of embodiment 5, wherein the interchain cysteine ​​substitutions by non-cysteine ​​include substitutions of cysteine ​​at position 220 in CH1 by non-cysteine, and substitutions of cysteine ​​at position 214 in CL by non-cysteine.

[0422] 7. The antibody or antigen-binding fragment thereof of embodiment 6, wherein the interchain cysteine ​​substitutions by non-cysteine ​​residues include C220S / A / V in CH1 and C214S / A / V in CL (EU numbering).

[0423] 8. The antibody or antigen-binding fragment thereof of embodiment 6, wherein the disulfide bond remodeling mutation comprises

[0424] (i) F126C and C220S in CH1, and Q124C and C214S in CL (EU numbering);

[0425] (ii) contains H168C and C220S in CH1 and T164C and C214S in CL (EU numbering);

[0426] (iii) contains F170C and C220S in CH1 and T164C and C214S in CL (EU numbering);

[0427] (iv) contains F170C and C220S in CH1 and S162C and C214S in CL (EU numbering);

[0428] (v) containing V173C and C220S in CH1 and S162C and C214S in CL (EU numbering);

[0429] (vi) contains V173C and C220S in CH1 and Q160C and C214S in CL (EU numbering);

[0430] (vii) contains L128C and C220S in CH1, and contains F118C and C214S in CL (EU numbering);

[0431] (viii) Contains S134C and C220S in CH1 and F116C and C214S in CL (EU numbering);

[0432] (ix) containing S136C and C220S in CH1 and S114C and C214S in CL (EU numbering);

[0433] (x) containing P171C and C220S in CH1 and S162C and C214S in CL (EU numbering);

[0434] (xi) Contains T139C and C220S in CH1, and contains F116C and C214S in CL (EU numbering).

[0435] 9. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 8, wherein the CH1 region is human IgG CH1, e.g., human IgG1 CH1, human IgG2 CH1, human IgG3 CH1 or human IgG4 CH1, e.g., the CH1 region comprises the amino acid sequence of SEQ ID NO: 116;

[0436] and / or the CL region is a human kappa light chain CL region or a human lambda light chain CL region, for example, the CL region comprises the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55.

[0437] 10. The antibody or antigen-binding fragment thereof according to embodiment 8 or 9, wherein CH1

[0438] (i) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 129, and comprises F126C and C220S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 129;

[0439] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 133, and comprises H168C and C220S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 133;

[0440] (iii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 135, and comprises F170C and C220S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 135;

[0441] (iv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 137, and comprises V173C and C220S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 137;

[0442] (v) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 139, and comprises L128C and C220S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 139;

[0443] (vi) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 141, and comprises S134C and C220S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 141;

[0444] (vii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 143, and comprises S136C and C220S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 143;

[0445] (viii) comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 145, and comprises P171C and C220S; or comprises or consists of the amino acid sequence of SEQ ID NO: 145; or

[0446] (ix) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 147, and comprises T139C and C220S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 147.

[0447] 11. The antibody or antigen-binding fragment thereof according to any one of embodiments 8-10, wherein CL

[0448] (i) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 128, and comprises Q124C and C124S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 128;

[0449] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 149, and comprises T164C and C124S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 149;

[0450] (iii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 151, and comprises S162C and C124S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 151;

[0451] (iv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 35, and comprises Q160C and C124S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 35;

[0452] (v) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 37, and comprises F118C and C124S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 37;

[0453] (vii) comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 98, and comprises F116C and C124S; or comprises or consists of the amino acid sequence of SEQ ID NO: 98; or

[0454] (viii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 100, and comprises S114C and C124S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 100.

[0455] 12. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-11, wherein the position of the amino acid pair on the interface of the heavy chain variable region and the light chain variable region is selected from: position 39 of the heavy chain variable region and position 38 of the light chain variable region, position 45 of the heavy chain variable region and position 87 of the light chain variable region, position 45 of the heavy chain variable region and position 44 of the light chain variable region, position 91 of the heavy chain variable region and position 43 of the light chain variable region, or position 91 of the heavy chain variable region and position 44 of the light chain variable region (Kabat numbering).

[0456] 13. The antibody or antigen-binding fragment thereof of any one of embodiments 1-12, wherein the position of an amino acid pair at the interface between the heavy and light chain variable regions in at least one first antigen-binding region is the same as that in at least one second antigen-binding region. 14. The antibody or antigen-binding fragment thereof of embodiment 13, wherein the mutated amino acid at a position in the amino acid pair in the first antigen-binding region has an opposite charge to the mutated amino acid at the same position in the second antigen-binding region.

[0457] 15. The antibody or antigen-binding fragment thereof of embodiment 14, wherein

[0458] (1) in the first antigen-binding region, the amino acid at position 39 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 38 of the light chain variable region is substituted with a negatively charged amino acid; and in the second antigen-binding region, the amino acid at position 39 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 38 of the light chain variable region is substituted with a positively charged amino acid;

[0459] (2) in the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 87 of the light chain variable region is substituted with a negatively charged amino acid; and in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 87 of the light chain variable region is substituted with a positively charged amino acid;

[0460] (3) in the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a negatively charged amino acid; and in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a positively charged amino acid;

[0461] (4) in the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 43 of the light chain variable region is substituted with a negatively charged amino acid; and in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 43 of the light chain variable region is substituted with a positively charged amino acid; or

[0462] (5) in the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a negatively charged amino acid; and in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a positively charged amino acid.

[0463] 16. The antibody or antigen-binding fragment thereof of embodiment 15, wherein the positively charged amino acid is selected from K, R or H, and / or the negatively charged amino acid is selected from E or D.

[0464] 17. The antibody or antigen-binding fragment thereof of embodiment 16, wherein

[0465] (i) In the first antigen-binding region, the amino acid at position 39 of the heavy chain variable region is substituted with K, and the amino acid at position 38 of the light chain variable region is substituted with D. Simultaneously, in the second antigen-binding region, the amino acid at position 39 of the heavy chain variable region is substituted with D, and the amino acid at position 38 of the light chain variable region is substituted with K;

[0466] (ii) In the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with K, and the amino acid at position 87 of the light chain variable region is substituted with D. Simultaneously, in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with D, and the amino acid at position 87 of the light chain variable region is substituted with K;

[0467] (iii) In the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with K, and the amino acid at position 44 of the light chain variable region is substituted with D. Simultaneously, in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with D, and the amino acid at position 44 of the light chain variable region is substituted with K.

[0468] (iv) In the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with K, and the amino acid at position 43 of the light chain variable region is substituted with D. Simultaneously, in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with D, and the amino acid at position 43 of the light chain variable region is substituted with K.

[0469] (v). In the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with K, and the amino acid at position 44 of the light chain variable region is substituted with D. Simultaneously, in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with D, and the amino acid at position 44 of the light chain variable region is substituted with K.

[0470] 18. The antibody or antigen-binding fragment thereof of any one of embodiments 1-17, wherein the multispecific antibody comprises a charge mutation and a disulfide bond remodeling mutation.

[0471] 19. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-18, wherein one or more or all antigen-binding regions that bind to the same antigen comprise the same mutation.

[0472] 20. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-19, wherein the first antigen-binding region comprises a heavy chain variable region, a heavy chain constant region CH1, a light chain variable region and a light chain constant region CL, and the second antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL.

[0473] 21. The antibody or antigen-binding fragment thereof of embodiment 20, wherein

[0474] 1) The first antigen binding region comprises a disulfide bond remodeling mutation as defined in any one of embodiments 1-17; or

[0475] 2) the first antigen binding region comprises a charge mutation as defined in any one of embodiments 1-17;

[0476] 3) the first antigen-binding region comprises a disulfide bond remodeling mutation and a charge mutation as defined in any one of embodiments 1-17; or

[0477] 4) The first antigen-binding region comprises a disulfide bond remodeling mutation and a charge mutation as defined in any one of embodiments 1-17; and the second antigen-binding region comprises a charge mutation as defined in any one of embodiments 1-17.

[0478] 22. The antibody or antigen-binding fragment thereof of embodiment 21, wherein

[0479] 1) The first antigen-binding region comprises a disulfide bond remodeling mutation as defined in any one of embodiments 2-8;

[0480] 2) the first antigen binding region comprises a charge mutation as defined in any one of embodiments 12-17;

[0481] 3) the first antigen binding region comprises a disulfide bond remodeling mutation as defined in any one of embodiments 2-8 and a charge mutation as defined in any one of embodiments 12-17; or

[0482] 4) The first antigen-binding region and the second antigen-binding region comprise a charge mutation as defined in any one of embodiments 12-17, and the first antigen-binding region further comprises a disulfide bond remodeling mutation as defined in any one of embodiments 2-8.

[0483] 23. The antibody or antigen-binding fragment thereof according to embodiment 22, wherein the first antigen-binding region of the multispecific antibody comprises a combination of mutations selected from the group consisting of:

[0484] Optionally, the second antigen binding region further comprises a combination of mutations selected from the following:

[0485] 24. The antibody or antigen-binding fragment thereof according to embodiment 23, wherein the first antigen-binding region and the second antigen-binding region of the multispecific antibody each comprise the following mutation combinations:

[0486] 25. An antibody or antigen-binding fragment thereof, comprising one or more first antigen-binding regions that specifically bind to a first antigen and one or more second antigen-binding regions that specifically bind to a second antigen, wherein at least one of the antigen-binding regions comprises a heavy chain variable region and a light chain variable region, and the amino acid pairs at the interface between the heavy chain variable region and the light chain variable region comprise charge mutations, wherein the charge mutations are mutations of the two amino acids on the amino acid pairs to amino acids with opposite charges, wherein the positions of the amino acid pairs at the interface between the heavy chain variable region and the light chain variable region are selected from the group consisting of: position 45 of the heavy chain variable region and position 87 of the light chain variable region, position 45 of the heavy chain variable region and position 44 of the light chain variable region, position 91 of the heavy chain variable region and position 43 of the light chain variable region, or position 91 of the heavy chain variable region and position 44 of the light chain variable region (Kabat numbering).

[0487] 26. The antibody or antigen-binding fragment thereof of embodiment 25, wherein the positions of the amino acid pairs at the interface between the heavy chain variable region and the light chain variable region in at least one first antigen-binding region are the same as the positions in at least one second antigen-binding region, wherein the first antigen and the second antigen are the same or different.

[0488] 27. The antibody or antigen-binding fragment thereof of embodiment 26, wherein the mutated amino acid at the position of the amino acid pair in the first antigen-binding region and the mutated amino acid at the same position in the second antigen-binding region have opposite charges.

[0489] 28. The antibody or antigen-binding fragment thereof of embodiment 27, wherein

[0490] (1) in the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 87 of the light chain variable region is substituted with a negatively charged amino acid; and in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 87 of the light chain variable region is substituted with a positively charged amino acid;

[0491] (2) in the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a negatively charged amino acid; and in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a positively charged amino acid;

[0492] (3) in the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 43 of the light chain variable region is substituted with a negatively charged amino acid; and in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 43 of the light chain variable region is substituted with a positively charged amino acid; or

[0493] (4) in the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a negatively charged amino acid; and in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a positively charged amino acid.

[0494] 29. The antibody or antigen-binding fragment thereof of embodiment 28, wherein the positively charged amino acid is selected from K, R or H, and / or the negatively charged amino acid is selected from E or D.

[0495] 30. The antibody or antigen-binding fragment thereof of embodiment 29, wherein

[0496] (i) In the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with K, and the amino acid at position 87 of the light chain variable region is substituted with D. Simultaneously, in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with D, and the amino acid at position 87 of the light chain variable region is substituted with K;

[0497] (ii) In the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with K, and the amino acid at position 44 of the light chain variable region is substituted with D. Simultaneously, in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with D, and the amino acid at position 44 of the light chain variable region is substituted with K.

