Mutants that promote homologous pairing of the heavy and light chains of a multispecific antibody

Abstract

The present disclosure relates to multispecific antibodies, such as bispecific antibodies, comprising mutant amino acid pairs that can promote the homologous pairing of the heavy and light chains of the multispecific antibody.

Description

Technical Field

[0001] The present disclosure relates to multispecific antibodies, such as bispecific antibodies, comprising mutated amino acid pairs that can promote the homologous pairing of the heavy and light chains of the multispecific antibody.

Background Art

[0002] Antibody molecules occupy a dominant position in the current biotherapy market. Bispecific antibodies (bsAbs) are single molecules that can simultaneously bind two different antigens, or two different epitopes of the same antigen, and are different from naturally occurring immunoglobulin G (IgG) monospecific antibodies (mAbs). Due to their further targeting ability, bsAbs provide improved clinical benefits in the treatment of complex diseases such as cancer and immune diseases, which are related to various cell surface receptors or ligands. Considerable efforts have been made to engineer mAbs into bsAbs, and more than 60 forms of bsAbs have been obtained. Many bsAbs can be engineered by linking antibody fragments such as single-chain variable fragments (scFvs), antigen-binding fragments (Fabs), and heavy-chain (VH) and light-chain (VL) variable domains. However, these new forms, which are different from the conventional IgG structure, typically have poor physiochemical properties such as low solubility, aggregation susceptibility, difficulty in large-scale production, poor thermal stability, poor pharmacokinetics, and the potential for immunogenicity. The significant production challenges of bispecific antibodies in terms of quantity, quality, and stability have hindered their wider clinical application and acceptance.

[0003] To assist in the production of bispecific antibodies, heterodimeric Fc technology has been developed. For example, Carter et al. adopted the "knob-into-hole" (Kih) model when modifying some amino acids of the antibody heavy chain and achieved relatively successful results in the production of bispecific antibodies (Ridgway, Presta et al. 1996; Carter 2001). They mutated small amino acids with side chains on the CH3 region of the first heavy chain of Fc into large amino acids with side chains to create a "knob" (such as T366Y), and mutated specific amino acids on the CH3 of the second heavy chain into small amino acids with side chains to create a "hole" (such as Y407T), that is, they used steric structural complementarity to achieve the formation of heterodimers (WO9627011). Subsequently, the proportion of heterodimers (WO1998050431) was increased by methods such as random mutagenesis-phage display. At the same time, disulfide bonds were introduced into the CH3 domain to increase the proportion of heterodimers, but the ability to prevent homodimer formation was still insufficient. Therefore, the proportion of the whole heterodimer was approximately 70 - 80%.

[0004] Alternatively, strand exchange engineered domain (SEED) heterogenicity represents another spatial mutation-based design strategy that forms complementary structures using exchangeable amino acid sequences within IgG and IgA CH3 domains (AG SEED CH3 and GA SEED CH3). IgG and IgA CH3 derivatives generate complementary sequences. Thus, two complementary heavy chains can be assembled to produce heterodimers, thereby eliminating the possibility of producing homodimers 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).

[0005] In addition to the aforementioned spatial variation, electrostatic interactions are also widely used to promote heterodimer formation in H. This method involves mutating individual amino acids within the CH3 domain of one heavy chain to Lys carrying a positive charge, and substituting individual amino acids within the CH3 of the other heavy chain with Asp or Glu carrying a negative charge. In this way, the charged amino acids readily form heterodimers by electrostatic attraction. Gunasekaran et al. first introduced charged amino acids to form Fc-heterodimer bispecific antibodies (Gunasekaran, K. et al., Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG. J. Biol. Chem., 2010, 285, 19637-19646.).

[0006] CN104011221B provides a bispecific antibody having a modified polypeptide chain containing disulfide bond reformation mutations within the variable and constant regions. However, this mutation is still insufficient to achieve the complete and correct pairing of more than 90% of the bispecific antibodies expressed by a single cell line. The amino acid pairs selected for disulfide bond reformation in this patent are not properly optimized, and some of them are molecules that cannot form disulfide bonds.

[0007] CN109475627A provides a method for preparing an antibody mixture, which relates to the preparation of a mixture of two types of bispecific antibodies. Charge mutations are mainly introduced into the constant region, and a large number of mutations need to be introduced to achieve the optimal pairing effect.

[0008] Therefore, there is still a need for new bispecific antibody platforms in this field, which require only the introduction of minor mutations to minimize expression mismatches on the resulting bispecific antibodies while maintaining affinity and expression levels. At the same time, due to the resulting bispecific antibodies having fewer mutations, this results in lower immunogenicity and enables the expression and recombination of antibodies on the bispecific antibody platform to be carried out in cell lines. Summary of the Invention

[0009] The present disclosure provides a new multispecific antibody (such as a new bispecific antibody) platform for obtaining multispecific antibodies by introducing charge mutations into the variable region of a bispecific antibody while introducing disulfide bond reformation mutations into the CH1 / CL region.

[0010] (1) Few mismatches and a high proportion of correctly paired molecules. (2) The immunogenic risk is equivalent to that of natural IgG. (3) Have an affinity similar to or the same as the antibody before the introduction of mutations. (4) Have a relatively high expression level, for example, an expression level equivalent to or higher than that of the antibody before the introduction of mutations or bispecific antibodies. (5) Avoid expression and in vitro recombination in different cell lines and enable expression in cell lines.

[0011] At the same time, the mutations and amino acid positions introduced into the multispecific antibody platform of the present disclosure are relatively conservative. For this reason, this is applicable to various multispecific antibodies (bispecific antibodies). Brief Description of the Drawings

[0012]

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[0013] Definition: It should be understood that the specific methodologies, solutions, and reagents described herein may be changed and are not limited thereto. It is understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the disclosure, 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 one of ordinary skill in the art to which this disclosure pertains.

[0014] For the purposes of this specification, the following definitions are used. As appropriate, terms used in the singular may also include the plural and vice versa. It is understood that the terms used herein are for the purpose of describing particular embodiments and are not intended to be limiting.

[0015] The term "about", when used in combination with a numerical value, means a value within a range of 5% below the specified numerical value and 5% above the specified numerical value.

[0016] As used herein, the term "and / or" means any one of the alternatives, or two or more or all of the alternatives.

[0017] When "first" and "second" are referred to herein, they are for the purpose of distinguishing two domains or strands and do not indicate the positions of the two domains in any way.

[0018] As used herein, the terms "comprise" and "include" mean including all elements, integers, or steps, but do not exclude any other element, integer, or step. When the terms "comprise" and "include" are used herein, unless otherwise specified, they also cover combinations of elements, integers, or steps. For example, when referring to "comprising" a variable region of an antibody of a specific particular sequence, this also covers the variable region of an antibody formed by that specific particular sequence.

[0019] As used herein, all amino acid positions within the variable regions of the heavy and light chains are numbered according to the Kabat numbering scheme described by Kabat et al., Sequences of Proteins of Immunological Interest, Version 5. Public Health Service, National Institutes of Health, Bethesda, MD (1991), and referred to herein as "Kabat numbers".

[0020] As used herein, amino acid positions within domains outside the variable regions of the antibodies referred to (e.g., constant regions, e.g., the Fc region) are numbered according to the EU numbering scheme described by Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969), and referred to herein as "EU numbers". When position numbers and / or amino acid residues are assigned to a specific antibody isotype, this is applicable to the corresponding positions and / or amino acid residues of any other antibody isotype, which is known to those skilled in the art.

[0021] General information on the nucleotide sequences of the light and heavy chains of immunoglobulins is provided in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, Version 5, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0022] The term "amino acid substitution" or "amino acid mutation" refers to the replacement of at least one amino acid residue in a given parent amino acid sequence with a different "substituent" amino acid residue. This substituent residue or residues may be "naturally occurring amino acid residues" (i.e., those encoded by the genetic code), and may be selected from 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 substitution with one or more non-naturally occurring amino acid residues. "Non-naturally occurring amino acid residues" refer to residues that, apart from the above-mentioned naturally occurring amino acid residues, can covalently bond to adjacent amino acid residues within a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, Aib, and other amino acid residue analogs.

[0023] The term "CH1 region" refers to the portion extending from EU position 118 to EU position 220 (EU numbering scheme) in the antibody heavy chain polypeptide. In one embodiment, the CH1 domain comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 116). In one embodiment, internal disulfide bonds are removed by mutating Cys to non-Cys in CH1, or new disulfide bonds are re-formed by mutating non-Cys to Cys.

[0024] The term "CH2 region" refers to the portion extending from EU position 231 to EU position 340 (EU numbering scheme) in the antibody heavy chain polypeptide. In one embodiment, the CH2 domain comprises the amino acid sequence APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 117).

[0025] The term "CH3 region" refers to the portion extending from EU position 341 to EU position 447 in the antibody heavy chain polypeptide. In one embodiment, the CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 118).

[0026] The term "hinge region" refers to the heavy chain polypeptide portion of the antibody that links the CH1 region and the CH2 region in the sequence of D221 - P230 according to EU numbering, for example, in the heavy chain of a wild-type antibody, such as the IgG1 hinge region. The hinge regions of other IgG subtypes may be identified by aligning the Cys residues within the hinge region of the IgG1 subtype sequence.

[0027] The hinge region is typically a dimeric molecule formed by two polypeptides having the same amino acid sequence. In one embodiment, the hinge region has the amino acid sequence DKTHTCPXCP (SEQ ID NO: 119), where X is S or P in the sequence. In one embodiment, the hinge region comprises the amino acid sequence HTCPXCP (SEQ ID NO: 120), where X is S or P in the sequence. In one embodiment, the hinge region comprises the amino acid sequence CPXCP (SEQ ID NO: 121), where X is S or P in the sequence. In one embodiment, the disulfide bonds within the hinge region are removed by mutating Cys within the hinge region to non-Cys.

[0028] The multispecific antibodies of the present disclosure may include a linker. As used herein, the term "linker" refers to any molecule that can directly link different portions of a multispecific antibody. Examples of linkers that establish covalent bonds between different portions of a multispecific antibody include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, or copolymers of polyoxyethylene or polyethylene glycol, or polypropylene glycol. In some embodiments, the term "peptide linker" according to the present disclosure refers to an amino acid sequence that links together the amino acid sequences of various portions of a multispecific antibody. Preferably, the peptide linker has a length sufficient to link the two entities in such a manner as to maintain their conformations relative to each other, so as to prevent interference with the desired activity. Peptide linkers may or may not primarily contain the following amino acid residues: Gly, Ser, Ala, or Thr. Useful linkers include Gly-Ser 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 Gly-Ala polymers, Ala-Ser polymers, and other flexible linkers.

[0029] The terms "Fc domain" or "Fc region" are used herein to define the C-terminal region of at least one portion of the constant region contained in immunoglobulin H. This term includes native sequence Fc regions and variant Fc regions. The native immunoglobulin "Fc domain" includes two or three constant domains, namely, the CH2 domain, the CH3 domain, and optionally the CH4 domain. For example, in native antibodies, the immunoglobulin Fc domain includes the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD 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 antibodies. Unless otherwise specified herein, the amino acid residue numbers within the Fc region or the heavy chain constant region are numbered according to the EU numbering scheme (also referred to as the EU index) in Kabat et al., Sequences of Proteins of Immunological Interest, Version 5, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal Lys (Lys447) within the Fc region may or may not be present. Two Fc regions may undergo dimerization to form dimeric Fc, and two different Fc [regions] may undergo heterodimerization to form heterodimeric Fc. The terms "Fc region", "Fc portion", and "dimeric Fc (e.g., heterodimeric Fc)" herein do not include the heavy chain variable region VH and the light chain variable region VL, and the heavy chain constant region CH1 and the light chain constant region CL of immunoglobulins, but may optionally include the N-terminal hinge region of the heavy chain constant region. In one embodiment, the human IgG heavy chain Fc region extends from Asp221, Cys226, or Asp231 to the C-terminus of H.

[0030] In one embodiment, the human IgG1 Fc region polypeptide (including the hinge region) includes the following amino acid sequence: [Chem.]

[0031] The human IgG4 Fc region polypeptide (including the hinge region) contains the following amino acid sequence: PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 123).

[0032] The antibodies produced by the methods reported herein contain an Fc region, and in one embodiment, the Fc region is derived from a human-derived Fc region. In one embodiment, the Fc region contains all parts of the 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 subtype. In one embodiment, the Fc region is of the human IgG1 subtype.