[0498] (iii) In the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with K, and the amino acid at position 43 of the light chain variable region is substituted with D, and in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with D, and the amino acid at position 43 of the light chain variable region is substituted with K; or

[0499] (iv). In the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with K, and the amino acid at position 44 of the light chain variable region is substituted with D. Simultaneously, in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with D, and the amino acid at position 44 of the light chain variable region is substituted with K.

[0500] 31. The antibody or antigen-binding fragment thereof of any one of embodiments 1-30, wherein all first antigen-binding regions comprise the same mutation, and all second antigen-binding regions comprise the same mutation.

[0501] 32. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-31, wherein the first antigen-binding region is a Fab fragment of the antibody, and / or the second antigen-binding region is a Fab fragment of the antibody.

[0502] 33. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-32, wherein the light chain constant region comprised by one or more or all of the first antigen-binding regions of the antibody is a Kappa light chain constant region, and the light chain constant region comprised by one or more or all of the second antigen-binding regions is a Lambda light chain constant region, or wherein the light chain constant region comprised by one or more or all of the first antigen-binding regions is a Lambda light chain constant region, and the light chain constant region comprised by one or more or all of the second antigen-binding regions is a Kappa light chain constant region.

[0503] 34. The antibody or antigen-binding fragment thereof of any one of embodiments 1-33, wherein the antibody further comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different.

[0504] 35. The antibody or antigen-binding fragment thereof of embodiment 34, wherein the first Fc region and the second Fc region are respectively human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 122 or 123, or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or more identity thereto.

[0505] 36. The multispecific antibody of embodiment 34 or 35, wherein corresponding Knob mutations and Hole mutations are introduced into the first Fc region and the second Fc region based on Knob-in-Hole technology.

[0506] 37. The antibody or antigen-binding fragment thereof of embodiment 36, wherein

[0507] a) one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises T366S, L368A and Y407V (numbering according to the EU index), or

[0508] b) One Fc region comprises amino acid substitutions S354C and T366W, and the other Fc region comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering is according to the EU index).

[0509] 38. The multispecific antibody of embodiment 36, wherein

[0510] a) one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 125, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 124;

[0511] b) one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 125, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 124;

[0512] c) one Fc-region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 125 and comprises the mutations S354C and T366W, and the other Fc-region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 124 and comprises the mutations Y349C, T366S, L368A, and Y407V;

[0513] d) one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 20 or 131, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 101 or 130;

[0514] e) one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 20 or 131, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 101 or 130; or

[0515] f) one Fc-region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 20 or 131 and comprises the mutation T366W, and the other Fc-region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 101 or 130 and comprises the mutations T366S, L368A, and Y407V.

[0516] 39. The antibody or antigen-binding fragment thereof according to embodiment 34 or 35, wherein mutations are introduced into the first Fc region and the second Fc region based on Innobody technology to promote heterodimerization of the first Fc region and the second Fc region.

[0517] 40. The antibody or antigen-binding fragment thereof of embodiment 39, wherein the CH3 of one Fc region comprises S364R and D399K mutations, and the CH3 mutations of the other Fc region comprise Y349T, K370S and K409D mutations.

[0518] 41. The antibody or antigen-binding fragment thereof of embodiment 40, wherein

[0519] a) one Fc region polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 126, and the other Fc region polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 127;

[0520] b) one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 126, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 127; or

[0521] c) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence of amino acids set forth in SEQ ID NO: 126 and comprises the mutations S364R and D399K, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence of amino acids set forth in SEQ ID NO: 127 and comprises the mutations Y349T, K370S, and K409D.

[0522] 42. The antibody or antigen-binding fragment thereof of any one of embodiments 34-41, wherein the first and / or second Fc region comprises L234A / L235A mutations.

[0523] 43. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-42, wherein the antigen bound by the antibody or antigen-binding fragment is selected from one or more of the following: CD3, BCMA, CD20, Her2, Her3, B7H3, EGFR, Hel, cMET, Axl, GPRC5D, immune checkpoint molecules such as PD-1, PD-L1, CD47, and immune activation molecules such as 4-1BB, CD40, and OX40.

[0524] 44. The antibody or antigen-binding fragment thereof of any one of embodiments 1-43, wherein the first antigen and the second antigen are different and the antibody is a bispecific antibody.

[0525] 45. The antibody or antigen-binding fragment thereof of embodiment 44, wherein the bispecific antibody binds to two antigens selected from the group consisting of CD3 / BCMA, CD3 / CD20, CD3 / Her2, Her2 / Her3, B7H3 / EGFR, PD-L1 / CD47, PD-L1 / CD40, PD-L1 / 4-1BB, EGFR / Her3, Her2 / Hel, Her2 / PD-1, Her2 / PD-L1, Her2 / CD47, Her2 / cMet, or CD3 / GPRC5D.

[0526] 46. ​​The antibody or antigen-binding fragment thereof of embodiment 44 or 45, wherein the bispecific antibody comprises a first antigen-binding region that specifically binds to a first antigen and a second antigen-binding region that specifically binds to a second antigen, wherein the first antigen-binding region comprises a first Fab and the second antigen-binding region comprises a second Fab, wherein the first Fab is connected at the C-terminus of its CH1 to the N-terminus of the first Fc region (via or without a linker, such as a hinge region), and the second Fab is connected at the C-terminus of its CH1 to the N-terminus of the second Fc region (via or without a linker, such as a hinge region).

[0527] 47. The antibody or antigen-binding fragment thereof according to embodiment 46, wherein the bispecific antibody is an IgG-like antibody having the configuration shown in Figure 1.

[0528] 48. The antibody or antigen-binding fragment thereof of embodiment 46 or 47, comprising

[0529] Heavy chain 1: comprises or consists of the following from N-terminus to C-terminus: a first Fab heavy chain variable region-a heavy chain constant region CH1-a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region);

[0530] Light chain 1: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the first Fab - the light chain constant region;

[0531] Heavy chain 2: comprises or consists of the following from N-terminus to C-terminus: a second Fab heavy chain variable region - a heavy chain constant region CH1 - a second Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region);

[0532] Light chain 2: comprises or consists of the following from N-terminus to C-terminus: the light chain variable region of the second Fab - the light chain constant region.

[0533] 49. The antibody or antigen-binding fragment thereof of embodiment 48; wherein

[0534] (1) The first antigen-binding region specifically binds to Her2, wherein the heavy chain 1 and the light chain 1 respectively comprise the amino acid sequence shown in the following SEQ ID NO: or comprise an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown, or consist of the sequence shown in the following SEQ ID NO:

[0535] i) SEQ ID NO:43 and SEQ ID NO:45;

[0536] ii) SEQ ID NO:48 and SEQ ID NO:49;

[0537] iii) SEQ ID NO: 21 and SEQ ID NO: 22;

[0538] iv) SEQ ID NO: 25 and SEQ ID NO: 26;

[0539] v) SEQ ID NO: 25 and SEQ ID NO: 29;

[0540] vi) SEQ ID NO: 31 and SEQ ID NO: 32; or

[0541] vii) SEQ ID NO: 31 and SEQ ID NO: 29;

[0542] and / or

[0543] (2) The second antigen-binding region specifically binds to Hel, and the heavy chain 2 and the light chain 2 respectively comprise the amino acid sequence shown in the following SEQ ID NO: or comprise an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown, or consist of the sequence shown in the following SEQ ID NO:

[0544] i) SEQ ID NO:41 and SEQ ID NO:42;

[0545] ii) SEQ ID NO:23 and SEQ ID NO:24;

[0546] iii) SEQ ID NO: 27 and SEQ ID NO: 28;

[0547] iv) SEQ ID NO: 27 and SEQ ID NO: 30;

[0548] v) SEQ ID NO: 33 and SEQ ID NO: 34; or

[0549] vi) SEQ ID NO: 33 and SEQ ID NO: 30;

[0550] (2)-1 The second antigen-binding region specifically binds to PD-1, and the heavy chain 2 and light chain 2 respectively comprise the amino acid sequence set forth in the following SEQ ID NO:, or comprise an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth, or consist of the sequence set forth in the following SEQ ID NO: 46 and SEQ ID NO: 47;

[0551] (2)-2 The second antigen-binding region specifically binds to CD47, and the heavy chain 2 and light chain 2 respectively comprise the amino acid sequence set forth in the following SEQ ID NO:, or comprise an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth, or consist of the sequence set forth in the following SEQ ID NO: 50 and SEQ ID NO: 51;

[0552] (3)-2 The second antigen-binding region specifically binds to c-Met, and the heavy chain 2 and the light chain 2 respectively comprise the amino acid sequence shown in the following SEQ ID NO:, or comprise an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the shown amino acid sequence, or consist of the sequence shown in the following SEQ ID NO: 52 and SEQ ID NO: 53.

[0553] 50. The multispecific antibody of embodiment 44 or 45, wherein the bispecific antibody comprises one first antigen-binding region that specifically binds to a first antigen and two second antigen-binding regions that specifically bind to a second antigen, wherein the first antigen-binding region comprises a first Fab, the second antigen-binding region comprises a second Fab, wherein one of the second Fabs is connected at the C-terminus of its CH1 to the N-terminus of the first Fc region (via or without a linker, such as a hinge region), one of the first Fabs is connected at the C-terminus of its CH1 to the N-terminus of the second Fc region (via or without a linker, such as a hinge region), and the other second Fab is connected at the C-terminus of its CH1 to the N-terminus of the heavy chain variable region of the first Fab (via or without a linker).

[0554] 51. The antibody or antigen-binding fragment thereof according to embodiment 50, wherein the bispecific antibody is a 2+1 N-terminal configuration, which has the configuration shown in Figure 5A.

[0555] 52. The antibody or antigen-binding fragment thereof of embodiment 51, wherein the bispecific antibody comprises

[0556] Heavy chain 1: from N-terminus to C-terminus, comprises or consists of: a second Fab heavy chain variable region-heavy chain constant region CH1-a first Fab heavy chain variable region-heavy chain constant region CH1-a second Fc region, wherein the first Fab heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region), and the second Fab is connected at the C-terminus of its CH1 to the N-terminus of the heavy chain variable region of the first Fab (via or without a linker);

[0557] Light chain 1: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the first Fab - the light chain constant region;

[0558] Heavy chain 2: comprises or consists of the following from N-terminus to C-terminus: a second Fab heavy chain variable region - a heavy chain constant region CH1 - a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region);

[0559] Light chain 2: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the second Fab - the light chain constant region,

[0560] The bispecific antibody comprises two light chains 2.

[0561] 53. The antibody or antigen-binding fragment thereof of embodiment 52, wherein the first antigen-binding region binds CD3 and the second antigen-binding region binds GPRC5D, for example, wherein

[0562] 1) Heavy chain 1 comprises the amino acid sequence of SEQ ID NO: 58, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 58;

[0563] Light chain 1 comprises the amino acid sequence of SEQ ID NO:59, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:59;

[0564] Heavy chain 2 comprises the amino acid sequence of SEQ ID NO: 57, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 57; and / or

[0565] Light chain 2 comprises the amino acid sequence of SEQ ID NO:56, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:56; or

[0566] 2) heavy chain 1 comprises the amino acid sequence of SEQ ID NO: 62, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 62, or consists of such an amino acid sequence;

[0567] Light chain 1 comprises the amino acid sequence of SEQ ID NO:63, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:63;

[0568] Heavy chain 2 comprises the amino acid sequence of SEQ ID NO: 61, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 61; and / or

[0569] Light chain 2 comprises the amino acid sequence shown in SEQ ID NO:60, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:60, or consists of the amino acid sequence.

[0570] 54. The multi-antibody or antigen-binding fragment thereof of embodiment 44 or 45, wherein the bispecific antibody comprises one first antigen-binding region that specifically binds to a first antigen and two second antigen-binding regions that specifically bind to a second antigen, wherein the first antigen-binding region comprises a first Fab, the second antigen-binding region comprises a second Fab, wherein the two second Fabs are connected at the C-terminus of their CH1 to the N-terminus of the first Fc region and the N-terminus of the second Fc region, respectively (via or without a linker, such as a hinge region), and the first Fab is connected at the N-terminus of the heavy chain variable region to the C-terminus of the second Fc region (via or without a linker).

[0571] 55. The antibody or antigen-binding fragment thereof according to embodiment 54, wherein the bispecific antibody is a 2+1 C-terminal configuration, which has the configuration shown in Figure 5B.

[0572] 56. The antibody or antigen-binding fragment thereof of embodiment 55, wherein the bispecific antibody comprises

[0573] Heavy chain 1: comprises or consists of the following from N-terminus to C-terminus: a second Fab heavy chain variable region-a heavy chain constant region CH1-a second Fc region-a first Fab heavy chain variable region-a heavy chain constant region CH1, wherein the second Fab is linked to the N-terminus of the second Fc region at the C-terminus of its CH1 (via or without a linker, such as a hinge region), and the first Fab is linked to the C-terminus of the second Fc region at the N-terminus of the heavy chain variable region (via or without a linker);

[0574] Light chain 1: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the first Fab - the light chain constant region;

[0575] Heavy chain 2: comprises or consists of the following from N-terminus to C-terminus: a second Fab heavy chain variable region - a heavy chain constant region CH1 - a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region);

[0576] Light chain 2: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the second Fab - the light chain constant region,

[0577] The bispecific antibody comprises two light chains 2.

[0578] 57. The antibody or antigen-binding fragment thereof of embodiment 56, wherein the first antigen-binding region binds CD3 and the second antigen-binding region binds GPRC5D, for example, wherein

[0579] 1) Heavy chain 1 comprises the amino acid sequence of SEQ ID NO: 64, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 64;

[0580] Light chain 1 comprises the amino acid sequence of SEQ ID NO:59, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:59;

[0581] Heavy chain 2 comprises the amino acid sequence of SEQ ID NO: 57, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 57; and / or

[0582] Light chain 2 comprises the amino acid sequence of SEQ ID NO:56, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:56; or

[0583] 2) Heavy chain 1 comprises the amino acid sequence of SEQ ID NO: 65, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 65;

[0584] Light chain 1 comprises the amino acid sequence of SEQ ID NO:63, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:63;

[0585] Heavy chain 2 comprises the amino acid sequence of SEQ ID NO: 61, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 61; and / or

[0586] Light chain 2 comprises the amino acid sequence shown in SEQ ID NO:60, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:60, or consists of the amino acid sequence.

[0587] 58. The antibody or antigen-binding fragment thereof of embodiment 44 or 45, wherein the bispecific antibody comprises two first antigen-binding regions that specifically bind to a first antigen and two second antigen-binding regions that specifically bind to a second antigen, wherein the first antigen-binding region comprises a first Fab and the second antigen-binding region comprises a second Fab, wherein the two second Fabs are respectively connected at the C-terminus of their CH1 to the N-termini of two Fc regions (via or without a linker, such as a hinge region) (the Fc regions may be the same or different), and the two first Fabs are respectively connected at the N-terminus of the heavy chain variable region to the C-termini of the two Fc regions (via or without a linker).

[0588] 59. The antibody or antigen-binding fragment thereof of embodiment 58, wherein the bispecific antibody is a 2+2 C-terminal configuration, which has the configuration shown in Figure 5C.

[0589] 60. The antibody or antigen-binding fragment thereof of embodiment 59, wherein the bispecific antibody comprises

[0590] Heavy chain: from N-terminus to C-terminus, comprising or consisting of: a second Fab heavy chain variable region-heavy chain constant region CH1-Fc region-a first Fab heavy chain variable region-heavy chain constant region CH1, wherein the second Fab is linked to the N-terminus of the Fc region at the C-terminus of its CH1 (via or without a linker, such as a hinge region), and the first Fab is linked to the C-terminus of the Fc region at the N-terminus of the heavy chain variable region (via or without a linker);

[0591] Light chain 1: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the first Fab - the light chain constant region;

[0592] Light chain 2: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the second Fab - the light chain constant region,

[0593] The bispecific antibody comprises two heavy chains.

[0594] 61. The antibody or antigen-binding fragment thereof of embodiment 60, wherein the first antigen-binding region binds CD3 and the second antigen-binding region binds GPRC5D, for example, wherein

[0595] 1) The heavy chain comprises the amino acid sequence of SEQ ID NO: 66, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 66;

[0596] Light chain 1 comprises the amino acid sequence of SEQ ID NO: 59, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 59; and / or

[0597] Light chain 2 comprises the amino acid sequence of SEQ ID NO:56, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:56; or

[0598] 2) the heavy chain comprises the amino acid sequence of SEQ ID NO: 67, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 67;

[0599] Light chain 1 comprises the amino acid sequence of SEQ ID NO: 63, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 63, or consists of the amino acid sequence; and / or

[0600] Light chain 2 comprises the amino acid sequence shown in SEQ ID NO:60, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:60, or consists of the amino acid sequence.

[0601] 62. The antibody or antigen-binding fragment thereof of any one of embodiments 1-43, wherein the first antigen and the second antigen are different, and the antibody further comprises an antigen-binding region that specifically binds to one or more other antigens.

[0602] 63. The antibody or antigen-binding fragment thereof of embodiment 62, wherein the antibody is a trispecific antibody.

[0603] 64. The antibody or antigen-binding fragment thereof of embodiment 63, wherein the trispecific antibody comprises a first antigen-binding region that specifically binds to a first antigen, a second antigen-binding region that specifically binds to a second antigen, and a third antigen-binding region that specifically binds to a third antigen, wherein the first antigen-binding region comprises a first Fab, the second antigen-binding region comprises a second Fab, wherein the first Fab is connected at the C-terminus of its CH1 to the N-terminus of the first Fc region (via or without a linker, such as a hinge region), the second Fab is connected at the C-terminus of its CH1 to the N-terminus of the second Fc region (via or without a linker, such as a hinge region), and the third antigen-binding region comprises an scFv (e.g., VH-VL or VL-VH), which is connected at the N-terminus or C-terminus of its VH or VL (e.g., the scFv is VL-VH, such as the N-terminus of VL) to the C-terminus of the second Fc region via or without a linker.

[0604] 65. The antibody or antigen-binding fragment thereof according to embodiment 64, wherein the configuration of the trispecific antibody is a 2+1 C-terminal configuration, as shown in Figure 7A or B.

[0605] 66. The antibody or antigen-binding fragment thereof of embodiment 65, wherein the multispecific antibody comprises

[0606] Heavy chain 1: comprises or consists of the following from N-terminus to C-terminus: a first Fab heavy chain variable region-a heavy chain constant region CH1-a first Fc region, wherein the C-terminus of the first Fab is connected to the N-terminus of the first Fc region (via or without a linker, such as a hinge region);

[0607] Light chain 1: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the first Fab - the light chain constant region;

[0608] Heavy chain 2: comprises or consists of the following from N-terminus to C-terminus: a second Fab heavy chain variable region-a heavy chain constant region CH1-a second Fc region-scFv, wherein the heavy chain constant region CH1 is linked at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region), and the scFv is linked at the N-terminus or C-terminus of its VH or VL (e.g., the scFv is VL-VH, e.g., the N-terminus of VL) to the C-terminus of the second Fc region via or without a linker;

[0609] Light chain 2: comprises or consists of the following from N-terminus to C-terminus: the light chain variable region of the second Fab - the light chain constant region.

[0610] 67. The antibody or antigen-binding fragment thereof of embodiment 66, wherein the first antigen-binding region specifically binds GPRC5D, the second antigen-binding region specifically binds CD3, and / or the third antigen-binding region specifically binds BCMA, e.g., wherein

[0611] Heavy chain 1 comprises the amino acid sequence of SEQ ID NO:71, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:71;

[0612] Light chain 1 comprises the amino acid sequence of SEQ ID NO:72, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:72;

[0613] Heavy chain 2 comprises the amino acid sequence of SEQ ID NO: 73, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 73; and / or

[0614] Light chain 2 comprises the amino acid sequence shown in SEQ ID NO:74, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:74, or consists of the amino acid sequence.

[0615] 68. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 67, or any one or more chains thereof.

[0616] 69. An expression vector comprising the nucleic acid of embodiment 68.

[0617] 70. A host cell comprising the nucleic acid of embodiment 68 or the expression vector of embodiment 69.

[0618] 71. A method for preparing the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 67, comprising:

[0619] a) introducing nucleic acids encoding each chain of the antibody or antigen-binding fragment thereof into a host cell;

[0620] b) expressing and assembling the antibody or antigen-binding fragment thereof in a host cell;

[0621] Optionally, the antibody or antigen-binding fragment thereof is purified, for example, by Protein A purification.

[0622] 72. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1-67.

[0623] 73. A fusion protein comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1-67.

[0624] 74. A T cell receptor comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1-67.

[0625] II. Preparation of Binding Molecules

[0626] The present invention also provides an in vitro method for producing a binding molecule such as an antibody. In some embodiments, the binding molecule is an antibody or a fusion protein. In some embodiments, the antibody is a multispecific antibody, such as a bispecific antibody.

[0627] In one aspect, a method for preparing a binding molecule, such as an antibody, of the invention comprises:

[0628] a) introducing nucleic acids encoding each chain of a binding molecule, such as an antibody, into a host cell;

[0629] b) expressing and assembling binding molecules, such as antibodies, in host cells;

[0630] Optionally, the binding molecule, eg, antibody, is purified, eg, by Protein A purification.

[0631] In yet another embodiment, a compound, such as L-arginine, is added to the culture medium during the production of binding molecules, such as antibodies. In one embodiment, the arginine is used to stabilize the protein.

[0632] In some embodiments, the products of the present invention have the same expression level and affinity as the binding molecules, such as antibodies, before mutation.

[0633] In some embodiments, the methods of the present invention provide stable binding molecules, eg, antibodies, such as multispecific antibodies (eg, bispecific antibodies), in high yield.

[0634] In some embodiments, the methods of the present invention enable expression and assembly of each chain of a binding molecule, such as an antibody, such as a multispecific antibody (eg, a bispecific antibody), in one cell line, preferably avoiding expression in two cell lines and in vitro recombination.

[0635] III Nucleic Acids and Host Cells

[0636] The present invention provides nucleic acids encoding any chain or any monomer or domain of a binding molecule of the present invention, such as an antibody, such as a multispecific antibody, such as a bispecific antibody. Polynucleotide sequences encoding each chain can be generated using methods well known in the art. In addition, the polynucleotides and nucleic acids of the present invention can include a segment encoding a secretory signal peptide and be operably linked to an antibody or protein encoding a binding molecule of the present invention, such as an antibody, such as a multispecific antibody, such as a bispecific antibody, thereby directing secretory expression of the binding molecule of the present invention, such as an antibody, such as a multispecific antibody, such as a bispecific antibody, and each chain thereof.

[0637] The present invention also provides vectors comprising the nucleic acids of the present invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In a preferred embodiment, the expression vector of the present invention is a pCNDA vector, such as the pCNDA3.1 expression vector.

[0638] The present invention also provides host cells comprising the nucleic acid or the vector. Host cells suitable for replicating and supporting the expression of binding molecules of the present invention, such as antibodies, multispecific antibodies, such as bispecific antibodies, are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and large quantities of vector-containing cells can be grown for inoculation of large-scale fermenters, thereby obtaining sufficient quantities of multispecific antibodies, such as bispecific antibodies, for clinical use.

[0639] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293 cells, eg, Expi293 cells). Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells, as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT060562), TRI cells (as described, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr-CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NSO, P3X63 and Sp2 / 0. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphocyte (e.g., Y0, NSO, Sp20 cell).

[0640] IV. Other Molecules Comprising the Binding Molecules of the Invention or Fragments thereof

[0641] In some embodiments, the first and / or second target binding region in the binding molecules of the invention can be conjugated to a drug, prodrug or toxin or contain an acceptor group for a drug, prodrug or toxin. Such an acceptor group can be, for example, an unnatural amino acid.