[0033] As used herein, "heterodimeric Fc scaffold" refers to a scaffold containing two different Fc regions formed after dimerization or two different Fc regions. This may be linked to an antigen-binding domain at its N-terminus or C-terminus (e.g., the heavy and / or light chain variable regions of an antibody capable of binding to a target molecule, an antigen-binding fragment of an antibody, or the soluble portion of a ligand or receptor capable of binding to a target molecule), and is used to construct multispecific antibodies, such as bispecific antibodies.

[0034] Amino acid mutations are expressed using (original amino acid, amino acid position, mutated amino acid). For example, when the mutation site is located in the V region, "A43D" refers to the substitution of the Ala amino acid located at Kabat position 43 with Asp (D), and when the mutation site is located in the C region, "Q124C" refers to the substitution of Gln located at EU position 124 with Cys (C). When referring to a mutation combination, the mutations in the combination are connected using the plus symbol (+). "A43D+Q124C" indicates that this combination contains both the mutations A43D and Q124C. If there are multiple possible mutations at a specific position, this is represented in this specification by the symbol " / ". For example, "K370T / S mutation" indicates that the residue K at position 370 may be substituted with a T or S residue.

[0035] When an amino acid position is referred to in this disclosure, unless otherwise specified, this refers to the amino acid position according to the IgG1 heavy chain or kappa light chain number, that is, it encompasses the amino acid position based on the IgG1 heavy chain or kappa light chain number and the amino acid positions corresponding to other heavy chains or light chains. For example, when referring to C220, this encompasses the amino acid of the IgG1 heavy chain at position 220 of the EU numbering and the corresponding amino acids in other heavy chains. For example, it is the amino acid at position 131 within IgG2, IgG3, or IgG4.

[0036] When describing a mutation, it should be noted that the original amino acid at a specific position may be the described amino acid or another amino acid at the corresponding position. For example, when describing a mutation, this may include the situation where the Ala at position 43 in the variable region mutates to Asp as long as the original amino acid corresponds to position 43 in the variable region, or if Ala does not correspond to position 43, the corresponding amino acid at position 43 mutates to Asp. The terms "host cell", "host cell line", and "host cell culture product" are used interchangeably and refer to a cell into which a foreign nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells" that include the primary transformed cells and progeny derived therein, regardless of the number of passages. The progeny may not be identical to the parental cells in terms of nucleic acid content and may even contain mutations. Such mutant progeny are included herein and have similar functions or biological activities that are screened or selected in the initial transformed cells.

[0037] As used herein, the term "vector" refers to a nucleic acid molecule that is capable of propagating another nucleic acid linked thereto. This term includes vectors having self-replicating nucleic acid structures and vectors that have integrated into the genome of a host cell into which they have been introduced. Some vectors are capable of inducing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

[0038] The term "binding molecule" refers to any molecule that can specifically bind to a target, such as an antibody, or an antigen-binding fragment or fusion protein thereof.

[0039] The term "target" refers to the object to be bound by a binding molecule. The target may be an antigen, or may also be a ligand or a receptor. The term "antigen" refers to a molecule that induces an immune response. Such an immune response may relate to antibody production or specific immune cell activation, or both. One of ordinary skill in the art will understand that essentially any macromolecule, including all proteins or peptides, can be used as an antigen. In addition, an antigen may be derived from recombinant DNA or genomic DNA. As used herein, the term "epitope" refers to a portion within an antigen that has specific interaction with an antibody molecule. In some embodiments, the antigen is a tumor-associated antigen (i.e., an antigen related to the development and progression of a tumor) or a T cell engager. When referring to the target-binding region of a binding molecule derived from an antibody of the present disclosure, the terms "target" and "antigen" may be used interchangeably.

[0040] As used herein, the term "target-binding region" refers to a portion of a binding molecule, such as a multispecific or bispecific binding molecule, that binds to a specific target or antigen. The target-binding region may be, for example, an antibody, an immunoglobulin, or an antibody fragment. Such target-binding regions may or may not have a tertiary structure independent of the rest of the binding molecule and may be used as an independent entity to bind or not bind to its target. The target-binding region may also be a receptor or a ligand, or a domain of a receptor that can bind to a ligand. The "target-binding region" of a multispecific or bispecific antibody may also be referred to as an "antigen-binding region".

[0041] As used herein, the term "antigen-binding region" refers to an antibody or an antigen-binding fragment thereof, e.g., any portion of a multispecific or bispecific antibody that binds to a particular target or antigen. The antigen-binding region may be, for example, an antibody, an immunoglobulin, or an antibody fragment. Such antigen-binding regions may or may not have a tertiary structure independent of the remainder of the multispecific or bispecific antibody and may be used as independent entities to bind or not bind to their antigen / epitope. When referring to a target-binding region of a binding molecule of the present disclosure that is derived from an antibody, the terms "target-binding region" and "antigen-binding region" may be used interchangeably.

[0042] The term "multispecific binding molecule" refers to a multispecific binding molecule that is at least bispecific, e.g., a bispecific binding molecule, i.e., a molecule that includes at least a first target-binding region and a second target-binding region, wherein the first target-binding region binds to one type of target or antigen and the second target-binding region binds to another antigen or target. The multispecific binding molecules according to the present disclosure also encompass multispecific molecules that include multiple target-binding regions / binding sites, such as trispecific binding molecules. In some embodiments, the multispecific binding molecules of the present disclosure are multispecific antibodies. In some embodiments, the bispecific binding molecules of the present disclosure are bispecific antibodies. In some embodiments, the multispecific binding molecules of the present disclosure are fusion proteins that may include, for example, a target-binding region from an antibody and a target-binding region from a receptor or ligand.

[0043] As used herein, the term "monospecific" refers to a polypeptide / protein molecule having one or more target-binding sites, wherein each of the sites binds to the same site or structure of the same target, or the same epitope of the same antigen.

[0044] As used herein, the term "multispecific" binding molecule, e.g., an antigen, refers to the presence of at least two target binding sites, and each target binding site of the at least two target binding sites binds to a different site / structure of the same target or different targets. A multispecific binding molecule is a binding molecule having binding specificity for at least two different targets or sites / structures. In one embodiment, such bispecific binding molecules are provided herein, which have binding specificity for a first target and a second target.

[0045] As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen-binding sites, and each antigen-binding site of the at least two antigen-binding sites binds to a different epitope of the same antigen or a different epitope of a different antigen. A multispecific antibody is an antibody having binding specificity for at least two different antigens / epitopes. In one embodiment, such bispecific antibodies are provided herein, which have binding specificity for a first antigen and a second antigen.

[0046] The "first antigen-binding region", when referred to in relation to a multispecific antibody or a bispecific antibody, refers to the binding region for binding to the first antigen and is not intended to limit the number of antigen-binding regions contained in the antibody. For example, a multispecific antibody or a bispecific antibody may include one or more first antigen-binding regions. For example, a bispecific antibody includes a first antigen-binding region and a second antigen-binding region, but may also include one or more first antigen-binding regions and one or more second antigen-binding regions.

[0047] When referring to a "first target binding region" with respect to a binding molecule, it refers to a binding region for binding to a 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 bispecific antibody may contain one or more first target binding regions. For example, a bispecific antibody contains a first target binding region and a second target binding region, but may also contain one or more first target binding regions and one or more second target binding regions.

[0048] When referring to a "first antigen binding region" with respect to an antibody, it refers to a binding region for binding to a first antigen, and is not intended to limit the number of 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 also contain one or more first antigen binding regions and one or more second antigen binding regions.

[0049] When it is described that a "target or antigen binding region is derived from an antibody", this refers to a binding domain that constitutes a target / antigen binding region derived from or derived from a specific antigen binding domain of an antibody. For example, a specific Fab of an antigen binding region is derived from or derived from a corresponding fragment of an antibody such as Fab, or the heavy chain variable region and / or light chain variable region of the antigen binding region is derived from or derived from the heavy chain variable region and / or light chain variable region of the antibody, or 1, 2, 3, 4, 5, or 6 CDRs of the antigen binding region are the CDRs of the antibody.

[0050] The term "derived from" means that a fragment within an antigen binding region is basically the same as the antibody fragment from which it is derived, but involves mutations, such as substitutions, deletions, or additions, at one or more sites. In a particular embodiment, the mutation does not exist in the CDR of the antibody.

[0051] The terms "whole antibody" or "full-length antibody" may be used interchangeably herein and refer to an antibody molecule having a native immunoglobulin molecular structure. A conventional full-length four-chain IgG antibody comprises two heavy chains (H) and two light chains (L) linked to each other by disulfide bonds. A full-length heavy-chain antibody having only heavy chains and lacking light chains comprises two heavy chains linked to each other by disulfide bonds. The heavy chains of a conventional full-length four-chain IgG antibody typically consist of a heavy-chain variable region (abbreviated as VH herein) and a heavy-chain constant region, and the heavy-chain constant region includes at least three domains, namely, CH1, CH2, and CH3. The light chain of a full-length antibody consists of a light-chain variable region (abbreviated as VL herein) and CL, and CL is composed of the domain CL. Each heavy-chain variable region VH and each light-chain variable region VL are composed of three CDRs and four FRs and are arranged in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody fragment" includes a part of a complete antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0052] The term "antigen-binding fragment" of an antibody is a molecule distinct from the full-length antibody, contains a part of the full-length antibody, and is an antigen that can bind to the antigen to which the full-length antibody binds, or an antigen that competitively binds to the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived). Antigen-binding fragments of a complete antibody may be prepared by recombinant DNA technology or by enzymatic or chemical cleavage. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), and nanobody. For example, a Fab fragment may be obtained from papain digestion of a full-length antibody. In addition, F(ab’)2 is produced from pepsin digestion of the whole antibody under disulfide bonds in the hinge region. F(ab’)2 is a dimer of Fab’ and is a bivalent antibody fragment. F(ab’)2 can be reduced under neutral conditions by disrupting the disulfide bond within the hinge region and converting the F(ab’)2 dimer to Fab’ monomer. The Fab’ monomer is basically a Fab fragment with a hinge region. The Fv fragment is composed of the single arms VL and VH domains of an antibody. The two domains of the Fv fragment, namely VL and VH, may be independently genetically encoded or may be recombined, and these two domains may be linked to a single protein chain using a synthetic linker peptide, and the VL region and VH region within the single protein chain pair to form a single-chain Fv (scFv).

[0053] "Fab fragment" or "Fab" may be used interchangeably herein and is used to refer to an immunoglobulin fragment composed of two polypeptide chains containing the immunoglobulin heavy chain variable domain VH, the heavy chain constant domain CH1, the light chain variable domain VL, and the light chain constant domain CL, where one polypeptide chain contains VH and one constant region selected from CH1 and CL from the N-terminus to the C-terminus, and the other polypeptide chain contains VL and another constant region selected from CL and CH1 from the N-terminus to the C-terminus, and the VH domain and the VL domain pair to form an antigen-binding site. The Fab polypeptide chain containing the heavy chain constant region CH1 herein is also referred to as the "Fab heavy chain", and correspondingly, the Fab polypeptide chain containing the light chain constant region CL is also referred to as the "Fab light chain".

[0054] "Complementary determining region" or "CDR" is a region within the variable domain of an antibody where the sequences are highly variable, form structurally defined loops ("hypervariable loops"), and / or contain antigen - contacting residues ("antigen - contact points"). CDRs are mainly involved in the binding to an antigen / epitope. The CDRs of the heavy and light chains are typically designated as CDR1, CDR2, and CDR3 and are numbered starting from the N - terminus. The CDRs located in the variable domain of the antibody heavy chain are designated as HCDR1, HCDR2, and HCDR3, while the CDRs located in the variable domain of the antibody light chain are designated as LCDR1, LCDR2, and LCDR3. In a given light - chain variable region or heavy - chain variable region amino - acid sequence, the exact amino - acid sequence boundaries of each CDR may be determined using any one or combination of various known antibody CDR assignment schemes, such as Chothia (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 (Kabat et al., Sequences of Proteins of Immunological Interest, Version 4, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (web: imgt.cines.fr / ) based on antibody sequence diversity, and North based on affinity propagation clustering using a large number of crystal structures. Unless otherwise specified, the terms "CDR" or "CDR sequence" in the present disclosure encompass CDR sequences identified by any of the above - mentioned methods.The CDR may also be identified based on a reference CDR sequence having the same Kabat number position (such as any exemplary CDR of the present disclosure).

[0055] The sequence identity between the sequences is calculated as follows.