[0642] Therefore, the present invention also relates to immunoconjugates (ADCs) comprising a binding molecule, eg, an antibody, such as a multispecific antibody (eg, a bispecific antibody), of the present invention.

[0643] In some embodiments, the binding molecules of the present invention or fragments thereof can also be constructed into a T cell receptor (TCR). In some embodiments, the variable region of the TCR comprises a mutation or combination of mutations described herein, such as a disulfide bond remodeling mutation, and / or a charge mutation.

[0644] In some embodiments, the binding molecules of the present invention or fragments thereof can be fused with other proteins to form fusion proteins. In some embodiments, examples of other proteins include, for example, ligands or receptors, such as granule-associated cytokines or their receptors, such as tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF) or transforming growth factor (TGF, such as TGF-β) or its receptor, and fragments thereof.

[0645] V. Products and Applications

[0646] In some embodiments, the present invention also relates to a composition (eg, a pharmaceutical composition) comprising the binding molecule, eg, an antibody, eg, a multispecific antibody, eg, a bispecific antibody.

[0647] In one embodiment, the composition further comprises a pharmaceutical excipient.In one embodiment, a composition, eg, a pharmaceutical composition, comprises a binding molecule of the invention in combination with one or more other therapeutic agents.

[0648] The compositions of the present invention may also include suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients as known in the art, including buffers. As used herein, "pharmaceutical carriers" include any and all solvents, dispersion media, isotonic agents, or absorption delay agents that are physiologically compatible. For the use and purposes of pharmaceutical excipients, see also "Handbook of Pharmaceutical Excipients", Eighth Edition, RC Rowe, PJ Eskey and SCO Wen, Pharmaceutical Press, London, Chicago. The compositions of the present invention may be in various forms. These forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. The medicine comprising the binding molecule described herein can be prepared by mixing the binding molecule of the present invention with the desired purity with one or more optional pharmaceutical excipients, for example, in the form of a lyophilized formulation or an aqueous solution.

[0649] In some embodiments, the present invention relates to a method of treating a disease, such as a tumor, such as cancer, using the binding molecules, such as antibodies, such as multispecific antibodies, such as bispecific antibodies, or a use for the treatment, or a use for preparing a medicament for the treatment.

[0650] Depending on its therapeutic use, the binding molecules of the present invention, such as antibodies, such as multispecific antibodies, such as bispecific antibodies, can also be combined with other therapeutic agents. For example, when the binding molecules are used to treat tumors, the therapeutic agents are various therapeutic agents such as chemotherapeutics, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists).

[0651] In some embodiments, the present invention also provides pharmaceutical combinations or pharmaceutical combination products comprising a binding molecule of the present invention and one or more other therapeutic agents.

[0652] Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, whereby dosage units can be provided according to a dosing regimen or a drug administration interval.

[0653] In one embodiment, the kit of parts of the present invention comprises in the same package:

[0654] - a first container containing a pharmaceutical composition comprising a binding molecule of the invention;

[0655] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent. Example

[0656] Example 1. Design of mutant antibodies

[0657] Disulfide bond remodeling

[0658] One strategy that can effectively force the pairing of homologous light and heavy chains and avoid mispairing between the light and heavy chains of two antibodies is to reshape the disulfide bond connecting the light and heavy chains of one antibody and change the position of the disulfide bond so that the mispaired light and heavy chains cannot effectively form a disulfide bond. We screened the amino acid pairs at the contact surface of the heavy and light chains and selected the amino acid pairs with a distance between the two α carbon atoms less than Better than The amino acid pairs are selected such that the side chains of the amino acids point toward each other. The selected amino acid pairs are shown in Table 1 (EU numbering):

[0659] Table 1: Selection of disulfide bond remodeling positions

[0660] Charge mutation

[0661] Another strategy that can effectively force homologous light and heavy chain pairing and avoid mismatching between the light and heavy chains of two antibodies is to find amino acid pairs on the contact surface of the heavy chain and light chain, mutate them into amino acids with opposite charges, and use the principle of opposite charges attracting each other to promote homologous pairing. The selection criteria are that the conserved amino acid pairs on the interface between the heavy chain variable region and the light chain variable region, and the distance between the alpha carbon atoms of the amino acid pairs is less than The side chains are oriented toward each other. The selected amino acid pairs are shown in Table 2 (Kabat code):

[0662] Table 2: Charge mutation combinations

[0663] The final mutations employed for bispecific antibody expression in one cell line were a combination of disulfide bond remodeling and charge mutations, as shown in Table 3:

[0664] Table 3: Combinations of V region charge mutations and disulfide bond remodeling mutations

[0665] When antibody 1 is an anti-Her2 monoclonal antibody and antibody 2 is an anti-Hel monoclonal antibody, the sequences of exemplary bispecific antibody full-length chains are shown in Table 3 Combination 1 to Combination 5 in the sequence listing, i.e., the antibodies of Combination 1 to Combination 5 in Table 9.

[0666] Replacement of the light chain constant region

[0667] To further facilitate downstream purification and remove trace amounts of mismatched antibodies, the light chain constant region of one of the antibodies can be replaced from C kappa to C lambda (if one of the antibodies already has a lambda light chain, replacement is not necessary). This allows the two antibody Fabs to be one kappa type and one lambda type, and downstream purification can be performed using an affinity medium selective for kappa and lambda light chains.

[0668] Example 2. Expression of mutants in HEK293 cells and purification of mutants

[0669] Each chain of each antibody of the present invention was sent to Jinweizhi for gene synthesis and constructed onto the pCDNA3.1 template plasmid, and a certain amount of plasmid was prepared. The sequences of the chains of each antibody used in the examples are summarized in Table 4.

[0670] Filter with a 0.22 μm filter for transient expression of cells. Subculture Expi293 cells (Invitrogen) according to the required transfection volume and adjust the cell density to 3×10 the day before transfection. 6 cells / ml. On the day of transfection, the cell density was adjusted to 3×10 6 cells / ml. Use Opti-MEM medium (Gibco catalog number: 31985-070) at 1 / 10 (v / v) of the final volume as transfection buffer. Add the expression plasmid at a ratio of 1:1:1:1 (or other suitable ratio). Mix thoroughly. Add the appropriate polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid from the previous step (plasmid:PEI mass ratio of 1:3), mix thoroughly, and incubate at room temperature for 10 minutes to obtain a DNA / PEI mixture. The DNA / PEI mixture was gently poured into HEK293 cells and mixed thoroughly. After incubation at 37°C, 8% CO₂ for 24 hours, VPA (Sigma, Catalog No. P4543-100G) was added to a final concentration of 2 mM, along with 2% (v / v) Feed (1 g / L Phytone Peptone + 1 g / L Difco Select Phytone). Culture was continued for another 6 days. The culture was harvested and centrifuged at 4000 rpm for 30 minutes. The cell supernatant was filtered through a 0.45 μM filter and purified by Protein A affinity chromatography. Samples with special requirements were further purified by ion exchange chromatography, KappaSelect affinity chromatography, and LambdaFabSelect affinity chromatography.

[0671] The specific Protein A affinity chromatography purification procedure is as follows: the supernatant is purified using a prepacked column, Hitrap Mabselect Sure (GE, 11-0034-95). The procedure is as follows: Before purification, the column is equilibrated with 5 column volumes of equilibration buffer (20mM Tris, 150mM NaCl, pH 7.2); the collected supernatant is passed through the column, and the column is then washed with 10 column volumes of equilibration buffer to remove nonspecifically bound proteins; the column is rinsed with 5 column volumes of elution buffer (100mM sodium citrate, pH 3.5), and the eluate is collected. 2M Tris is added to neutralize the pH to 6.5. The purified bispecific antibody is then quantified for the correct pairing ratio using liquid chromatography-mass spectrometry (LC-MS).

[0672] The specific ion exchange chromatography purification procedure is as follows: using ultrafiltration concentrator tubes (MILLIPORE, Catalog No.: UFC901096) to exchange into pH 6.0 low-salt PB buffer (10mM phosphate, pH 6.0), and fine purification using Capto HiRes S 10 / 100 (GE, Catalog No. 29275879). Before purification, the packed column was equilibrated with 5 column volumes of pH 6.0 low-salt PB buffer; the sample after the exchange was passed through the column, and the packed column was then washed with 10 column volumes of pH 6.0 low-salt PB buffer to remove non-specifically bound proteins; during elution, the concentration of pH 6.0 high-salt PB buffer (10mM phosphate, pH 6.0, 1M NaCl) was linearly increased (linear gradient: increasing the proportion of pH 6.0 high-salt PB buffer from 30% to 100% within 30 column volumes), and the main elution peak was collected. The purified bispecific antibody was then exchanged into PBS buffer (Gibco, Catalog No. 70011-044) using ultrafiltration concentrators (MILLIPORE, Catalog No. UFC901096). The correct pairing ratio of the purified bispecific antibody was quantitatively determined using liquid chromatography-mass spectrometry (LC-MS).

[0673] The specific KappaSelect affinity chromatography purification procedure is as follows: the supernatant is purified using a prepacked HiTrap KappaSelect column (GE, 17-5458-11). The procedure is as follows: Before purification, the column is equilibrated with 5 column volumes of equilibration buffer (PBS, pH 7.4); the collected supernatant is passed through the column, and the column is then washed with 10 column volumes of equilibration buffer to remove nonspecifically bound proteins; the column is rinsed with 5 column volumes of elution buffer (0.1 M glycine buffer, pH 2.5), and the eluate is collected. 2 M Tris is added to neutralize the pH to 6.5. The purified bispecific antibody is then quantified for the correct pairing ratio using liquid chromatography-mass spectrometry (LC-MS).

[0674] The specific LambdaFabSelect affinity chromatography purification procedure is as follows: the supernatant is purified using a prepacked HiTrap LambdaFabSelect column (GE, 17-5482-11). The procedure is as follows: Before purification, the column is equilibrated with 5 column volumes of equilibration buffer (PBS, pH 7.4); the collected supernatant is passed through the column, and the column is then washed with 10 column volumes of equilibration buffer to remove nonspecifically bound proteins; the column is rinsed with 5 column volumes of elution buffer (0.1 M acetate buffer, pH 3.5), and the eluate is collected. 2 M Tris is added to neutralize the pH to 6.5. The purified bispecific antibody is then quantified for the correct pairing ratio using liquid chromatography-mass spectrometry (LC-MS).

[0675] Example 3 Screening of mutant amino acids in the present invention

[0676] Screening for mutation sites that can correctly form disulfide bonds

[0677] Using trastuzumab as a model antibody, the disulfide bond between the native heavy and light chains was mutated (heavy chain C220S, light chain C214S). The amino acid pairs selected in Table 1 were then mutated to cysteine. The mutant mAbs were expressed in HEK293 cells, purified with protein A, and subjected to non-reducing polyacrylamide gel electrophoresis, as shown in Figures 9A-9C. These mutant mAbs maintained intact molecules on non-reducing polyacrylamide gel electrophoresis, with only minimal light chain loss. The selected disulfide bond mutation sites essentially all formed normal disulfide bonds.

[0678] Furthermore, these disulfide-bonded antibodies were subjected to denaturing non-reducing capillary electrophoresis (nrCE-SDS) (Figures 9D-9M) to quantify the integrity of the disulfide bond formation. As can be seen, the integrity of the disulfide-bond-remodeled antibodies was above 98%, further demonstrating that the remodeled disulfide bonds were correctly formed.

[0679] Effects of disulfide bond remodeling mutations on the correct pairing ratio

[0680] The effect of disulfide bond remodeling mutations on the correct pairing ratio was tested using the F126C / Q124C disulfide bond remodeling mutations in Table 1 as an example. The mutant antibodies of the present invention and the wild-type antibody sequences were used as controls. After expression in HEK293 cells, purification was performed using only protein A affinity chromatography to avoid the removal of mismatched molecules by other purification methods. LC-MS was used to test the correct pairing ratio when the anti-Her2 / anti-Hel antibody pair and the anti-Her2 / anti-PD-1 antibody pair formed a bispecific antibody. Heterologous pairing was promoted by the knob-in-hole between the heavy chains.

[0681] Table 5 Bispecific antibodies with disulfide bond remodeling and correct pairing ratios

[0682] As shown in the table above, the F126C / Q124C mutation significantly increases the correct pairing rate of bispecific antibodies. For example, the correct pairing rate for wild-type Her2 / PD-1 antibody pairs reached 55.60%. With the addition of the F126C / Q124C mutation, the correct pairing rate increased to 76.93%.

[0683] It can be seen that the F126C / Q124C mutation forms the highest proportion of correctly paired molecules, which is significantly higher than the wild-type antibody sequence and also higher to a certain extent than the mutation F126C / S121C published in CN104011221B.

[0684] Furthermore, if the mutated cysteine ​​pairs do not form disulfide bonds correctly, the intact cysteine ​​residues will often react with free cysteine ​​residues in cells or solutions to form disulfide bonds, resulting in cysteine ​​modification of the protein molecule. This modification can be detected by LC-MS.

[0685] Table 6 Cysteine ​​modification test of disulfide bond remodeling bispecific antibodies

[0686] In both the Her2 / Hel bispecific antibody and the Her2 / PD-1 bispecific antibody, the F126C / Q124C mutation showed virtually no free cysteine ​​modifications, further demonstrating a high percentage of correctly formed disulfide bonds. In contrast, the Her2 / PD-1 bispecific antibody incorporating the F126C / S121C mutations disclosed in CN104011221B still showed approximately 10% free cysteine ​​modifications, indicating that some F126C / S121C disulfide bonds were not fully formed.

[0687] Disulfide bond remodeling mutations combined with V region charge mutations

[0688] The disulfide bond remodeling mutations (F126C / Q124C) were combined with charge mutations (Q39K / Q38D). After expression in HEK293 cells, the protein was purified using only protein A affinity chromatography to avoid the removal of mispaired molecules by other purification methods. LC-MS was used to determine the correct pairing ratio of the anti-Her2 / anti-Hel antibody pair in the bispecific antibody (Figure 2). A knob-in-hole was used between the heavy chains to facilitate heterologous pairing.

[0689] Table 7 Correct pairing ratio of bispecific antibodies with disulfide bond remodeling combined with charge mutation

[0690] As can be seen, the correct pairing rate of bispecific antibodies significantly improved after disulfide bond remodeling. After removing the antibody's native disulfide bonds (Table 7, combination 2, Figure 2B), the correct pairing rate was further improved compared to simply adding disulfide bonds (Table 7, combination 1, Figure 2A). Furthermore, after adding charge mutations to the V region (Table 7, combination 3, Figure 2C), the correct pairing rate was further improved.

[0691] Four different antibody pairs were used to construct bispecific antibodies. After expression in HEK293 cells, the antibodies were purified using protein A affinity chromatography alone to avoid the removal of mismatched molecules that might be removed by other purification methods. LC-MS was used to verify the correct pairing ratio in the resulting bispecific antibodies (Figure 3). The combination of disulfide bond remodeling and charge mutations was validated for universality across different antibodies. Antibody expression levels were also monitored.

[0692] Table 8-1 Antibody sequences and correct pairing ratios after introducing disulfide bond remodeling and charge mutations in different antibody pairs

[0693] All four antibody combinations maintained a correct pairing ratio of approximately 90%, demonstrating the universality of disulfide bond remodeling combined with charge mutations across different antibodies. In terms of expression levels, all four antibody combinations reached approximately 200 mg / L, achieving high expression levels comparable to monoclonal antibodies.

[0694] Effects of disulfide bond remodeling mutations combined with charge mutations at different positions on the correct pairing ratio

[0695] The natural process of IgG assembly and secretion in cells is: after the heavy chain is expressed in the cell, CH1 binds to the molecular chaperone BiP and remains in an incompletely folded state until it encounters the light chain. After encountering the light chain, the light chain first binds to VH through VL, then CL replaces BiP and binds to CH1. With the help of CL, CH1 is completely folded, and the assembly of light and heavy chains is completed. (Matthias J. Feige, An Unfolded CH1 Domain Controls the Assembly and Secretion of IgG Antibodies, Molecular Cell. 2009 Jun 12; 34(5): 569-79). From this biological process, it can be seen that the mutual recognition of VH and VL is the first process that drives the assembly of light and heavy chains. Therefore, the charge mutations of the present invention are mainly designed in the VH and VL regions. To further demonstrate the superiority of our design, we also introduced charge between CH1 and CL to compare the effect of charge mutations in the V region and C region on the correct pairing ratio. The Her2 / Hel bispecific antibody comprising any one of the mutations in Table 8-2 differs in sequence from the antibody comprising combination 3 in Table 7 only in the listed mutation combination; the other sequences remain unchanged.

[0696] Table 8-2 Effects of disulfide bond remodeling mutations combined with charge mutations at different positions on the correct pairing ratio of light and heavy chains

[0697] As can be seen, based on the same disulfide bond remodeling mutations, combining the Q39K / Q38D charge mutations in the V region with the H171K+T190K / N137D and K216E / E123R charge mutations in the C region, the charge mutations in the V region resulted in a higher proportion of correctly paired molecules, demonstrating the superiority of the design with charges located in the V region.

[0698] Other combinations of V region charge mutations and disulfide bond remodeling

[0699] In addition to Q39K and Q38D, other discovered V region charge mutations were combined with the disulfide bond-remodeling mutations F126C / Q124C to form combinations 1 through 5 (Table 3). After expression in HEK293 cells, purification was performed using protein A affinity chromatography alone to avoid the removal of mismatched molecules by other purification methods. LC-MS was used to assess the correct pairing ratio in the resulting bispecific antibody (Figure 4). Knob-in-holes were used between the heavy chains to facilitate heterologous pairing. Antibody expression levels were also monitored.

[0700] Table 9-1 Proportion of molecules correctly paired after V region mutation combined with disulfide bond remodeling

[0701] Mutation combinations 1 through 5 all achieved a correct pairing rate exceeding 90%. Combinations 1 through 3 achieved expression levels similar to those of monoclonal antibodies (200-300 mg / L). Combinations 4 and 5 achieved slightly lower expression levels, but still reached approximately 100 mg / L. Therefore, bispecific antibodies containing the mutation combinations of the present invention can achieve relatively good expression levels.

[0702] Combination of V region charge mutations with other disulfide bond remodeling mutations

[0703] In addition to F126C / Q124C, other discovered disulfide bond remodeling mutations were combined with the V region charge mutations Q39K and Q38D to form combinations 6 to 14 in Table 3. The Her2 / Hel bispecific antibodies comprising any one of the mutations in combinations 6 to 14 in Table 3 differ in sequence from those comprising any one of combinations 1 to 5 in Table 3 only in the listed combination of mutations, and the other sequences remain unchanged.

[0704] After expression in HEK293 cells, the antibody was purified using only protein A affinity chromatography to avoid the removal of mismatched molecules that may occur with other purification methods. LC-MS was used to verify the correct pairing ratio in the bispecific antibody. A knob-in-hole was used between the heavy chains to facilitate heterologous pairing. Antibody expression levels were also monitored.

[0705] Table 9-2 Proportions of molecules correctly paired after V region charge mutations combined with disulfide bond remodeling mutations

[0706] Mutation combinations 6 through 14 resulted in bispecific antibodies with a correct pairing rate of 79.46% to 98.90%, and expression levels similar to those of monoclonal antibodies (200-300 mg / L). Therefore, bispecific antibodies containing the mutation combinations of the present invention achieved good correct pairing rates and expression levels.

[0707] Example 4. Affinity determination experiment of the antibody of the present invention

[0708] The equilibrium dissociation constant (KD) of the bispecific antibodies of the present invention binding to their respective antigens was determined using biolayer interferometry (BLI). BLI affinity determination was performed according to existing methods (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5(2): p. 270-8).

[0709] Half an hour before the start of the experiment, take an appropriate number of ProA (ForteBio, 18-5010) sensors based on the number of samples and soak them in SD buffer (PBS 1×, BSA 0.1%, Tween-20 0.05%). Take 100 μl of SD buffer and antigen fusion protein and add them to a 96-well black polystyrene half-volume microplate (Greiner, 675076). Arrange the plate according to the sample position and select the sensor position. The instrument setting parameters are as follows: Run steps: Baseline, Loading ~ 1nm, Baseline, Association and Dissociation; the running time of each step depends on the sample binding and dissociation rate, the rotation speed is 400 rpm, and the temperature is 30 degrees. Use ForteBio analysis software to analyze KD values.

[0710] Using anti-Her2 antibody as a template, after introducing the mutations of combination 1 to combination 5 in Table 3, the affinity data for its antigen Her2 are shown in the table below.

[0711] Table 10. Affinity test of exemplary antibodies to human Her2

[0712] As can be seen from the above charts, there is no significant difference in affinity between the mutants and the wild-type antibody. The mutations and disulfide bond changes introduced into the light and heavy chain binding surfaces of the V region of the present invention will not affect the antibody affinity.

[0713] Example 5. Multivalent configuration expansion

[0714] In addition to the monovalent 1+1 configuration (Figure 1), the bispecific antibodies of the present invention can also be expanded to include multivalent 2+1 N-terminal configurations (Figure 5A), 2+1 C-terminal configurations (Figure 5B), and 2+2 C-terminal configurations (Figure 5C). In addition to the tandem Fab configuration, which does not require heavy chain heterologous pairing, heterologous pairing between heavy chains is promoted through our company's Innobody mutation.

[0715] According to the method of Example 2, multivalent 2+1 N-terminal configuration, 2+1 C-terminal configuration, and 2+2 C-terminal configuration were expressed and purified, and the correct pairing ratio was detected by mass spectrometry (Figure 6). The results are summarized in Table 11.

[0716] Table 11 Correct pairing ratio of multivalent configuration molecules

[0717] As shown in the results in the table above, the mutations claimed in this patent can maintain a high correct pairing ratio when applied to multivalent molecules. Furthermore, when applied to multivalent molecules, it can be seen that the combination of disulfide bond remodeling and charge mutations significantly improves the correct pairing ratio compared to disulfide bond remodeling alone.

[0718] Example 6. Cell killing experiment

[0719] The BCMA / GPRC5D / CD3 triple antibody (wild-type and incorporating the mutations claimed in this patent, see Figure 7 for configuration and sequence in the sequence table) simultaneously binds to BCMA or GPRC5D on the surface of H929 cells and CD3 on the surface of T cells in primary PBMCs, activating T cells and mediating T cell killing of BCMA or GPRC5D-positive tumor cells. This study used the lactate dehydrogenase (LDH) assay to detect the level of LDH released by dead cells in the supernatant collected 24 hours after the addition of the example antibodies to PBMCs co-cultured with BCMA-positive H929 cells and GPRC5D-positive H929 cells, thereby evaluating the antibody-mediated T cell killing ability against BCMA tumor cells and GPRC5D tumor cells. The effects of the mutations claimed in this patent on the triple antibody molecule were evaluated by comparing the BCMA / GPRC5D / CD3 triple antibody without the charge mutation and disulfide bond remodeling mutation of the present invention (Figure 7A) and the BCMA / GPRC5D / CD3 triple antibody with the mutations claimed in this patent (Figure 7B).

[0720] PBMCs (Sai ​​Li Bio, SLB-HP100A) were removed from a liquid nitrogen tank and rapidly thawed at 37°C. The cells were then added dropwise to preheated 1640 medium (Gibco, 22400-071) (containing 0.1% DNase) to obtain a 10 ml mixture. The mixture was centrifuged at 400 g for 5 minutes, resuspended in 10 ml of 1640 medium, and 10 μl of DNase was added. The cells were cultured overnight at 37°C, 5% CO2, and allowed to adhere. The suspended cells were aspirated and centrifuged at 400 g for 5 minutes, resuspended in 1640 medium, counted, and adjusted to a cell density of 4 × 106 cells / ml to serve as effector cells. BCMA- or GPRC5D-positive H929 cells (ATCC, CRL-9068) were used as target cells. The cells were centrifuged at 400 g for 5 minutes, resuspended in 1640 medium, counted, and adjusted to a cell density of 2 × 105 cells / ml. Target cells at adjusted cell density were added to a 96-well plate at 100 μl per well. A serially diluted antibody was added to the plate at 50 μl per well. Finally, PBMC effector cells were added to the plate at 50 μl per well. The final effector-target ratio was 10:1. The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 24 hours. The supernatant was collected and the LDH level released by dead cells was measured using the CytoTox96 non-radioactive cytotoxicity kit (Promega). The percentage of MM cell killing by the example antibody was then calculated.