[0056] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal comparison, gaps may be introduced into one or both of the first and second amino acid sequences or nucleic acid sequences, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being compared is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. Thereafter, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are 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, the molecules at this position are the same.

Mode for Carrying Out the Invention

[0057] I. Binding Molecule The present disclosure relates to a type of binding molecule that includes a first target binding region and a second target binding region, and optionally one or more additional target binding regions, where one or two or more of the target binding regions include one or more mutations that are preferred for preventing mismatches and / or promoting heterodimerization. In some embodiments, the binding molecule also includes one or more mutations for facilitating heterodimer purification. In some embodiments, the mutations included in the binding molecules of the present disclosure are selected from disulfide bond reformation mutations, charge mutations, and / or one or more other mutations that are preferred for preventing mismatches 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.

[0058] In some embodiments, the binding molecules of the present disclosure may be fusion proteins that include, for example, a target binding region from an antibody and a target binding region from a receptor or ligand. In some embodiments, exemplary examples of ligands or receptors include granule-associated cytokines or their receptors, such as tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), or transforming growth factor (TGF, e.g., TGF-β) or its receptor, and fragments thereof.

[0059] In some embodiments, the binding molecule is an antibody. In some embodiments, the present disclosure relates to antibodies containing mutations, such as single - specificity or multispecific antibodies, e.g., bispecific antibodies, containing 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 contain one or more mutations that are preferred for preventing mismatches and / or promoting heterodimerization. In some embodiments, the antibody also contains one or more mutations for facilitating heterodimer purification. In some embodiments, the mutations included in the binding molecules of the present disclosure are selected from disulfide - bond re - formation mutations, charge mutations, and / or one or more other mutations that are preferred for preventing mismatches 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.

[0060] 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.

[0061] In some embodiments, the CH1 region is a human IgG CH1, such as 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 has an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 99%, or 100% identity therewith, or is composed thereof.

[0062] 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 the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55, or has an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99%, or 100% identity thereto, or is composed of them.

[0063] In the present disclosure, the limitation of the antigen-binding region is equally applicable to the target-binding region.

[0064] I-1 disulfide bond reformation mutation The disulfide bond reformation mutation is an effective strategy to prevent mismatches between the light and heavy chains of different target-binding regions of the present binding molecule or multispecific antibody by forcing homologous light and heavy chain pairing. Reform the disulfide bonds that link the light and heavy chains, or one or more target-binding regions, and change the positions of the disulfide bonds so that mismatched light and heavy chains cannot effectively form disulfide bonds.

[0065] Thus, in one embodiment, in the binding molecule of the present disclosure, such as an antibody, at least one target-binding region or antigen-binding region comprises CH1-CL, and the disulfide bond at the CH1-CL interface is reformed. For example, the disulfide bond reformation mutation is present on the CH1-CL interface. In one embodiment, in the binding molecule of the present disclosure, such as an antibody, two or more target-binding regions comprise a disulfide bond reformation mutation at the CH1-CL1 interface.

[0066] As used herein, "disulfide bond reformation mutation" refers to a mutation at the position of an amino acid pair at the interface, where a non-Cys at the position of the amino acid pair is replaced with Cys. For example, two non-Cys at the position of the amino acid pair are replaced with Cys, enabling the formation of an interchain disulfide bond.

[0067] In some embodiments, the disulfide bond reformation mutations of the present disclosure are mutations at the positions of amino acid pairs at the CH1-CL interface of the antigen-binding region (e.g., an antigen-binding region comprising a Fab), where the non-Cys at the position of the amino acid pair is replaced with Cys. In some embodiments, two non-Cys at the position of the amino acid pair are replaced with Cys, enabling the formation of an inter-chain disulfide bond.

[0068] In some embodiments, this mutation refers to an amino acid within a non-Cys amino acid pair at the CH1-CL interface that has mutated to Cys and the distance between the α-carbon atoms is less than 8 Å, more preferably less than 6 Å. In some embodiments, the amino acid side chains of the amino acid pair face each other. In some embodiments, the amino acids in the non-Cys amino acid pair at the CH1-CL interface with a distance between the α-carbon atoms less than 8 Å, more preferably less than 6 Å, mutate to Cys, and the amino acid side chains in the non-Cys amino acid pair face each other.

[0069] In some embodiments, the non-Cys amino acids in the heavy chain CH1 within the antigen-binding region (e.g., IgG1, IgG2, IgG3, or IgG4) are replaced with Cys amino acids at positions 126, 128, 134, 136, 139, 168, 170, 171, or 173 (EU numbering). In some embodiments, the non-Cys amino acids in the light chain CL within the antigen-binding region are replaced with Cys amino acids at positions 114, 116, 118, 124, 160, 162, or 164 (EU numbering).

[0070] In some embodiments, the non-Cys amino acids in the heavy chain CH1 at the CH1-CL [interface] of the antigen-binding region (e.g., IgG1, IgG2, IgG3, or IgG4) are replaced with Cys amino acids at position 126 (EU numbering), and the non-Cys amino acids in the light chain CL are replaced with Cys amino acids at position 124 (EU numbering).

[0071] In some embodiments, the non-Cys amino acid in the heavy chain CH1 at the CH1-CL [interface] of the antigen-binding region (e.g., IgG1, IgG2, IgG3, or IgG4) is substituted with a Cys amino acid at position 168 (EU number), and the non-Cys amino acid in the light chain CL is substituted with a Cys amino acid at position 164 (EU number).

[0072] In some embodiments, the non-Cys amino acid in the heavy chain CH1 at the CH1-CL [interface] of the antigen-binding region (e.g., IgG1, IgG2, IgG3, or IgG4) is substituted with a Cys amino acid at position 170 (EU number), and the non-Cys amino acid in the light chain CL is substituted with a Cys amino acid at position 162 (EU number).

[0073] In some embodiments, the non-Cys amino acid in the heavy chain CH1 at the CH1-CL [interface] of the antigen-binding region (e.g., IgG1, IgG2, IgG3, or IgG4) is substituted with a Cys amino acid at position 173 (EU number), and the non-Cys amino acid in the light chain CL is substituted with a Cys amino acid at position 160 or 162 (EU number).

[0074] In some embodiments, the non-Cys amino acid in the heavy chain CH1 at the CH1-CL [interface] of the antigen-binding region (e.g., IgG1, IgG2, IgG3, or IgG4) is substituted with a Cys amino acid at position 128 (EU number), and the non-Cys amino acid in the light chain CL is substituted with a Cys amino acid at position 118 (EU number).

[0075] In some embodiments, the non-Cys amino acid in the heavy chain CH1 at the CH1-CL [interface] of the antigen-binding region (e.g., IgG1, IgG2, IgG3, or IgG4) is substituted with a Cys amino acid at position 134 (EU number), and the non-Cys amino acid in the light chain CL is substituted with a Cys amino acid at position 116 (EU number).

[0076] In some embodiments, non-Cys amino acids in the CH1 chain at the CH1-CL [interface] of the antigen-binding region (e.g., IgG1, IgG2, IgG3, or IgG4) are replaced with Cys amino acids at position 136 (EU number), and non-Cys amino acids in the CL chain are replaced with Cys amino acids at position 114 (EU number).

[0077] In some embodiments, non-Cys amino acids in the heavy-chain CH1 at the CH1-CL [interface] of the antigen-binding region (e.g., IgG1, IgG2, IgG3, or IgG4) are replaced with Cys amino acids at position 171 (EU number), and non-Cys amino acids in the light-chain CL are replaced with Cys amino acids at position 162 (EU number).

[0078] In some embodiments, non-Cys amino acids in the heavy-chain CH1 at the CH1-CL [interface] of the antigen-binding region (e.g., IgG1, IgG2, IgG3, or IgG4) are replaced with Cys amino acids at position 139 (EU number), and non-Cys amino acids in the light-chain CL are replaced with Cys amino acids at position 116 (EU number).

[0079] Non-Cys amino acids include, in some aspects, naturally occurring and / or non-classical amino acids. Naturally occurring non-Cys amino acids include Gly, Ala, Val, Leu, Ile, Pro, Ser, Thr, Met, His, Lys, Arg, Glu, Asp, Gln, Asp, Phe, Tyr, and Trp. 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, pyridylalanine, thienylalanine, naphthylalanine, and phenylglycine.

[0080] In some embodiments, the disulfide bond reformation mutation includes F126C in the CH1 of the antigen-binding region and Q124C (EU number) in the CL.

[0081] In some embodiments, the disulfide bond reformation mutation comprises H168C in CH1 of the antigen-binding region and T164C (EU number) in CL.

[0082] In some embodiments, the disulfide bond reformation mutation comprises HF170C in CH1 of the antigen-binding region and T164C (EU number) in CL.

[0083] In some embodiments, the disulfide bond reformation mutation comprises HF170C in CH1 of the antigen-binding region and S162C (EU number) in CL.

[0084] In some embodiments, the disulfide bond reformation mutation comprises V173C in CH1 of the antigen-binding region and S162C (EU number) in CL.

[0085] In some embodiments, the disulfide bond reformation mutation comprises V173C in CH1 of the antigen-binding region and Q160C (EU number) in CL.

[0086] In some embodiments, the disulfide bond reformation mutation comprises L128C in CH1 of the antigen-binding region and F118C (EU number) in CL.

[0087] In some embodiments, the disulfide bond reformation mutation comprises S134C in CH1 of the antigen-binding region and F116C (EU number) in CL.

[0088] In some embodiments, the disulfide bond reformation mutation comprises S136C in CH1 of the antigen-binding region and S114C (EU number) in CL.

[0089] In some embodiments, the disulfide bond reformation mutation comprises P171C in CH1 of the antigen-binding region and S162C (EU number) in CL.

[0090] In some embodiments, the disulfide bond reformation mutations include T139C in CH1 of the antigen-binding region and F116C (EU number) in CL.

[0091] Accordingly, in one embodiment of the present disclosure, the present disclosure 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, (i) the first CH1 comprises an F126C mutation and the first CL comprises a Q124C mutation (EU number), (ii) the first CH1 comprises an H168C mutation and the first CL comprises a T164C mutation (EU number), (iii) the first CH1 comprises an F170C mutation and the first CL comprises a T164C mutation (EU number), (iv) the first CH1 comprises an F170C mutation and the first CL comprises an S162C mutation (EU number), (v) the first CH1 comprises a V173C mutation and the first CL comprises an S162C mutation (EU number), (vi) the first CH1 comprises a V173C mutation and the first CL comprises a Q160C mutation (EU number), (vii) the first CH1 comprises an L128C mutation and the first CL comprises an F118C mutation (EU number), (viii) the first CH1 comprises an S134C mutation and the first CL comprises an F116C mutation (EU number), (ix) the first CH1 comprises an S136C mutation and the first CL comprises an S114C mutation (EU number), (x) the first CH1 comprises a P171C mutation and the first CL comprises an S162C mutation (EU number), (xi) the first CH1 comprises a T139C mutation and the first CL comprises an F116C mutation (EU number). In some embodiments, CH1 is derived from IgG1, IgG2, IgG3, or IgG4, preferably from IgG1 or IgG4. In some embodiments, CL is derived from a kappa light chain or a lambda light chain.

[0092] In some embodiments, disulfide bond reformation mutations also include replacing native Cys with non-Cys. In some embodiments, the interchain Cys at the heavy chain constant region-light chain constant region interface of one or two or more antigen-binding regions is replaced with non-Cys. In some embodiments, the Cys amino acid of CH1 within the antigen-binding region is replaced with a non-Cys amino acid at position 220 (EU number). In some embodiments, the Cys amino acid of IgG heavy chain CH1 within the antigen-binding region is replaced with a non-Cys amino acid at position 220 (EU number), or the Cys of IgG2, IgG3, or IgG4 heavy chain CH1 within the antigen-binding region is replaced with a non-Cys amino acid at position 131 (EU number). In some embodiments, the Cys amino acid of light chain CL within the antigen-binding region is replaced with a non-Cys amino acid at position 214 (EU number). In some embodiments, the Cys amino acid of IgG1 heavy chain CH1 within the antigen-binding region is replaced with a non-Cys amino acid at position 220 (EU number), and the Cys amino acid of light chain CL is replaced with a non-Cys amino acid at position 214, or the Cys amino acid within the IgG2, IgG3, or IgG4 heavy chain CH1 region is replaced with a non-Cys amino acid at position 131 (EU number), and the Cys amino acid of light chain CL is replaced with a non-Cys amino acid at position 214 (EU number). Exemplary mutations from Cys to non-Cys are described, for example, in CN104011221B. In some embodiments, Cys is replaced with Ser, Ala, or Val, for example, Ser.