[0721] In experiments performed as described in the above assays, the exemplified antibodies were able to induce dose-dependent killing of H929 cells by human PBMCs (see Figure 8).

[0722] In experiments conducted as described in the assay above, the cytotoxicity of the example antibodies against BCMA- or GPRC5D-positive H929 cell lines induced by PBMCs from the same donor was compared. The introduction of the mutations claimed in this patent had no effect on the cytotoxicity of the example antibodies. However, the introduction of the mutations claimed in this patent allowed the example antibodies to be expressed and produced in a single cell line, eliminating the need for expression and production in two cell lines and in vitro recombination.

Claims

1. An antibody or an antigen-binding fragment thereof, comprising one or more first antigen-binding regions that specifically bind to a first antigen and one or more second antigen-binding regions that specifically bind to a second antigen, wherein at least one or more of the antigen-binding regions comprises a heavy chain CH1 and a light chain CL, and comprises a disulfide bond remodeling mutation at the CH1-CL interface, and / or At least one or more of the antigen binding regions comprises a heavy chain variable region and a light chain variable region, and an amino acid pair at the interface between the heavy chain variable region and the light chain variable region comprises a charge mutation, wherein the charge mutation is to mutate two amino acids in the amino acid pair into amino acids with opposite charges; wherein the first antigen and the second antigen may be the same or different; Preferably, the disulfide bond remodeling mutations are only contained at the CH1-CL interface of one antigen binding region.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the disulfide bond remodeling mutation comprises (x) a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 126 of the heavy chain CH1 (EU numbering), and a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 124 of the light chain CL (EU numbering); (xi) a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 168 of the heavy chain CH1 (EU numbering), and a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 164 of the light chain CL (EU numbering); (xii) a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 170 of the heavy chain CH1 (EU numbering), and a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 162 or 164 of the light chain CL (EU numbering); (xiii) a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 173 of the heavy chain CH1 (EU numbering), and a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 160 or 162 of the light chain CL (EU numbering); (xiv) a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 128 of the heavy chain CH1 (EU numbering) and a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 118 of the light chain CL (EU numbering); (xv) a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 134 of the heavy chain CH1 (EU numbering) and a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 116 of the light chain CL (EU numbering); (xvi) a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 136 of the heavy chain CH1 (EU numbering) and a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 114 of the light chain CL (EU numbering); (xvii) a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 171 of the heavy chain CH1 (EU numbering) and a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 162 of the light chain CL (EU numbering); (xviii) a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 139 of the heavy chain CH1 (EU numbering), and a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 116 of the light chain CL (EU numbering).

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the disulfide bond remodeling mutation comprises (xii) contains F126C in CH1 and Q124C in CL (EU numbering); (xiii) contains H168C in CH1 and T164C in CL (EU numbering); (xiv) contains F170C in CH1 and T164C in CL (EU numbering); (xv) containing F170C in CH1 and S162C in CL (EU numbering); (xvi) containing V173C in CH1 and S162C in CL (EU numbering); (xvii) contains V173C in CH1 and Q160C in CL (EU numbering); (xviii) contains L128C in CH1 and F118C in CL (EU numbering); (xix) containing S134C in CH1 and F116C in CL (EU numbering); (xx) containing S136C in CH1 and S114C in CL (EU numbering); (xxi) P171C in CH1 and S162C in CL (EU numbering); (xxii) contains T139C in CH1 and F116C in CL (EU numbering).

4. An antibody or an antigen-binding fragment thereof, comprising one or more first antigen-binding regions that specifically bind to a first antigen and one or more second antigen-binding regions that specifically bind to a second antigen, wherein at least one of the antigen-binding regions comprises a heavy chain CH1 and a light chain CL, and comprises a disulfide bond remodeling mutation at the CH1-CL interface, wherein the disulfide bond remodeling mutation comprises T139C in CH1 and F116C in CL, wherein the first antigen-binding region and the second antigen-binding region may be the same or different.

5. The antibody or antigen-binding fragment thereof according to any one of claims 2 to 4, wherein the disulfide bond remodeling mutation further comprises substitution of an interchain cysteine ​​with a non-cysteine ​​within the heavy chain constant region-light chain constant region interface.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the interchain cysteine ​​is substituted with a non-cysteine, including the cysteine ​​at position 220 in CH1 being substituted with a non-cysteine, and the cysteine ​​at position 214 in CL being substituted with a non-cysteine.

7. The antibody or antigen-binding fragment thereof of claim 6, wherein the interchain cysteine ​​is substituted with a non-cysteine ​​residue comprising C220S / A / V in CH1 and comprising C214S / A / V in CL (EU numbering).

8. The antibody or antigen-binding fragment thereof of claim 6, wherein the disulfide bond remodeling mutation comprises (xii) F126C and C220S in CH1, and Q124C and C214S in CL (EU numbering); (xiii) containing H168C and C220S in CH1 and T164C and C214S in CL (EU numbering); (xiv) F170C and C220S in CH1 and T164C and C214S in CL (EU numbering); (xv) F170C and C220S in CH1 and S162C and C214S in CL (EU numbering); (xvi) containing V173C and C220S in CH1 and S162C and C214S in CL (EU numbering); (xvii) contains V173C and C220S in CH1 and Q160C and C214S in CL (EU numbering); (xviii) containing L128C and C220S in CH1 and F118C and C214S in CL (EU numbering); (xix) containing S134C and C220S in CH1 and F116C and C214S in CL (EU numbering); (xx) containing S136C and C220S in CH1 and S114C and C214S in CL (EU numbering); (xxi) P171C and C220S in CH1 and S162C and C214S in CL (EU numbering); (xxii) contains T139C and C220S in CH1, and contains F116C and C214S in CL (EU numbering).

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the CH1 region is human IgG CH1, for example, human IgG1 CH1, human IgG2 CH1, human IgG3 CH1 or human IgG4 CH1, for example, the CH1 region comprises the amino acid sequence shown in SEQ ID NO: 116; And / or the CL region is a human Kappa light chain CL region or a human Lambda light chain CL region, for example, the CL region comprises the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO:

55.

10. The antibody or antigen-binding fragment thereof according to claim 8 or 9, wherein CH1 (i) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 129, and comprises F126C and C220S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 129; (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 133, and comprises H168C and C220S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 133; (iii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 135, and comprises F170C and C220S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 135; (iv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 137, and comprises V173C and C220S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 137; (v) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 139, and comprises L128C and C220S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 139; (vi) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 141, and comprises S134C and C220S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 141; (vii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 143, and comprises S136C and C220S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 143; (viii) comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 145, and comprises P171C and C220S; or comprises or consists of the amino acid sequence of SEQ ID NO: 145; or (ix) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 147, and comprises T139C and C220S; or comprising or consisting of the amino acid sequence of SEQ ID NO:

147.

11. The antibody or antigen-binding fragment thereof according to any one of claims 8 to 10, wherein CL (i) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 128, and comprises Q124C and C124S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 128; (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 149, and comprises T164C and C124S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 149; (iii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 151, and comprises S162C and C124S; or comprising or consisting of the amino acid sequence of SEQ ID NO: 151; (iv) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:35, and comprises Q160C and C124S; or comprising or consisting of the amino acid sequence of SEQ ID NO:35; (v) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, An amino acid sequence with 94%, 95%, 96%, 97%, 98% or 99% identity, and comprising F118C and C124S; or comprising or consisting of the amino acid sequence shown in SEQ ID NO: 37; (vii) comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:98, and comprises F116C and C124S; or comprises or consists of the amino acid sequence of SEQ ID NO:98; or (viii) comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 100, and comprises S114C and C124S; or comprises or consists of the amino acid sequence of SEQ ID NO:

100.

12. The antibody or antigen-binding fragment thereof of any one of claims 1 to 11, wherein the position of the amino acid pair on the interface of the heavy chain variable region and the light chain variable region is selected from: position 39 of the heavy chain variable region and position 38 of the light chain variable region, position 45 of the heavy chain variable region and position 87 of the light chain variable region, position 45 of the heavy chain variable region and position 44 of the light chain variable region, position 91 of the heavy chain variable region and position 43 of the light chain variable region, or position 91 of the heavy chain variable region and position 44 of the light chain variable region (Kabat numbering).

13. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the positions of the amino acid pairs at the interface between the heavy chain variable region and the light chain variable region in at least one first antigen-binding region are the same as those in at least one second antigen-binding region.

14. The antibody or antigen-binding fragment thereof of claim 13, wherein the mutated amino acid at the position of the amino acid pair in the first antigen-binding region and the mutated amino acid at the same position in the second antigen-binding region have opposite charges.

15. The antibody or antigen-binding fragment thereof of claim 14, wherein (1) in the first antigen-binding region, the amino acid at position 39 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 38 of the light chain variable region is substituted with a negatively charged amino acid; and in the second antigen-binding region, the amino acid at position 39 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 38 of the light chain variable region is substituted with a positively charged amino acid; (2) in the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 87 of the light chain variable region is substituted with a negatively charged amino acid; and in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 87 of the light chain variable region is substituted with a positively charged amino acid; (3) in the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a negatively charged amino acid; and in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a positively charged amino acid; (4) in the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 43 of the light chain variable region is substituted with a negatively charged amino acid; and in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 43 of the light chain variable region is substituted with a positively charged amino acid; or (5) in the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a negatively charged amino acid; And in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a positively charged amino acid.

16. The antibody or antigen-binding fragment thereof of claim 15, wherein the positively charged amino acid is selected from K, R or H, and / or the negatively charged amino acid is selected from E or D.

17. The antibody or antigen-binding fragment thereof of claim 16, wherein (vi). In the first antigen-binding region, the amino acid at position 39 of the heavy chain variable region is substituted with K, and The amino acid at position 38 of the light chain variable region is substituted with D, and at the same time, in the second antigen-binding region, the amino acid at position 39 of the heavy chain variable region is substituted with D, and the amino acid at position 38 of the light chain variable region is substituted with K; (vii). In the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with K, and the amino acid at position 87 of the light chain variable region is substituted with D, and at the same time, in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with D, and the amino acid at position 87 of the light chain variable region is substituted with K; (viii). In the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with K, and the amino acid at position 44 of the light chain variable region is substituted with D, and at the same time, in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with D, and the amino acid at position 44 of the light chain variable region is substituted with K; (ix) In the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with K, and the amino acid at position 43 of the light chain variable region is substituted with D, and at the same time, in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with D, and the amino acid at position 43 of the light chain variable region is substituted with K; (x). In the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with K, and the amino acid at position 44 of the light chain variable region is substituted with D, while in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with D, and the amino acid at position 44 of the light chain variable region is substituted with K.

18. The antibody or antigen-binding fragment thereof of any one of claims 1-17, wherein the multispecific antibody comprises a charge mutation and a disulfide bond remodeling mutation.

19. The antibody or antigen-binding fragment thereof of any one of claims 1 to 18, wherein one or more or all antigen-binding regions that bind the same antigen comprise the same mutation.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, wherein The first antigen-binding region comprises a heavy chain variable region, a heavy chain constant region CH1, a light chain variable region, and a light chain constant region CL, and the second antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL.