[0093] In some embodiments, the disulfide bond reformation mutations include C220S / A / V in CH1 of the antigen-binding region (including C131S / A / V at the corresponding position of IgG2, IgG3, or IgG4, or IgG1 subtype), and C214S / A / V (EU number) in CL.

[0094] Thus, in one embodiment of the present disclosure, the present disclosure relates to a bsAb comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region comprises CH1 and CL, (i) The first CH1 contains the F126C mutation, and the first CL contains the Q124C mutation (EU number). (ii) The first CH1 contains the H168C mutation, and the first CL contains the T164C mutation (EU number). (iii) The first CH1 contains the F170C mutation, and the first CL contains the T164C mutation (EU number). (iv) The first CH1 contains the F170C mutation, and the first CL contains the S162C mutation (EU number). (v) The first CH1 contains the V173C mutation, and the first CL contains the S162C mutation (EU number). (vi) The first CH1 contains the V173C mutation, and the first CL contains the Q160C mutation (EU number). (vii) The first CH1 contains the L128C mutation, and the first CL contains the F118C mutation (EU number). (viii) The first CH1 contains the S134C mutation, and the first CL contains the F116C mutation (EU number). (ix) The first CH1 contains the S136C mutation, and the first CL contains the S114C mutation (EU number). (x) The first CH1 contains the P171C mutation, and the first CL contains the S162C mutation (EU number). (xi) The first CH1 contains the T139C mutation, and the first CL contains the F116C mutation (EU number). CH1 also contains the C220S / A / V mutation (or the corresponding C131S / A / V), and CL also contains the C214S / A / V mutation (EU number). Preferably, CH1 also contains C220S (EU number), and CL also contains C214S (EU number).

[0095] In some embodiments, the first antigen-binding region contains CH1 and CL. CH1 contains the T139C and C220S mutations (or the corresponding C131S), and CL contains the F116C and C214S mutations (EU number).

[0096] In some embodiments, the first antigen-binding region comprises CH1 and CL, wherein CH1 comprises the F126C and C220S mutations (or the corresponding C131S), and CL comprises the Q124C and C214S mutations (EU numbering).

[0097] In some embodiments, the first antigen-binding region comprises CH1 and CL, wherein CH1 comprises the H168C and C220S mutations (or the corresponding C131S), and CL comprises the T164C and C214S mutations (EU numbering).

[0098] In some embodiments, the first antigen-binding region comprises CH1 and CL, wherein CH1 comprises the F170C and C220S mutations (or the corresponding C131S), and CL comprises the T164C and C214S mutations (EU numbering).

[0099] In some embodiments, the first antigen-binding region comprises CH1 and CL, wherein CH1 comprises the F170C and C220S mutations (or the corresponding C131S), and CL comprises the S162C and C214S mutations (EU numbering).

[0100] In some embodiments, the first antigen-binding region comprises CH1 and CL, wherein CH1 comprises the V173C and C220S mutations (or the corresponding C131S), and CL comprises the S162C and C214S mutations (EU numbering).

[0101] In some embodiments, the first antigen-binding region comprises CH1 and CL, wherein CH1 comprises the V173C and C220S mutations (or the corresponding C131S), and CL comprises the Q160C and C214S mutations (EU numbering).

[0102] In some embodiments, the first antigen-binding region comprises CH1 and CL, wherein CH1 comprises the L128C and C220S mutations (or the corresponding C131S), and CL comprises the F118C and C214S mutations (EU numbering).

[0103] In some embodiments, the first antigen-binding region comprises CH1 and CL, wherein CH1 comprises the S134C and C220S mutations (or the corresponding C131S), and CL comprises the F116C and C214S mutations (EU numbering).

[0104] In some embodiments, the first antigen-binding region comprises CH1 and CL, wherein CH1 comprises the S136C and C220S mutations (or the corresponding C131S), and CL comprises the S114C and C214S mutations (EU numbering).

[0105] In some embodiments, the first antigen-binding region comprises CH1 and CL, wherein CH1 comprises the P171C and C220S mutations (or the corresponding C131S), and CL comprises the S162C and C214S mutations (EU numbering).

[0106] In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 1 and comprises F126C. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 129 and comprises F126C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 129.

[0107] In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 132 and comprises H168C. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 132. In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 133 and comprises H168C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 133.

[0108] In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 134 and comprises F170C. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 134. In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 135 and comprises F170C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 135.

[0109] In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 136 and includes V173C. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 136. In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 137 and includes V173C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 137.

[0110] In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 138 and includes L128C. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 138. In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 139 and includes L128C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 139.

[0111] In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 140 and comprises S134C. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 140. In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 141 and comprises S134C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 141.

[0112] In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 142 and comprises S136C. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 142. In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 143 and comprises S136C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 143.

[0113] In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 144 and comprises P171C. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 144. In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 145 and comprises P171C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 145.

[0114] In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 146 and comprises T139C. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 146. In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 147 and comprises T139C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 147.

[0115] In some embodiments, CL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 2 and comprises Q124C. In some embodiments, CL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, CL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 128 and comprises Q124C and C124S. In some embodiments, CL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 128.

[0116] In some embodiments, CL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 148 and comprises T164C. In some embodiments, CL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 148. In some embodiments, CL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 149 and comprises T164C and C124S. In some embodiments, CL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 149.

[0117] In some embodiments, CL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity 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 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity 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.

[0118] In some embodiments, CL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 152 and comprises Q160C. In some embodiments, CL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 152. In some embodiments, CL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 35 and comprises Q160C and C124S. In some embodiments, CL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35.

[0119] In some embodiments, CL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity 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 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity 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.

[0120] In some embodiments, CL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity 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 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity 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.

[0121] In some embodiments, CL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 99 and comprises S114C. In some embodiments, CL comprises or consists of the amino acid sequence shown in SEQ ID NO: 99. In some embodiments, CL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 100 and comprises S114C and C124S. In some embodiments, CL comprises or consists of the amino acid sequence shown in SEQ ID NO: 100.

[0122] I-2 Charge Variation Charge variation is an effective strategy to prevent mismatches between the light and heavy chains of two target binding regions by forcing homologous light and heavy chain pairing, where amino acid pairs are found on the contact surfaces of the heavy and light chains and are mutated to amino acids carrying opposite charges to promote homologous pairing based on the principle of opposite charge attraction.

[0123] In one embodiment, the binding molecules of the present disclosure, such as antibodies (e.g., multispecific antibodies, e.g., bsAbs), comprise a charge variation. In some embodiments, the charge variation is a mutation of an amino acid pair on the contact surfaces of the heavy and light chains of at least one antigen-binding region, and 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: 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. b. The distance between the α-carbon atoms of the amino acid pair is less than 12 Å, and / or c. The side chains of the amino acid pair face each other.

[0124] In some embodiments, the charge mutation comprises mutating two amino acids on an amino acid pair selected from the following positions to amino acids carrying opposite charges: 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).

[0125] In some embodiments, the charge mutation comprises mutating two amino acids on an amino acid pair selected from the following positions to amino acids carrying opposite charges: position Q39 of the heavy chain variable region and position Q38 of the light chain variable region, position L45 of the heavy chain variable region and position Y87 of the light chain variable region, position L45 of the heavy chain variable region and position P44 of the light chain variable region, position Y91 of the heavy chain variable region and position A43 of the light chain variable region, or position Y91 of the heavy chain variable region and position P44 of the light chain variable region (Kabat numbering).

[0126] In some embodiments, the positions of one or more amino acid pairs in the antigen-binding regions for binding to different antigens each contain a charge variation. In some embodiments, the amino acid positions containing charge variations in the antigen-binding regions for binding to different antigens may be the same or different. When the amino acid positions containing charge variations are the same, the mutated amino acids of the amino acid pairs in the antigen-binding regions for binding to different antigens are different. For example, in some embodiments, the same positions of one or more amino acid pairs in the first antigen-binding region and the second antigen-binding region contain charge variations, but the charge variation for the amino acid pair in one antigen-binding region is different from the charge variation for the amino acid pair in another antigen-binding region. For example, at the same position, the charge carried by the mutated amino acid in the first antigen-binding region is opposite to the charge carried by 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 mutates to an amino acid carrying a positive charge, and the light-chain amino acid at position X2 mutates to an amino acid carrying a negative charge. At the same time, in the second antigen-binding region, the heavy-chain amino acid at position X1 mutates to an amino acid carrying a negative charge, and the light-chain amino acid at position X2 mutates to an amino acid carrying a positive charge, where the amino acid carrying a positive charge in the first antigen-binding region may be the same as or different from, for example, the amino acid carrying a positive charge in the second antigen-binding region, and the amino acid carrying a negative charge in the first antigen-binding region may be the same as or different from, for example, the amino acid carrying a negative charge in the second antigen-binding region.

[0127] In some embodiments, the amino acids in the heavy-chain variable region within the amino acid pair are substituted with amino acids carrying a positive charge, and the amino acids in the light-chain variable region are substituted with amino acids carrying a negative charge. In some embodiments, the amino acids in the heavy-chain variable region within the amino acid pair are substituted with amino acids carrying a negative charge, and the amino acids in the light-chain variable region are substituted with amino acids carrying a positive charge.

[0128] In some embodiments, in the first antigen-binding region, the amino acid at position 39 of the heavy-chain variable region is substituted with an amino acid carrying a positive charge, the amino acid at position 38 of the light-chain variable region is substituted with an amino acid carrying a negative charge, and / or in the second antigen-binding region, the amino acid at position 39 of the heavy-chain variable region is substituted with an amino acid carrying a negative charge, and the amino acid at position 38 of the light-chain variable region is substituted with an amino acid carrying a positive charge.

[0129] In some embodiments, in the first antigen-binding region, the amino acid at position 45 of the heavy-chain variable region is substituted with an amino acid carrying a positive charge, the amino acid at position 87 of the light-chain variable region is substituted with an amino acid carrying a negative charge, and / or in the second antigen-binding region, the amino acid at position 45 of the heavy-chain variable region is substituted with an amino acid carrying a negative charge, and the amino acid at position 87 of the light-chain variable region is substituted with an amino acid carrying a positive charge.

[0130] In some embodiments, in the first antigen-binding region, the amino acid at position 45 of the heavy-chain variable region is substituted with an amino acid carrying a positive charge, the amino acid at position 44 of the light-chain variable region is substituted with an amino acid carrying a negative charge, and / or in the second antigen-binding region, the amino acid at position 45 of the heavy-chain variable region is substituted with an amino acid carrying a negative charge, and the amino acid at position 44 of the light-chain variable region is substituted with an amino acid carrying a positive charge.

[0131] In some embodiments, in the first antigen-binding region, the amino acid at position 91 of the heavy-chain variable region is substituted with an amino acid carrying a positive charge, the amino acid at position 43 of the light-chain variable region is substituted with an amino acid carrying a negative charge, and / or in the second antigen-binding region, the amino acid at position 91 of the heavy-chain variable region is substituted with an amino acid carrying a negative charge, and the amino acid at position 43 of the light-chain variable region is substituted with an amino acid carrying a positive charge.

[0132] In some embodiments, in the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with an amino acid carrying a positive charge, the amino acid at position 44 of the light chain variable region is substituted with an amino acid carrying a negative charge, and / or in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with an amino acid carrying a negative charge, and the amino acid at position 44 of the light chain variable region is substituted with an amino acid carrying a positive charge.

[0133] In some embodiments, the amino acid carrying a positive charge is selected from K, R, and H, and / or the amino acid carrying a negative charge is selected from E or D. In some embodiments, the amino acid carrying a positive charge is selected from K and R, and / or the amino acid carrying a negative charge is selected from E or D. In some embodiments, the amino acid carrying a positive charge is K, and / or the amino acid carrying a negative charge is D.

[0134] In some embodiments, in the 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 the 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.

[0135] In some embodiments, in the 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 the 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.

[0136] In some embodiments, in the 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 the 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.

[0137] In some embodiments, in the 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 the 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.

[0138] In some embodiments, in the 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 the 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.

[0139] In some embodiments, the binding molecules of the present disclosure, such as multispecific antibodies, e.g., bsAbs, comprise a first antigen-binding region and a second antigen-binding region comprising substitutions selected from the following: (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; 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 heavy-chain variable region is substituted with K. (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; 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. (iii). In the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with K, the amino acid at position 44 of the light chain variable region is substituted with D, 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. (iv). In the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with K, the amino acid at position 43 of the light chain variable region is substituted with D, 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. (v). In the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with K, the amino acid at position 44 of the light chain variable region is substituted with D, 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.