21. The antibody or antigen-binding fragment thereof of claim 20, wherein 1) The first antigen binding region comprises a disulfide bond remodeling mutation as defined in any one of claims 1 to 17; or 2) the first antigen binding region comprises a charge mutation as defined in any one of claims 1 to 17; 3) the first antigen binding region comprises a disulfide bond remodeling mutation and a charge mutation as defined in any one of claims 1 to 17; or 4) The first antigen-binding region comprises a disulfide bond remodeling mutation and a charge mutation as defined in any one of claims 1-17; and the second antigen-binding region comprises a charge mutation as defined in any one of claims 1-17.

22. The antibody or antigen-binding fragment thereof of claim 21, wherein 1) The first antigen binding region comprises a disulfide bond remodeling mutation as defined in any one of claims 2 to 8; 2) the first antigen binding region comprises a charge mutation as defined in any one of claims 12 to 17; 3) the first antigen binding region comprises a disulfide bond remodeling mutation as defined in any one of claims 2 to 8 and comprises a charge mutation as defined in any one of claims 12 to 17; or 4) The first antigen-binding region and the second antigen-binding region comprise a charge mutation as defined in any one of claims 12-17, and the first antigen-binding region further comprises a disulfide bond remodeling mutation as defined in any one of claims 2-8.

23. The antibody or antigen-binding fragment thereof of claim 22, wherein the first antigen-binding region of the multispecific antibody comprises a combination of mutations selected from the group consisting of: Optionally, the second antigen binding region further comprises a combination of mutations selected from the following:

24. The antibody or antigen-binding fragment thereof of claim 23, wherein the first antigen-binding region and the second antigen-binding region of the multispecific antibody respectively comprise the following mutation combinations:

25. An antibody or an antigen-binding fragment thereof, comprising one or more first antigen-binding regions that specifically bind to a first antigen and one or more second antigen-binding regions that specifically bind to a second antigen, wherein at least one of the antigen-binding regions comprises a heavy chain variable region and a light chain variable region, and the amino acid pairs on the interface of the heavy chain variable region and the light chain variable region comprise a charge mutation, wherein the charge mutation is the mutation of two amino acids on the amino acid pair to amino acids with opposite charges, respectively, wherein the position of the amino acid pair on the interface of the heavy chain variable region and the light chain variable region is selected from: position 45 of the heavy chain variable region and position 87 of the light chain variable region, position 45 of the heavy chain variable region and position 44 of the light chain variable region, position 91 of the heavy chain variable region and position 43 of the light chain variable region, or position 91 of the heavy chain variable region and position 44 of the light chain variable region (Kabat numbering).

26. The antibody or antigen-binding fragment thereof of claim 25, wherein the position of the amino acid pairs at the interface of the heavy chain variable region and the light chain variable region in at least one first antigen-binding region is the same as the position of at least one second antigen-binding region, wherein the first antigen and the second antigen are the same or different.

27. The antibody or antigen-binding fragment thereof of claim 26, wherein the mutated amino acid at the position of the amino acid pair in the first antigen-binding region has an opposite charge to the mutated amino acid at the same position in the second antigen-binding region.

28. The antibody or antigen-binding fragment thereof of claim 27, wherein (1) in the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 87 of the light chain variable region is substituted with a negatively charged amino acid; and in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 87 of the light chain variable region is substituted with a positively charged amino acid; (2) in the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a negatively charged amino acid; and in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a positively charged amino acid; (3) in the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 43 of the light chain variable region is substituted with a negatively charged amino acid; and in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 43 of the light chain variable region is substituted with a positively charged amino acid; or (4) in the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a negatively charged amino acid; And in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 44 of the light chain variable region is substituted with a positively charged amino acid.

29. The antibody or antigen-binding fragment thereof of claim 28, wherein the positively charged amino acid is selected from K, R or H, and / or the negatively charged amino acid is selected from E or D.

30. The antibody or antigen-binding fragment thereof of claim 29, wherein (v) In the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with K, and the amino acid at position 87 of the light chain variable region is substituted with D, and at the same time, in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with D, and the amino acid at position 87 of the light chain variable region is substituted with K; (vi) In the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with K, and the amino acid at position 44 of the light chain variable region is substituted with D, and at the same time, in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with D, and the amino acid at position 44 of the light chain variable region is substituted with K; (vii). In the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with K, and the amino acid at position 43 of the light chain variable region is substituted with D, and at the same time, in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with D, and the amino acid at position 43 of the light chain variable region is substituted with K; or (viii). In the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with K, and The amino acid at position 44 of the light chain variable region is substituted with D, and at the same time, in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with D, and the amino acid at position 44 of the light chain variable region is substituted with K.

31. The antibody or antigen-binding fragment thereof of any one of claims 1-30, wherein all first antigen-binding regions comprise the same mutation, and all second antigen-binding regions comprise the same mutation.

32. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 31, wherein the first antigen-binding region is a Fab fragment of the antibody, and / or the second antigen-binding region is a Fab fragment of the antibody.

33. The antibody or antigen-binding fragment thereof of any one of claims 1 to 32, wherein the light chain constant region comprised by one or more or all of the first antigen-binding regions of the antibody is a Kappa light chain constant region, and the light chain constant region comprised by one or more or all of the second antigen-binding regions is a Lambda light chain constant region, or wherein the light chain constant region comprised by one or more or all of the first antigen-binding regions is a Lambda light chain constant region, and the light chain constant region comprised by one or more or all of the second antigen-binding regions is a Kappa light chain constant region.

34. The antibody or antigen-binding fragment thereof of any one of claims 1-33, wherein the antibody further comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different.

35. The antibody or antigen-binding fragment thereof of claim 34, wherein the first Fc region and the second Fc region are respectively human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 122 or 123 or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or more identity thereto.

36. The multispecific antibody of claim 34 or 35, wherein corresponding Knob mutations and Hole mutations are introduced into the first Fc region and the second Fc region based on Knob-in-Hole technology.

37. The antibody or antigen-binding fragment thereof of claim 36, wherein a) one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises T366S, L368A and Y407V (numbering according to the EU index), or b) one Fc region comprises the amino acid substitutions S354C and T366W and the other Fc region comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering is according to EU index).

38. The multispecific antibody of claim 36, wherein a) one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 125, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 124; b) one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 125, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 124; c) one Fc-region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO: 125 and comprises the mutations S354C and T366W, and the other Fc-region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO: 124 and comprises the mutations Y349C, T366S, L368A and Y407V; d) one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 20 or 131, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 101 or 130; e) one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO: 20 or 131, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO: 101 or 130; or f) an Fc region polypeptide comprising an amino acid sequence having at least 85%, The invention further comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 101 or 130 and comprises the mutation T366W, and the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 101 or 130 and comprises the mutations T366S, L368A and Y407V.

39. The antibody or antigen-binding fragment thereof according to claim 34 or 35, wherein mutations are introduced into the first Fc region and the second Fc region based on the Innobody technology to promote heterodimerization of the first Fc region and the second Fc region.

40. The antibody or antigen-binding fragment thereof of claim 39, wherein the CH3 of one Fc region comprises S364R and D399K mutations, and the CH3 mutations of the other Fc region comprise Y349T, K370S and K409D mutations.

41. The antibody or antigen-binding fragment thereof of claim 40, wherein a) one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 126, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 127; b) one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 126, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 127; or c) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 126 and comprises the mutations S364R and D399K, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 127 and comprises the mutations Y349T, K370S and K409D.

42. The antibody or antigen-binding fragment thereof of any one of claims 34-41, wherein the first and / or second Fc region comprises a L234A / L235A mutation.

43. The antibody or antigen-binding fragment thereof of any one of claims 1-42, wherein the antigen bound by the antibody or antigen-binding fragment thereof is selected from one or more of the following: CD3, BCMA, CD20, Her2, Her3, B7H3, EGFR, Hel, cMET, Axl, GPRC5D, immune checkpoint molecules such as PD-1, PD-L1, CD47, immune activation molecules such as 4-1BB, CD40, OX40.

44. The antibody or antigen-binding fragment thereof of any one of claims 1-43, wherein the first antigen and the second antigen are different and the antibody is a bispecific antibody.

45. The antibody or antigen-binding fragment thereof of claim 44, wherein the bispecific antibody binds to two antigens selected from the group consisting of CD3 / BCMA, CD3 / CD20, CD3 / Her2, Her2 / Her3, B7H3 / EGFR, PD-L1 / CD47, PD-L1 / CD40, PD-L1 / 4-1BB, EGFR / Her3, Her2 / Hel, Her2 / PD-1, Her2 / PD-L1, Her2 / CD47, Her2 / cMet, or CD3 / GPRC5D.

46. ​​The antibody or antigen-binding fragment thereof of claim 44 or 45, wherein the bispecific antibody comprises a first antigen-binding region that specifically binds to a first antigen and a second antigen-binding region that specifically binds to a second antigen, wherein the first antigen-binding region comprises a first Fab and the second antigen-binding region comprises a second Fab, wherein the first Fab is connected at the C-terminus of its CH1 to the N-terminus of the first Fc region (via or without a linker, such as a hinge region), and the second Fab is connected at the C-terminus of its CH1 to the N-terminus of the second Fc region (via or without a linker, such as a hinge region).

47. The antibody or antigen-binding fragment thereof of claim 46, wherein the bispecific antibody is an IgG-like antibody having the configuration as shown in Figure 1.

48. The antibody or antigen-binding fragment thereof of claim 46 or 47, comprising Heavy chain 1: from N-terminus to C-terminus, comprises or consists of: a first Fab heavy chain variable region-a heavy chain constant region CH1-a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region); Light chain 1: from N-terminus to C-terminus, it comprises or consists of: the light chain variable region of the first Fab - the light chain constant region; Heavy chain 2: comprising or consisting of the following from N-terminus to C-terminus: a second Fab heavy chain variable region-a heavy chain constant region CH1-a second Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region); Light chain 2: comprises or consists of the following from N-terminus to C-terminus: the light chain variable region of the second Fab - the light chain constant region.

49. The antibody or antigen-binding fragment thereof of claim 48; wherein (1) The first antigen-binding region specifically binds to Her2, wherein the heavy chain 1 and the light chain 1 respectively comprise the amino acid sequence shown in the following SEQ ID NO:, or comprise an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown, or consist of the sequence shown in the following SEQ ID NO: i) SEQ ID NO:43 and SEQ ID NO:45; ii) SEQ ID NO:48 and SEQ ID NO:49; iii) SEQ ID NO:21 and SEQ ID NO:22; iv) SEQ ID NO:25 and SEQ ID NO:26; v) SEQ ID NO: 25 and SEQ ID NO: 29; vi) SEQ ID NO:31 and SEQ ID NO:32; or vii) SEQ ID NO:31 and SEQ ID NO:29; and / or (2) The second antigen-binding region specifically binds to Hel, and the heavy chain 2 and the light chain 2 respectively comprise the amino acid sequence shown in the following SEQ ID NO:, or comprise an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown, or consist of the sequence shown in the following SEQ ID NO: i) SEQ ID NO:41 and SEQ ID NO:42; ii) SEQ ID NO:23 and SEQ ID NO:24; iii) SEQ ID NO:27 and SEQ ID NO:28; iv) SEQ ID NO:27 and SEQ ID NO:30; v) SEQ ID NO:33 and SEQ ID NO:34; or vi) SEQ ID NO:33 and SEQ ID NO:30; (2)-1 The second antigen-binding region specifically binds to PD-1, and the heavy chain 2 and the light chain 2 respectively comprise the amino acid sequence shown in the following SEQ ID NO:, or comprise an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown, or consist of the sequence shown in the following SEQ ID NO: 46 and SEQ ID NO: 47; (2)-2 The second antigen binding region specifically binds to CD47, and the heavy chain 2 and the light chain 2 respectively comprise the amino acid sequence shown in the following SEQ ID NO:, or comprise an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown, or consist of the following SEQ ID NO: The sequence shown consists of: SEQ ID NO:50 and SEQ ID NO:51; (3)-2 The second antigen binding region specifically binds to c-Met, and the heavy chain 2 and the light chain 2 respectively comprise the amino acid sequence shown in the following SEQ ID NO:, or comprise an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the shown amino acid sequence, or consist of the sequence shown in the following SEQ ID NO: SEQ ID NO:52 and SEQ ID NO:

53.