[0140] I-3 Combinatorial Mutations In some embodiments, the binding molecules of the present disclosure, e.g., antibodies, e.g., multispecific antibodies, e.g., bispecific antibodies, comprise a first target-binding region (antigen-binding region) and a second target-binding region (antigen-binding region) that include disulfide bond reformation mutations and charge mutations.

[0141] In some embodiments, the present disclosure relates to types of antibodies, e.g., multispecific antibodies, e.g., bispecific antibodies, that comprise 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.

[0142] In some embodiments, the first antigen-binding region comprises a disulfide bond reformation mutation at the CH1-CL interface and charge mutations in VH and VL.

[0143] In some embodiments, the first antigen-binding region comprises a disulfide bond reformation mutation at the CH1-CL interface and charge mutations in VH and VL, and the second antigen-binding region comprises charge mutations in VH and VL.

[0144] In some embodiments, the first antigen-binding region comprises a combination of mutations selected from the following. [Table 1-1] [Table 1-2]

[0145] In some embodiments, the second antigen-binding region comprises a combination of mutations selected from the following. [Table 2]

[0146] In some embodiments, the present disclosure relates to a type of antibody comprising a first antigen-binding region and a second antigen-binding region, for example, a multispecific antibody, for example, a bispecific antibody, 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, and the antibody comprises the following combination of mutations. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0147] In some embodiments, in an antibody, e.g., a multispecific antibody (e.g., a bispecific antibody), one or more or all of the antigen-binding regions that bind to the same antigen contain the same mutation.

[0148] In some embodiments, in an antibody, e.g., a multispecific antibody (e.g., a bispecific antibody), one or more or all of the antigen-binding regions that bind to the same antigen contain different mutations.

[0149] I-4 Kappa light chain constant region substitution In some embodiments, a binding molecule of the present disclosure, e.g., an antibody such as a multispecific antibody, comprises a first target-binding region (antigen-binding region) and a second target-binding region (antigen-binding region), and the light chain constant region contained in one or more or all of the first target-binding regions (antigen-binding regions) is a kappa light chain constant region, and the light chain constant region contained in one or more or all of the second target-binding regions is a lambda light chain constant region, and the light chain constant region contained in one or more or all of the first target-binding regions (antigen-binding regions) is a lambda light chain constant region, and the light chain constant region contained in one or more or all of the second target-binding regions is a kappa light chain constant region.

[0150] I-5 Fc region mutation In some embodiments, one or more target-binding regions (antigen-binding regions) of a binding molecule of the present disclosure, e.g., an antibody such as a multispecific antibody (e.g., a bispecific antibody), comprise an Fc region, and the Fc regions comprised may be the same or different.

[0151] In some embodiments, the first Fc region and the second Fc region are different and may heterodimerize to form a heterodimeric Fc scaffold.

[0152] As used herein, the Fc region refers to the C-terminal region that includes at least a part of the constant region of the immunoglobulin heavy chain, and may include a native sequence Fc region and a variant Fc region. The native sequence Fc region encompasses various naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various IgG subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector function, 20 October 2014, doi:10.3389 / fimmu.2014.00520). In some embodiments, the Fc region of the present disclosure includes antibody CH2 and CH3. In some embodiments, the antibody Fc region may also carry an IgG hinge region or a part of the IgG hinge region at the N-terminus, such as the IgG1 hinge region or a part of the IgG1 hinge region, for example, the sequence of D221 - P230 according to the EU numbering. The hinge region may contain mutations.

[0153] Unless otherwise specified herein, the number of amino acid residues within the Fc region is based on the EU numbering scheme, also known as the EU index, such as those referred to in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, Version 5, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91 - 3,242.

[0154] In some embodiments, the Fc region is a human IgG Fc, such as 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 has at least 90% identity, such as 95%, 96%, 97%, 99%, or more identity thereto.

[0155] As will be understood by those skilled in the art, in order to facilitate the formation of the multispecific antibodies of the present disclosure as heterodimers, the Fc regions included in the multispecific antibodies of the present disclosure may include mutations that are favorable for the heterodimerization of the first Fc region and the second Fc region. In one embodiment, the mutations are introduced into the CH3 regions of the two Fc regions.

[0156] Methods known in the art for promoting Fc region heterodimerization. For example, the CH3 region of the first Fc region and the CH3 region of the second Fc region are manipulated in a complementary manner, such that as a result, each CH3 region (or the heavy chain containing it) can no longer homodimerize with itself and is forced to heterodimerize with the other CH3 region that has been manipulated complementarily (such that the CH3 regions of the first and second Fc regions heterodimerize and no homodimers are formed between two first CH3 regions or two second CH3 regions).

[0157] Preferably, based on the knob-into-hole technology, corresponding knob mutations and hole mutations are introduced into the first Fc region and the second Fc region. For this technology, see, for example, U.S. Patent No. 5,731,168, U.S. Patent No. 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996), and Carter, J Immunol Meth 248, 7-15 (2001).

[0158] In a particular embodiment, in the CH3 region of one Fc region, the Thr residue at position 366 is replaced with a Trp residue (T366W) (knob mutation), while in the CH3 region of the other Fc region, the Tyr residue at position 407 is replaced with a Val residue (Y407V) (hole mutation), and optionally, the Thr residue at position 366 is replaced with a Ser residue (T366S), and the Tyr residue at position 407 is replaced with a Val residue (Y407V) (numbering based on the EU index).

[0159] In another embodiment, in the CH3 region of one Fc region, the Thr residue at position 366 is replaced with a Trp residue (T366W), the Ser residue at position 354 is replaced with a Cys residue (S354C), or the Glu residue at position 356 is replaced with a Cys residue (E356C) (in particular, the Ser residue at position 354 is replaced with a Cys residue), while in the CH3 region of the other Fc region, the Tyr residue at position 407 is replaced with a Val residue (Y407V) (whole mutation), optionally, the Thr residue at position 366 is replaced with a Ser residue (T366S), the Leu residue at position 368 is replaced with an Ala residue (L368A) (numbering based on EU index), optionally, the Tyr residue at position 349 is replaced with a Cys residue (Y349C) (numbering based on EU index).

[0160] In a particular embodiment, one Fc region contains the amino acid substitution T366W and the other Fc region contains the amino acid substitutions T366S, L368A, and Y407V (numbering based on EU index).

[0161] In a particular embodiment, one Fc region contains the amino acid substitutions S354C and T366W and the other Fc region contains the amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering based on EU index).

[0162] The corresponding mutations may be introduced into the first Fc region and the second Fc region based on the Innobody technology. For this technology, see, for example, PCT / CN2021 / 143141.

[0163] In a particular embodiment The first CH3 region contains the S364R / K mutation (preferably S364R), and optionally one or more other mutations. In some embodiments, the second CH3 region contains the K370S / T / A / V mutation (preferably K370S), and optionally one or more other mutations. In some embodiments, the first CH3 region contains the S364R / K mutation and the second CH3 region contains the K370S / T / A / V mutation. In some embodiments, the first CH3 region contains the S364R mutation and the second CH3 region contains the K370S mutation.

[0164] In some embodiments, the first CH3 region contains the S364R / K (preferably S364R) and D399K / R (preferably D399K) mutations. In some embodiments, the second CH3 region contains the K370S / T / A / V mutation (preferably K370S), and the K409D / E (preferably K409D) mutation. In some embodiments, the first CH3 region contains S364R / K + D399K / R, and the second CH3 region contains K370S / T / A / V + Y349T / S / A / V. In some embodiments, the first CH3 region contains S364R + D399K, and the second CH3 region contains K370S + Y349T. In some embodiments, the first CH3 region also contains E375N / Q (preferably E375N), and / or T350V / A (preferably T350V). In some embodiments, the second CH3 region also contains K409D / E (preferably K409D), Q347D / E (preferably Q347D), and / or T350V / A (preferably T350V).

[0165] 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 also comprises E357N. In some embodiments, the second CH3 region also comprises Q347D. In some embodiments, the first CH3 region also comprises E357N, and the second CH3 region also comprises Q347D. In some embodiments, the first CH3 region and the second CH3 region each further comprise T350V, or both comprise T350V.

[0166] 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.

[0167] 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 also comprises T411R / K (preferably T411R). In some embodiments, the second CH3 region also comprises K370T / S / A / V (preferably K370T). In some embodiments, the [first] CH3 region comprises K409E / D + T411R / K, and the second CH3 region comprises D399K / R + K370T / S / A / V. In some embodiments, the [first] CH3 region comprises K409E + T411R, and the second CH3 region comprises D399K + K370T.

[0168] In some specific embodiments, the first and second CH3 regions have the following combination of mutations. [Table 4]

[0169] In one embodiment, the CH3 of one Fc region contains the S364R and D399K mutations, and the CH3 mutation of the other Fc region contains the Y349T, K370S, and K409D mutations.

[0170] Thus, in certain embodiments, the binding molecules of the present disclosure, such as antibodies such as bispecific antibodies, include heterodimerization of two Fc regions, wherein a) the polypeptide of one Fc region contains the mutation T366W, while the polypeptide of the other Fc region contains T366S, L368A, and Y407V, or b) the polypeptide of one Fc region contains the mutations S354C and T366W, while the polypeptide of the other Fc region contains Y349C, T366S, L368A, and Y407V, or c) the polypeptide of one Fc region contains the mutations S364R and D399K, while the polypeptide of the other Fc region contains Y349T, K370S, and K409D.

[0171] Thus, in certain embodiments, the binding molecules of the present disclosure, such as antibodies such as bispecific antibodies, include heterodimerization of two Fc regions, wherein a) the polypeptide of one Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 20 or 131, while the polypeptide of the other Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 101 or 130, b) the polypeptide of one Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 125, while the polypeptide of the other Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 124, or c) One Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 126, while the polypeptide of the other Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 127.

[0172] Thus, in certain embodiments, the binding molecules of the present disclosure, such as antibodies including bispecific antibodies, include heterodimerization of two Fc regions, where a) the polypeptide of one Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 20 or 131, while the polypeptide of the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 101 or 130, b) the polypeptide of one Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 125, while the polypeptide of the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 124, or c) one Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 126, while the polypeptide of the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 127.

[0173] Thus, in certain embodiments, the binding molecules of the present disclosure, such as antibodies including bispecific antibodies, include heterodimerization of two Fc regions, where a) The polypeptide of one Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 20 or 131, and includes the mutation T366W. On the other hand, the polypeptide of the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 101 or 130, and includes T366S, L368A, and Y407V, or b) The polypeptide of one Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 125, and includes the mutations S354C and T366W. On the other hand, the polypeptide of the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 124, and includes the mutations Y349C, T366S, L368A, and Y407V, or c) One Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 126, and includes the mutations S364R and D399K. On the other hand, the polypeptide of the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 127, and includes the mutations Y349T, K370S, and K409D.

[0174] In some embodiments, the Fc region also includes other mutations that are preferred for heterodimer purification.

[0175] The Fc region in the binding molecules of the present disclosure, such as antibodies, may also be mutated to obtain desired properties. Mutations of the Fc region are known in the art.

[0176] In one embodiment, the Fc region is modified to [modify] its effector function characteristics therein (e.g., the complement activation function in the Fc region). In one embodiment, the effector function is reduced or eliminated as compared to the Fc region of the wild-type isotype. In one embodiment, the effector function is reduced or eliminated via a method selected from: an Fc isotype in which the effector function is naturally reduced or eliminated, and Fc region modification.

[0177] In a preferred embodiment, the Fc region has a reduced Fc region-mediated effector function, such as a reduced ADCC or ADCP or CDC effector function, including mutations for achieving the above functions, for example.

[0178] As will be understood by those skilled in the art, in accordance with the expected use of the binding molecules of the present disclosure, such as antibody molecules, the binding molecules of the present disclosure, such as antibody molecules, may also include modifications within 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 includes mutations that reduce binding to the Fcγ receptor. For example, in some embodiments, the Fc region used in the present disclosure has an L234A / L235A mutation that reduces binding to the Fcγ receptor. In another preferred embodiment, the Fc fragment may have mutations that cause an increase in serum half-life, such as mutations that improve binding of the Fc fragment to FcRn.

[0179] I-6. Bispecific Antibody or Multispecific Antibody In some embodiments, the antibodies of the present disclosure are bispecific antibodies.

[0180] The term "bispecific antibody" refers to an antibody that includes a first antigen-binding region and a second antigen-binding region, where the first antigen-binding region binds to one type of antigen or epitope, and the second antigen-binding region binds to a different antigen or epitope. Thus, bispecific antibodies according to the present disclosure include specificity for two different types of antigens, or two different epitopes of one type of antigen. Bispecific antibody formats include IgG-like antibodies (Fan et al. (2015) Journal of Hematology & Oncology. 8:130). The most common type of IgG-like antibody includes two Fab regions and two Fc regions, and the heavy and light chains of each Fab may be from independent monoclonal antibodies.

[0181] The bispecific antibodies of the present disclosure can be prepared using bispecific antibody formats or techniques known in the art. For specific exemplary bispecific antibody formats used in the context of the present disclosure, see, 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 disclosure have the following formats: bispecific antibodies having a 2+1 N-terminal configuration (Figure 5A), bispecific antibodies having a 2+1 C-terminal configuration (Figure 5B), bispecific antibodies having a 2+2 C-terminal configuration (Figure 5C). In some embodiments, the bispecific antibodies of the present disclosure also include a 2+2 N configuration or a tandem Fab configuration.

[0182] In some embodiments, the bispecific antibody of the present disclosure 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. The first antigen-binding region comprises a first Fab, and the second antigen-binding region comprises a second Fab. The first Fab is linked at the C-terminus of its CH1 to the N-terminus of a first Fc region (with or without a linker, such as a hinge region), and the second Fab is linked at the C-terminus of its CH1 to the N-terminus of a second Fc region (with or without a linker, such as a hinge region). In certain embodiments, the bispecific antibody is an IgG-like antibody having the configuration shown in FIG. 1. In certain embodiments, the first Fab comprises charge mutations and disulfide bond reformation mutations, the second Fab comprises charge mutations, and optionally, the first Fc region and the second Fc region comprise Innobody mutations or knob-into-hole mutations. Preferably, the charge mutations comprised by the first Fab and the charge mutations comprised by the second Fab have the same positions, but the charges carried by the mutated amino acids at the same positions are opposite. In one embodiment, the bispecific antibody is Heavy chain 1: hereinafter, from the N-terminus to the C-terminus, comprising or consisting of the heavy chain variable region - heavy chain constant region CH1 - first Fc region of the first Fab, and the heavy chain constant region CH1 is linked at its C-terminus to the N-terminus of the first Fc region, with or without a linker (such as a hinge region), Light chain 1: hereinafter, from the N-terminus to the C-terminus, comprising or consisting of the light chain variable region - light chain constant region of the first Fab, Heavy chain 2: hereinafter, from the N-terminus to the C-terminus, comprising or consisting of the heavy chain variable region - heavy chain constant region CH1 - second Fc region of the second Fab, and the heavy chain constant region CH1 is linked at its C-terminus to the N-terminus of the second Fc region, with or without a linker (such as a hinge region), Light chain 2: hereinafter, from the N-terminus to the C-terminus, comprising or consisting of the light chain variable region - light chain constant region of the second Fab.

[0183] In some embodiments, the bispecific antibody of the present disclosure includes 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. The first antigen-binding region includes a first Fab, and the second antigen-binding region includes a second Fab. One second Fab is linked at the C-terminus of its CH1 to the N-terminus of the first Fc region (with or without a linker, such as a hinge region). One first Fab is linked at the C-terminus of its CH1 to the N-terminus of the second Fc region (with or without a linker, such as a hinge region). The other second Fab is linked at the C-terminus of its CH1 to the N-terminus of the heavy chain variable region of the first Fab (with or without a linker). In certain embodiments, the bispecific antibody has a 2+1 N-terminal configuration and the configuration shown in FIG. 5A. In certain embodiments, one or two of the second Fabs include a charge variant according to the present disclosure, the first Fab includes a charge variant and a disulfide bond reformation variant according to the present disclosure. Optionally, the variants are derived from the first Fc region and the second Fc region based on Innobody or knob-into-hole. Preferably, the two second Fabs include the same charge variant. Preferably, the charge variant included in the second Fab has the same position as the charge variant included in the first Fab, but the charge carried by the mutated amino acid at the same position is opposite. In one embodiment, the bispecific antibody is Heavy chain 1: hereinafter, from the N-terminus to the C-terminus, includes or consists of the heavy chain variable region of the second Fab - heavy chain constant region CH1 - the heavy chain variable region of the first Fab - heavy chain constant region CH1 - the second Fc region. The heavy chain constant region CH1 of the first Fab is linked at its C-terminus to the N-terminus of the second Fc region with or without a linker (such as a hinge region). The second Fab is linked at the C-terminus of its CH1 to the N-terminus of the heavy chain variable region of the first Fab with or without a linker. Light chain 1: hereinafter, from the N-terminus to the C-terminus, includes or consists of the light chain variable region - light chain constant region of the first Fab. Heavy chain 2: hereinafter, from the N-terminus to the C-terminus, comprising or consisting of the variable heavy chain region of the second Fab - the constant heavy chain region CH1 - the first Fc region, wherein the constant heavy chain region CH1 is linked at its C-terminus to the N-terminus of the first Fc region, with or without a linker (e.g., a hinge region), Light chain 2: hereinafter, from the N-terminus to the C-terminus, comprising or consisting of the variable light chain region of the second Fab - the constant light chain region, The bispecific antibody comprises two light chains 2.

[0184] In some embodiments, the bispecific antibody of the present disclosure 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, and the two second Fabs are each linked 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 (with or without a linker, e.g., a hinge region), and the N-terminus of the variable heavy chain region of the first Fab is linked to the C-terminus of the second Fc region (with or without a linker). In certain embodiments, the bispecific antibody has a 2+1 C-terminus configuration and the configuration shown in FIG. 5B. In certain embodiments, one or two of the second Fabs comprise a charge variant according to the present disclosure, the first Fab comprises a charge variant and a disulfide bond reformation variant according to the present disclosure, and optionally, the first Fc region and the second Fc region comprise an Innobody variant or a knob-into-hole variant. Preferably, the two second Fabs comprise the same charge variant. Preferably, the charge variant comprised by the first Fab and the charge variant comprised by the second Fab have the same position, but the charges carried by the variant amino acids at the same position are opposite. In one embodiment, the bispecific antibody is Heavy chain 1: hereinafter, from the N-terminus to the C-terminus, comprising or consisting of the variable heavy region of the second Fab - the constant heavy region CH1 of the heavy chain - the second Fc region - the variable heavy region of the first Fab - the constant heavy region CH1 of the heavy chain, wherein the second Fab is linked at the N-terminus of the second Fc region to the C-terminus of its CH1 (with or without a linker, such as a hinge region), and the N-terminus of the variable heavy region of the first Fab is linked to the C-terminus of the second Fc region (with or without a linker). Light chain 1: hereinafter, from the N-terminus to the C-terminus, comprising or consisting of the variable light region of the first Fab - the constant light region. Heavy chain 2: hereinafter, from the N-terminus to the C-terminus, comprising or consisting of the variable heavy region of the second Fab - the constant heavy region CH1 of the heavy chain - the first Fc region, wherein the constant heavy region CH1 is linked at the N-terminus of the first Fc region to the C-terminus thereof with or without a linker (such as a hinge region). Light chain 2: hereinafter, from the N-terminus to the C-terminus, comprising or consisting of the variable light region of the second Fab - the constant light region, and The bispecific antibody comprises two light chains 2.

[0185] In some embodiments, the bispecific antibody of the present disclosure includes 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. The first antigen-binding region includes a first Fab, and the second antigen-binding region includes a second Fab. The two second Fabs are linked at the C-terminus of their CH1 to the N-terminus of two Fc regions (with or without a linker, e.g., a hinge region) (the Fc regions may be the same or different). The N-terminus of the heavy chain variable regions of the two first Fabs is linked to the C-terminus of the two Fc regions (with or without a linker). In certain embodiments, the bispecific antibody has a 2+2 C-terminal configuration and the configuration shown in FIG. 5C. In certain embodiments, one or two of the second Fabs include a charge variant according to the present disclosure, and one or two of the first Fabs include a charge variant and a disulfide bond reformation variant according to the present disclosure. In certain embodiments, the two second Fabs include the same charge variant of the present disclosure, and / or the two first Fabs include a charge variant and a disulfide bond reformation variant according to the present disclosure. Preferably, the charge variant included in the first Fab and the charge variant included in the second Fab have the same position, but the charges carried by the mutated amino acids at the same position are opposite. In one embodiment, the bispecific antibody is Heavy chain: hereinafter, from the N-terminus to the C-terminus, includes or consists of the heavy chain variable region of the second Fab - the heavy chain constant region CH1 - the Fc region - the heavy chain variable region of the first Fab - the heavy chain constant region CH1. The second Fab is linked at the C-terminus of its CH1 to the N-terminus of the Fc region (with or without a linker, e.g., a hinge region), and the N-terminus of the heavy chain variable region of the first Fab is linked to the C-terminus of the Fc region (with or without a linker). Light chain 1: hereinafter, from the N-terminus to the C-terminus, includes or consists of the light chain variable region of the first Fab - the light chain constant region. Light chain 2: hereinafter, from the N-terminus to the C-terminus, includes or consists of the light chain variable region of the second Fab - the light chain constant region, and includes The bispecific antibody includes two heavy chains.

[0186] In some embodiments, the antibodies of the present disclosure are multispecific antibodies. In some embodiments, the multispecific antibodies of the present disclosure are trispecific antibodies.

[0187] In some embodiments, the trispecific antibodies of the present disclosure include 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. The first antigen-binding region includes a first Fab, the second antigen-binding region includes a second Fab, the first Fab is linked at the C-terminus of its CH1 to the N-terminus of a first Fc region (with or without a linker, such as a hinge region), the second Fab is linked at the C-terminus of its CH1 to the N-terminus of a second Fc region (with or without a linker, such as a hinge region), and the third antigen-binding region includes a scFv (such as VH-VL or VL-VH) that is linked, with or without a linker, to the C-terminus of the second Fc region at the N-terminus of its VH or VL (for example, the scFv is VL-VH, such as at the N-terminus of VL). In certain embodiments, the trispecific antibody configuration is a 2+1 C-terminus configuration (Figure 7A or B). In certain embodiments, the first Fab includes charge mutations and disulfide bond reformation mutations, the second Fab includes charge mutations, and optionally, the first Fc region and the second Fc region include Innobody mutations or knob-into-hole mutations. Preferably, the charge mutations included in the first Fab and the charge mutations included in the second Fab have the same positions, but the charges carried by the mutated amino acids at the same positions are opposite. In one embodiment, the multispecific antibody is Heavy chain 1: hereinafter, from the N-terminus to the C-terminus, includes or consists of the heavy chain variable region - heavy chain constant region CH1 - first Fc region of the first Fab, and the first Fab is linked at the C-terminus of its CH1 to the N-terminus of the first Fc region (with or without a linker, such as a hinge region), Light chain 1: hereinafter, from the N-terminus to the C-terminus, includes or consists of the light chain variable region - light chain constant region of the first Fab, Heavy chain 2: hereinafter, from the N-terminus to the C-terminus, comprising or consisting of the heavy chain variable region - heavy chain constant region CH1 - the second Fc region - scFv of the second Fab, wherein the heavy chain constant region CH1 is linked to the N-terminus of the second Fc region at the C-terminus of the heavy chain constant region CH1, either with or without a linker (e.g., hinge region), and the scFv is linked 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, e.g., at the N-terminus of VL), either with or without a linker. Light chain 2: hereinafter, from the N-terminus to the C-terminus, comprising or consisting of the light chain constant region - light chain constant region of the second Fab.

[0188] I-7. Antigen and antigen-binding region The binding molecules of the present disclosure, such as antibodies, e.g., multispecific antibodies, e.g., bispecific antibodies, can bind to antigens applicable to antibodies, or any target such as ligands or receptors.

[0189] In some embodiments, the antigen is a tumor-associated antigen (TA). In some embodiments, the TA is an immune checkpoint molecule.

[0190] In some embodiments, the target or antigen is selected from FAP, CEA, p95 HER2, BCMA, EpCAM, MSLN, MCSP, HER-1, HER-2, HER-3, CD19, CD20, CD22, CD33, CD38, CD52, Flt3, EpCAM, IGF-1R, FOLR1, Trop-2, CA-12-5, HLA-DR, MUC-1 (mucoprotein), GD2, A33 antigen, PSMA, PSCA, transferrin receptor, TNC (tenascin), CA-IX, CD3, B7H3, EGFR, Hel, cMET, Axl, GPRC5D, PD-1, PD-L1, CD47, and immune activation molecules such as 4-1BB, CD40, and OX40.