50. The multispecific antibody of claim 44 or 45, wherein the bispecific antibody comprises one first antigen-binding region that specifically binds to a first antigen and two second antigen-binding regions that specifically bind to a second antigen, wherein the first antigen-binding region comprises a first Fab, the second antigen-binding region comprises a second Fab, wherein one of the second Fabs is connected at the C-terminus of its CH1 to the N-terminus of the first Fc region (via or without a linker, such as a hinge region), one of the first Fabs is connected at the C-terminus of its CH1 to the N-terminus of the second Fc region (via or without a linker, such as a hinge region), and the other of the second Fab is connected at the C-terminus of its CH1 to the N-terminus of the heavy chain variable region of the first Fab (via or without a linker).

51. The antibody or antigen-binding fragment thereof of claim 50, wherein the bispecific antibody is a 2+1 N-terminal configuration having the configuration as shown in Figure 5A.

52. The antibody or antigen-binding fragment thereof of claim 51, wherein the bispecific antibody comprises Heavy chain 1: from N-terminus to C-terminus, comprises or consists of: a second Fab heavy chain variable region-heavy chain constant region CH1-a first Fab heavy chain variable region-heavy chain constant region CH1-a second Fc region, wherein the first Fab heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region), and the second Fab is connected at the C-terminus of its CH1 to the N-terminus of the heavy chain variable region of the first Fab (via or without a linker); Light chain 1: from N-terminus to C-terminus, it comprises or consists of: the light chain variable region of the first Fab - the light chain constant region; Heavy chain 2: from N-terminus to C-terminus, comprises or consists of: a second Fab heavy chain variable region-a heavy chain constant region CH1-a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region); Light chain 2: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the second Fab - the light chain constant region, The bispecific antibody comprises two light chains 2.

53. The antibody or antigen binding fragment thereof of claim 52, wherein the first antigen binding region binds CD3 and the second antigen binding region binds GPRC5D, e.g., wherein 1) Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:58, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:58, or consists of the amino acid sequence; Light chain 1 comprises the amino acid sequence shown in SEQ ID NO:59, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:59, or consists of the amino acid sequence; Heavy chain 2 comprises the amino acid sequence of SEQ ID NO:57, or comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence of SEQ ID NO:

57. an amino acid sequence, or consisting of said amino acid sequence; and / or Light chain 2 comprises the amino acid sequence of SEQ ID NO:56, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO:56, or consists of the amino acid sequence; or 2) heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:62, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:62, or consists of said amino acid sequence; Light chain 1 comprises the amino acid sequence of SEQ ID NO:63, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO:63, or consists of the amino acid sequence; Heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:61, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:61, or consists of the amino acid sequence; and / or Light chain 2 comprises the amino acid sequence shown in SEQ ID NO:60, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:60, or consists of the amino acid sequence.

54. The multi-antibody or antigen-binding fragment thereof of claim 44 or 45, wherein the bispecific antibody comprises one first antigen-binding region that specifically binds to a first antigen and two second antigen-binding regions that specifically bind to a second antigen, wherein the first antigen-binding region comprises a first Fab, and the second antigen-binding region comprises a second Fab, wherein the two second Fabs are connected at the C-terminus of their CH1 to the N-terminus of the first Fc region and the N-terminus of the second Fc region, respectively (via or without a linker, such as a hinge region), and the first Fab is connected at the N-terminus of the heavy chain variable region to the C-terminus of the second Fc region (via or without a linker).

55. The antibody or antigen-binding fragment thereof of claim 54, wherein the bispecific antibody is a 2+1 C-terminal configuration having the configuration shown in Figure 5B.

56. The antibody or antigen-binding fragment thereof of claim 55, wherein the bispecific antibody comprises Heavy chain 1: from N-terminus to C-terminus, comprises or consists of: a second Fab heavy chain variable region-heavy chain constant region CH1-a second Fc region-a first Fab heavy chain variable region-heavy chain constant region CH1, wherein the second Fab is connected to the N-terminus of the second Fc region at the C-terminus of its CH1 (via or without a linker, such as a hinge region), and the first Fab is connected to the C-terminus of the second Fc region at the N-terminus of the heavy chain variable region (via or without a linker); Light chain 1: from N-terminus to C-terminus, it comprises or consists of: the light chain variable region of the first Fab - the light chain constant region; Heavy chain 2: from N-terminus to C-terminus, comprises or consists of: a second Fab heavy chain variable region-a heavy chain constant region CH1-a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region); Light chain 2: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the second Fab - the light chain constant region, The bispecific antibody comprises two light chains 2.

57. The antibody or antigen binding fragment thereof of claim 56, wherein the first antigen binding region binds CD3 and the second antigen binding region binds GPRC5D, e.g., wherein 1) Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:64, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:64, or consists of the amino acid sequence; Light chain 1 comprises the amino acid sequence shown in SEQ ID NO:59, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:59, or consists of the amino acid sequence; Heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:57, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:57, or consists of the amino acid sequence; and / or Light chain 2 comprises the amino acid sequence of SEQ ID NO:56, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO:56, or consists of the amino acid sequence; or 2) heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:65, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:65, or consists of the amino acid sequence; Light chain 1 comprises the amino acid sequence of SEQ ID NO:63, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO:63, or consists of the amino acid sequence; Heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:61, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:61, or consists of the amino acid sequence; and / or Light chain 2 comprises the amino acid sequence shown in SEQ ID NO:60, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:60, or consists of the amino acid sequence.

58. The antibody or antigen-binding fragment thereof of claim 44 or 45, wherein the bispecific antibody comprises two first antigen-binding regions that specifically bind to a first antigen and two second antigen-binding regions that specifically bind to a second antigen, wherein the first antigen-binding region comprises a first Fab, and the second antigen-binding region comprises a second Fab, wherein the two second Fabs are respectively connected to the N-termini of two Fc regions at the C-termini of their CH1 (via or without a linker, such as a hinge region) (the Fc regions may be the same or different), and the two first Fabs are respectively connected to the C-termini of the two Fc regions at the N-termini of the heavy chain variable regions (via or without a linker).

59. The antibody or antigen-binding fragment thereof of claim 58, wherein the bispecific antibody is a 2+2 C-terminal configuration having the configuration shown in Figure 5C.

60. The antibody or antigen-binding fragment thereof of claim 59, wherein the bispecific antibody comprises Heavy chain: from N-terminus to C-terminus, it comprises or consists of: a second Fab heavy chain variable region-heavy chain constant region CH1-Fc region-a first Fab heavy chain variable region-heavy chain constant region CH1, wherein the second Fab is connected to the N-terminus of the Fc region at the C-terminus of its CH1 (via or without a linker, such as a hinge region), and the first Fab is connected to the heavy chain constant region CH1 at the C-terminus of the Fc region. The N-terminus of the variable chain region is connected to the C-terminus of the Fc region (with or without a linker); Light chain 1: from N-terminus to C-terminus, it comprises or consists of: the light chain variable region of the first Fab - the light chain constant region; Light chain 2: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the second Fab - the light chain constant region, The bispecific antibody comprises two heavy chains.

61. The antibody or antigen binding fragment thereof of claim 60, wherein the first antigen binding region binds CD3 and the second antigen binding region binds GPRC5D, e.g., wherein 1) The heavy chain comprises the amino acid sequence shown in SEQ ID NO:66, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:66, or consists of the amino acid sequence; Light chain 1 comprises the amino acid sequence shown in SEQ ID NO:59, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:59, or consists of the amino acid sequence; and / or Light chain 2 comprises the amino acid sequence of SEQ ID NO:56, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO:56, or consists of the amino acid sequence; or 2) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:67, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:67, or consists of the amino acid sequence; Light chain 1 comprises the amino acid sequence shown in SEQ ID NO:63, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:63, or consists of the amino acid sequence; and / or Light chain 2 comprises the amino acid sequence shown in SEQ ID NO:60, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:60, or consists of the amino acid sequence.

62. The antibody or antigen-binding fragment thereof of any one of claims 1-43, wherein the first antigen and the second antigen are different and the antibody further comprises an antigen binding region that specifically binds one or more additional antigens.

63. The antibody or antigen-binding fragment thereof of claim 62, wherein the antibody is a trispecific antibody.

64. The antibody or antigen-binding fragment thereof of claim 63, wherein the trispecific antibody comprises a first antigen-binding region that specifically binds to a first antigen, a second antigen-binding region that specifically binds to a second antigen, and a third antigen-binding region that specifically binds to a third antigen, wherein the first antigen-binding region comprises a first Fab, the second antigen-binding region comprises a second Fab, wherein the first Fab is connected at the C-terminus of its CH1 to the N-terminus of the first Fc region (via or without a linker, such as a hinge region), the second Fab is connected at the C-terminus of its CH1 to the N-terminus of the second Fc region (via or without a linker, such as a hinge region), and the third antigen-binding region comprises a scFv (e.g., VH-VL or VL-VH), which is connected at the N-terminus or C-terminus of its VH or VL (e.g., the scFv is VL-VH, such as the N-terminus of VL) to the C-terminus of the second Fc region via or without a linker.

65. The antibody or antigen-binding fragment thereof of claim 64, wherein the configuration of the trispecific antibody is a 2+1 C-terminal configuration, as shown in Figure 7A or B.

66. The antibody or antigen-binding fragment thereof of claim 65, wherein the multispecific antibody comprises Heavy chain 1: from N-terminus to C-terminus, comprises or consists of: a first Fab heavy chain variable region-a heavy chain constant region CH1-a first Fc region, wherein the first Fab is connected at the C-terminus of its CH1 to the N-terminus of the first Fc region (via or without a linker, such as a hinge region); Light chain 1: from N-terminus to C-terminus, it comprises or consists of: the light chain variable region of the first Fab - the light chain constant region; Heavy chain 2: from N-terminus to C-terminus, comprises or consists of: a second Fab heavy chain variable region-heavy chain constant region CH1-second Fc region-scFv, wherein the heavy chain constant region CH1 is connected to the N-terminus of the second Fc region via or without a linker (e.g., hinge region) at its C-terminus, and the scFv is connected to the C-terminus of the second Fc region via or without a linker at the N-terminus or C-terminus of its VH or VL (e.g., scFv is VL-VH, e.g., the N-terminus of VL); Light chain 2: comprises or consists of the following from N-terminus to C-terminus: the light chain variable region of the second Fab - the light chain constant region.

67. The antibody or antigen-binding fragment thereof of claim 66, wherein the first antigen-binding region specifically binds GPRC5D, the second antigen-binding region specifically binds CD3, and / or the third antigen-binding region specifically binds BCMA, e.g., wherein Heavy chain 1 comprises the amino acid sequence of SEQ ID NO:71, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO:71, or consists of the amino acid sequence; Light chain 1 comprises the amino acid sequence of SEQ ID NO:72, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO:72, or consists of the amino acid sequence; Heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:73, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:73, or consists of the amino acid sequence; and / or Light chain 2 comprises the amino acid sequence shown in SEQ ID NO:74, or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:74, or consists of the amino acid sequence.

68. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-67, or any one or more chains thereof.

69. An expression vector comprising the nucleic acid of claim 68.

70. A host cell comprising the nucleic acid of claim 68 or the expression vector of claim 69.

71. A method for preparing the antibody or antigen-binding fragment thereof of any one of claims 1 to 67, comprising a) introducing nucleic acids encoding each chain of the antibody or antigen-binding fragment thereof into a host cell; b) expressing and assembling the antibody or antigen-binding fragment thereof in a host cell; Optionally, the antibody or antigen-binding fragment thereof is purified, for example, by Protein A purification.

72. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-67.

73. A fusion protein comprising the antibody or antigen-binding fragment thereof of any one of claims 1-67.

74. A T cell receptor comprising the antibody or antigen binding fragment thereof of any one of claims 1-67.

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