[0191] In some embodiments, the target or antigen is selected from one or more of the following targets / antigens: 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, and different epitopes of the same antigen.

[0192] In some embodiments, the bispecific antibody of the present disclosure binds to two different epitopes of one antigen or binds to two antigens.

[0193] In some specific embodiments, the bispecific antibody of the present disclosure binds to two antigens selected from: 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, and CD3 / GPRC5D.

[0194] In some specific embodiments, the trispecific antibody of the present disclosure binds to BCMA, GPRC5D, and CD3.

[0195] The target binding region or antigen binding region of the binding molecule of the present disclosure, such as an antibody, may be derived from an antibody that binds to the above antigen.

[0196]

Number

[0197] In some embodiments, the antigen binding region comprises one, two, three, four, five, or six CDRs of a known antibody that specifically binds to Her2, such as trastuzumab.

[0198] In some embodiments, the antigen-binding region comprises one, two, and three heavy chain variable region CDRs of a known antibody that specifically binds to Her2, such as trastuzumab, i.e., HCDR1, HCDR2, and HCDR3.

[0199] In some embodiments, the antigen-binding region comprises one, two, and three light chain variable region CDRs of a known antibody that specifically binds to Her2, such as trastuzumab, i.e., LCDR1, LCDR2, and LCDR3.

[0200] 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 Her2, such as trastuzumab.

[0201] 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 a mutation according to the present disclosure.

[0202] In some embodiments, the antigen-binding region comprises the Fab of a known antibody that specifically binds to Her2, such as trastuzumab, and a mutation according to the present disclosure.

[0203]

Number

[0204] In some embodiments, the antigen-binding region comprises one, two, three, four, five, or six CDRs of a known antibody that specifically binds to PD-1, such as the PD-1 antibody disclosed in WO2017025016, such as sintilimab.

[0205] In some embodiments, the antigen-binding region comprises the one, two, and three 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 antibodies disclosed in WO2017025016, such as sintilimab.

[0206] In some embodiments, the antigen-binding region comprises the one, two, and three 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 antibodies disclosed in WO2017025016, such as sintilimab.

[0207] In some embodiments, the antigen-binding region comprises the three heavy chain variable region CDRs and the three light chain variable region CDRs of a known antibody that specifically binds to PD-1, such as the PD-1 antibodies disclosed in WO2017025016, such as sintilimab.

[0208] 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 PD-1, such as the PD-1 antibodies disclosed in WO2017025016, such as sintilimab, and mutations according to the present disclosure.

[0209] In some embodiments, the antigen-binding region comprises the Fab of a known antibody that specifically binds to PD-1, such as the PD-1 antibodies disclosed in WO2017025016, such as sintilimab, and mutations according to the present disclosure.

[0210]

Number

[0211] In some embodiments, the antigen-binding region comprises a known antibody that specifically binds to CD47, such as the antibodies disclosed in WO2019042285A1 that specifically bind to CD47, such as one, two, three, four, five, or six CDRs of ADI26630.

[0212] In some embodiments, the antigen-binding region comprises a known antibody that specifically binds to CD47, such as the antibodies disclosed in WO2019042285A1 that specifically bind to CD47, such as the three heavy chain variable region CDRs of ADI26630, namely, HCDR1, HCDR2, and HCDR3.

[0213] In some embodiments, the antigen-binding region comprises a known antibody that specifically binds to CD47, such as the antibodies disclosed in WO2019042285A1 that specifically bind to CD47, such as the three light chain variable region CDRs of ADI26630, namely, LCDR1, LCDR2, and LCDR3.

[0214] In some embodiments, the antigen-binding region comprises a known antibody that specifically binds to CD47, such as the antibodies disclosed in WO2019042285A1 that specifically bind to CD47, such as the three heavy chain variable region CDRs and the three light chain variable region CDRs of ADI26630.

[0215] In some embodiments, the antigen-binding region comprises a known antibody that specifically binds to CD47, such as the antibodies disclosed in WO2019042285A1 that specifically bind to CD47, such as the heavy chain variable region and the light chain variable region of ADI26630 disclosed herein, and a mutation according to the present disclosure.

[0216] In some embodiments, the antigen-binding region comprises a known antibody that specifically binds to CD47, such as the antibodies disclosed in WO2019042285A1 that specifically bind to CD47, such as ADI26630 disclosed herein, and a mutation according to the present disclosure.

[0217] [Number] Antigen-binding region that specifically binds to cMet In some embodiments, the antigen-binding region is derived from an antibody that specifically binds to cMet, such as, for example, olaratumab.

[0218] In some embodiments, the antigen-binding region comprises one, two, three, four, five, or six CDRs of a known antibody that specifically binds to cMet, such as, for example, olaratumab.

[0219] In some embodiments, the antigen-binding region comprises one, two, and three heavy chain variable region CDRs of a known antibody that specifically binds to cMet, such as, for example, olaratumab, namely, HCDR1, HCDR2, and HCDR3.

[0220] In some embodiments, the antigen-binding region comprises one, two, and three light chain variable region CDRs of a known antibody that specifically binds to cMet, such as, for example, olaratumab, namely, LCDR1, LCDR2, and LCDR3.

[0221] 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 cMet, such as, for example, olaratumab.

[0222] 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, for example, olaratumab, and mutations according to the present disclosure.

[0223] Some antigen-binding regions comprise the Fab of a known antibody that specifically binds to cMet, such as, for example, olaratumab, and mutations according to the present disclosure.

[0224] [Number] Antigen-binding region that specifically binds to GPRC5D In some embodiments, the antigen-binding region is derived from an antibody that specifically binds to GPRC5D, such as the antibody disclosed in PCT / CN2022 / 076832 that specifically binds to GPRC5D.

[0225] In some embodiments, the antibody that specifically binds to GPRC5D comprises three CDRs (HCDRs) from the heavy chain variable region, namely, HCDR1, HCDR2, and HCDR3, and HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 87.

[0226] In some embodiments, the antibody that specifically binds to GPRC5D comprises three CDRs (LCDRs) from the light chain variable region, namely, LCDR1, LCDR2, and LCDR3, and LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO: 83.

[0227] In some embodiments, HCDR1 of the antibody that specifically binds to GPRC5D 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 / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 86.

[0228] In some embodiments, an antibody that specifically binds to GPRC5D comprises or consists of three CDRs (HCDRs) from the heavy chain variable region, namely, HCDR1, HCDR2, and HCDR3, and three CDRs (LCDRs) from the light chain variable region, namely, 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 / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 86.

[0229] In some aspects, an antibody that specifically binds to GPRC5D comprises a heavy chain variable region (VH), and the VH (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 87, or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 87, or (iii) comprises or consists of an amino acid substitution having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, and 1 or fewer) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) in the amino acid sequence of SEQ ID NO: 87, and preferably, the amino acid changes do not occur in the CDRs.

[0230] In some aspects, an antibody that specifically binds to GPRC5D comprises a light chain variable region (VL), and the VL (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 83, or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 83, or (iii) comprising or consisting of an amino acid substitution having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) of SEQ ID NO: 83, preferably, the amino acid changes do not occur in the CDRs.

[0231] In some embodiments, an antibody that specifically binds to GPRC5D comprises VH and VL, wherein VH comprises or consists of the sequence shown in SEQ ID NO: 87, and / or VL comprises or consists of the sequence shown in SEQ ID NO: 83.

[0232] In some embodiments, the antigen-binding region comprises one, two, three, four, five, or six CDRs of a known or the above-described antibody that specifically binds to GPRC5D.

[0233] In some embodiments, the antigen-binding region comprises one, two, and three heavy chain variable region CDRs of a known or the above-described antibody that specifically binds to GPRC5D, namely, HCDR1, HCDR2, and HCDR3.

[0234] In some embodiments, the antigen-binding region comprises one, two, and three light chain variable region CDRs of a known or the above-described antibody that specifically binds to GPRC5D, namely, LCDR1, LCDR2, and LCDR3.

[0235] 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 the above-described antibody that specifically binds to GPRC5D.

[0236] In some embodiments, the antigen-binding region comprises the heavy chain variable region and the light chain variable region of a known or the above-described antibody that specifically binds to GPRC5D, and a mutation according to the present disclosure.

[0237] Some antigen-binding regions comprise a Fab of a known or the above-described antibody that specifically binds to GPRC5D and a mutation according to the present disclosure.

[0238]

Number

[0239] In some embodiments, the antigen-binding region is a known antibody that specifically binds to CD3, for example, an antibody disclosed in WO2022068809 that specifically binds to CD3, for example, comprises 1, 2, 3, 4, 5, or 6 CDRs of sp34.24 or sp34.87.

[0240] In some embodiments, the antigen-binding region is a known antibody that specifically binds to CD3, for example, an antibody disclosed in WO2022068809 that specifically binds to CD3, for example, comprises 1, 2, and 3 heavy-chain variable region CDRs of sp34.24 or sp34.87, namely, HCDR1, HCDR2, and HCDR3.

[0241] In some embodiments, the antigen-binding region is a known antibody that specifically binds to CD3, for example, an antibody disclosed in WO2022068809 that specifically binds to CD3, for example, comprises 1, 2, and 3 light-chain variable region CDRs of sp34.24 or sp34.87, namely, LCDR1, LCDR2, and LCDR3.

[0242] In some embodiments, the antigen-binding region is a known antibody that specifically binds to CD3, for example, an antibody disclosed in WO2022068809 that specifically binds to CD3, for example, comprises 3 heavy-chain variable region CDRs and 3 light-chain variable region CDRs of sp34.24 or sp34.87.

[0243] In some embodiments, the antigen-binding region comprises a known antibody that specifically binds to CD3, such as the antibodies disclosed in WO2022068809 that specifically bind to CD3, such as the heavy chain variable region and the light chain variable region of sp34.24 or sp34.87, and a mutation according to the present disclosure.

[0244] Some antigen-binding regions comprise a known antibody that specifically binds to CD3, such as the antibodies disclosed in WO2022068809 that specifically bind to CD3, such as the Fab of sp34.24 or sp34.87, and a mutation according to the present disclosure.

[0245]

Number

[0246] In some embodiments, the antibody that specifically binds to BCMA comprises three CDRs (HCDRs) from the heavy chain variable region, namely HCDR1, HCDR2, and HCDR3, and HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 103.

[0247] In some embodiments, the antibody that specifically binds to BCMA comprises three CDRs (LCDRs) from the light chain variable region, namely LCDR1, LCDR2, and LCDR3, and LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO: 107.

[0248] In some embodiments, the HCDR1 of an 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.

[0249] In some embodiments, an antibody that specifically binds to BCMA comprises three CDRs from the heavy chain variable region (HCDRs), namely, HCDR1, HCDR2, and HCDR3, and three CDRs from the light chain variable region (LCDRs), namely, LCDR1, LCDR2, and LCDR3, wherein the HCDR1 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.

[0250] In some aspects, an antibody that specifically binds to BCMA comprises a heavy chain variable region (VH), and the VH (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 103, or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 103, or (iii) one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, and 1 or fewer) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) of SEQ ID NO: 103, comprising or consisting of an amino acid substitution, preferably the amino acid change does not occur in the CDR.

[0251] In some embodiments, an antibody that specifically binds to BCMA comprises a light chain variable region (VL), and VL (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 107, or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 107, or (iii) one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, and 1 or fewer) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) of SEQ ID NO: 107, comprising or consisting of an amino acid substitution, preferably the amino acid change does not occur in the CDR.

[0252] In some embodiments, an antibody that specifically binds to BCMA comprises VH and VL, where VH comprises or consists of the sequence set forth in SEQ ID NO: 103 and / or VL comprises or consists of the sequence set forth in SEQ ID NO: 107.

[0253] In some embodiments, the antigen-binding region comprises one, two, three, four, five, or six CDRs of a known or the above-described antibody that specifically binds to BCMA.

[0254] In some embodiments, the antigen-binding region comprises one, two, and three heavy chain variable region CDRs of a known or the above-described antibody that specifically binds to BCMA, namely, HCDR1, HCDR2, and HCDR3.

[0255] In some embodiments, the antigen-binding region comprises one, two, and three light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of a known or the above-described antibody that specifically binds to BCMA.

[0256] 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 the above-described antibody that specifically binds to BCMA.

[0257] In some embodiments, the antigen-binding region comprises a heavy chain variable region and a light chain variable region of a known or the above-described antibody that specifically binds to BCMA, and a mutation according to the present disclosure.

[0258] Some antigen-binding regions comprise a Fab of a known or the above-described antibody that specifically binds to BCMA, and a mutation according to the present disclosure.

[0259] I.8. Binding Fragments of the Binding Molecule The present disclosure also relates to binding fragments herein, e.g., antibody fragments, particularly fragments of a target molecule bound by a specific binding molecule, e.g., antigen-binding fragments.

[0260] In some embodiments, the antigen-binding fragment is a Fab.

[0261] In some embodiments, the antigen-binding fragment comprises one or more Fabs, and the one or more Fabs comprise a mutation or combination of mutations according to the present disclosure.

[0262] I.9. Exemplary Embodiments 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 comprise a heavy chain CH1 and a light chain CL, and comprise a disulfide bond reformation mutation at the CH1-CL interface, and / or At least one or more antigen-binding regions comprise a heavy-chain variable region and a light-chain variable region, and the amino acid pair at the interface between the heavy-chain variable region and the light-chain variable region comprises a charge mutation that mutates the two amino acids on the amino acid pair to amino acids having opposite charges, and comprises at least one or more heavy-chain CH1 and light-chain CL, The first antigen and the second antigen may be the same or different, Preferably, only one antigen-binding region comprises a disulfide bond reformation mutation at the CH1-CL interface, an antibody or an antigen-binding fragment thereof. 2. The disulfide bond reformation mutation is (i) Substitution of a non-Cys amino acid at position 126 of the heavy-chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 124 of the light-chain CL with a Cys amino acid (EU number), (i) Substitution of a non-Cys amino acid at position 168 of the heavy-chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 164 of the light-chain CL with a Cys amino acid (EU number), (iii) Substitution of a non-Cys amino acid at position 170 of the heavy-chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 162 or 164 of the light-chain CL with a Cys amino acid (EU number), (iv) Substitution of a non-Cys amino acid at position 173 of the heavy-chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 160 or 162 of the light-chain CL with a Cys amino acid (EU number), (v) Substitution of a non-Cys amino acid at position 128 of the heavy-chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 118 of the light-chain CL with a Cys amino acid (EU number), (vi) Substitution of a non-Cys amino acid at position 134 of the heavy-chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 116 of the light-chain CL with a Cys amino acid (EU number), (vii) Substitution of a non-Cys amino acid at position 136 of the heavy-chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 114 of the light-chain CL with a Cys amino acid (EU number), (xiii) Substitution of a non-Cys amino acid at position 171 of the heavy-chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 162 of the light-chain CL with a Cys amino acid (EU number), (ix) Substitution of a non-Cys amino acid at position 139 of the heavy-chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 116 of the light-chain CL with a Cys amino acid (EU number), the antibody or antigen-binding fragment thereof according to Embodiment 1. 3. The disulfide bond reformation mutation is (i) F126C in CH1 and Q124C in CL (EU number), (ii) H168C in CH1 and T164C in CL (EU number), (iii) F170C in CH1 and T164C in CL (EU number), (iv) F170C in CH1 and S162C in CL (EU number), (v) V173C in CH1 and S162C in CL (EU number), (vi) V173C in CH1 and Q160C in CL (EU number), (vii) L128C in CH1 and F118C in CL (EU number), (viii) S134C in CH1 and F116C in CL (EU number), (ix) S136C in CH1 and S114C in CL (EU number), (x) P171C in CH1 and S162C in CL (EU number), (xi) T139C in CH1 and F116C in CL (EU number), the antibody or antigen-binding fragment thereof according to Embodiment 2. 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 contains a disulfide bond reformation mutation at the CH1-CL interface, the disulfide bond reformation mutation comprising T139C in CH1 and F116C in CL, and 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 embodiments 2 to 4, wherein the disulfide bond reformation mutation also includes a non-Cys substitution of an inter-chain Cys at the heavy chain constant region-light chain constant region interface. 6. The antibody or antigen-binding fragment thereof according to embodiment 5, wherein the non-Cys substitution of the inter-chain Cys includes a non-Cys substitution of Cys at position 220 in CH1 and a non-Cys substitution of Cys at position 214 in CL. 7. The antibody or antigen-binding fragment thereof according to embodiment 6, wherein the non-Cys substitution of the inter-chain Cys includes C220S / A / V in CH1 and S214S / A / V (EU number) in CL. 8. The disulfide bond reformation mutation is (i) F126C and C220S in CH1, and Q124C and C214S (EU number) in CL, (ii) H168C and C220S in CH1, and T164C and C214S (EU number) in CL, (iii) F170C and C220S in CH1, and T164C and C214S (EU number) in CL, (iv) F170C and C220S in CH1, and S162C and C214S (EU number) in CL, (v) V173C and C220S in CH1, and S162C and C214S (EU number) in CL, (vi) V173C and C220S in CH1, and Q160C and C214S (EU number) in CL, (vii) L128C and C220S in CH1, and F118C and C214S (EU number) in CL, (viii) The antibody or antigen-binding fragment thereof according to embodiment 6, comprising S134C and C220S in CH1, and F116C and C214S (EU number) in CL. (ix) The antibody or antigen-binding fragment thereof according to embodiment 6, comprising S136C and C220S in CH1, and S114C and C214S (EU number) in CL. (x) The antibody or antigen-binding fragment thereof according to embodiment 6, comprising P171C and C220S in CH1, and S162C and C214S (EU number) in CL. (xi) The antibody or antigen-binding fragment thereof according to embodiment 6, comprising T139C and C220S in CH1, and F116C and C214S (EU number) in CL. 9. The CH1 region is human IgG1 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. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 8. 10. CH1 is (i) Comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 129, comprises F126C and C220S, or comprises or consists of the amino acid sequence shown in SEQ ID NO: 129. (ii) Comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 133, comprises H168C and C220S, or comprises or consists of the amino acid sequence shown in SEQ ID NO: 133. (iii) Comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 135, comprises F170C and C220S, or comprises or consists of the amino acid sequence shown in SEQ ID NO: 135. (iv) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 137, or comprising V173C and C220S, or comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 137, (v) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 139, or comprising L128C and C220S, or comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 139, (vi) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 141, or comprising S134C and C220S, or comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 141, (vii) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 143, or comprising S136C and C220S, or comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 143, (viii) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 145, or comprising P171C and C220S, or comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 145, (ix) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 147, or comprising T139C and C220S, or comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 147, the antibody or antigen-binding fragment thereof according to Embodiment 8 or 9. 11. CL is (i) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 128, or comprising Q124C and C124S, or comprising or consisting of the amino acid sequence shown in SEQ ID NO: 128, (ii) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 149, or comprising T164C and C124S, or comprising or consisting of the amino acid sequence shown in SEQ ID NO: 149, (iii) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 151, or comprising S162C and C124S, or comprising or consisting of the amino acid sequence shown in SEQ ID NO: 151, (iv) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 35, or comprising Q160C and C124S, or comprising or consisting of the amino acid sequence shown in SEQ ID NO: 35, (v) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 37, or comprising F118C and C124S, or comprising or consisting of the amino acid sequence shown in SEQ ID NO: 37, (vii) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 98, or comprising F116C and C124S, or comprising or consisting of the amino acid sequence shown in SEQ ID NO: 98, (viii) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 100, including the S114C and C124S, or including or consisting of the amino acid sequence shown in SEQ ID NO: 100, an antibody or an antigen-binding fragment thereof according to any one of Embodiments 8 to 10. 12. The position of the amino acid pair at the interface between the heavy chain variable region and the light chain variable region is selected from the 39th position of the heavy chain variable region and the 38th position of the light chain variable region, the 45th position of the heavy chain variable region and the 87th position of the light chain variable region, the 45th position of the heavy chain variable region and the 44th position of the light chain variable region, the 91st position of the heavy chain variable region and the 43rd position of the light chain variable region, or the 91st position of the heavy chain variable region and the 44th position of the light chain variable region (Kabat number), an antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 11. 13. The position of the amino acid pair at the interface between 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 in at least one second antigen-binding region, an antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 12. 14. 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, an antibody or an antigen-binding fragment thereof according to Embodiment 13. 15. (1) In the first antigen-binding region, the amino acid at the 39th position of the heavy chain variable region is substituted with an amino acid carrying a positive charge, and the amino acid at the 38th position of the light chain variable region is substituted with an amino acid carrying a negative charge. In the second antigen-binding region, the amino acid at the 39th position of the heavy chain variable region is substituted with an amino acid carrying a negative charge, and the amino acid at the 38th position of the light chain variable region is substituted with an amino acid carrying a positive charge. (2) In the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with an amino acid carrying a positive charge, the amino acid at position 87 of the light chain variable region is substituted with an amino acid carrying a negative charge, in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with an amino acid carrying a negative charge, and the amino acid at position 87 of the light chain variable region is substituted with an amino acid carrying a positive charge. (3) In the first antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with an amino acid carrying a positive charge, the amino acid at position 44 of the light chain variable region is substituted with an amino acid carrying a negative charge, in the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is substituted with an amino acid carrying a negative charge, and the amino acid at position 44 of the light chain variable region is substituted with an amino acid carrying a positive charge. (4) In the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with an amino acid carrying a positive charge, the amino acid at position 43 of the light chain variable region is substituted with an amino acid carrying a negative charge, in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with an amino acid carrying a negative charge, and the amino acid at position 43 of the light chain variable region is substituted with an amino acid carrying a positive charge, or (5) In the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with an amino acid carrying a positive charge, the amino acid at position 44 of the light chain variable region is substituted with an amino acid carrying a negative charge, in the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is substituted with an amino acid carrying a negative charge, and the amino acid at position 44 of the light chain variable region is substituted with an amino acid carrying a positive charge, the antibody or antigen-binding fragment thereof according to Embodiment 14. 16. The antibody or antigen-binding fragment thereof according to Embodiment 15, wherein the amino acid carrying a positive charge is selected from K, R, or H, and / or the amino acid carrying a negative charge is selected from E or D. 17. (i) In the first antigen-binding region, the amino acid at position 39 of the heavy-chain variable region is substituted with K, the amino acid at position 38 of the light-chain variable region is substituted with D, 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. (ii) In the first antigen-binding region, the amino acid at position 45 of the heavy-chain variable region is substituted with K, the amino acid at position 87 of the light-chain variable region is substituted with D, 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. (iii) In the first antigen-binding region, the amino acid at position 45 of the heavy-chain variable region is substituted with K, the amino acid at position 44 of the light-chain variable region is substituted with D, 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. (iv) In the first antigen-binding region, the amino acid at position 91 of the heavy-chain variable region is substituted with K, the amino acid at position 43 of the light-chain variable region is substituted with D, 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. (v) An antibody or antigen-binding fragment thereof according to embodiment 16, wherein in the first antigen-binding region, the amino acid at position 91 of the heavy-chain variable region is substituted with K, the amino acid at position 44 of the light-chain variable region is substituted with D, 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. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 17, wherein the multispecific antibody comprises a charge mutation and a disulfide bond reformation mutation. 19. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 18, wherein one or more or all of the antigen-binding regions that bind to the same antigen comprise the same mutation. 20. The antibody or antigen-binding fragment thereof according to any one of Embodiments 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. 1) The first antigen-binding region comprises a disulfide bond reformation mutation defined in any one of Embodiments 1 to 17, or 2) The first antigen-binding region comprises a charge mutation defined in any one of Embodiments 1 to 17, 3) The first antigen-binding region comprises a disulfide bond reformation mutation and a charge mutation defined in any one of Embodiments 1 to 17, or 4) The first antigen-binding region comprises a disulfide bond reformation mutation and a charge mutation defined in any one of Embodiments 1 to 17, and the second antigen-binding region comprises a charge mutation defined in any one of Embodiments 1 to 17. The antibody or antigen-binding fragment thereof according to Embodiment 20. 22. 1) The second antigen-binding region comprises a disulfide bond reformation mutation defined in any one of Embodiments 2 to 8, 2) The first antigen-binding region comprises a charge mutation defined in any one of Embodiments 12 to 17, 3) The first antigen-binding region comprises a disulfide bond reformation mutation defined in any one of Embodiments 2 to 8 and a charge mutation defined in any one of Embodiments 12 to 17, or 4) The first antigen-binding region and the second antigen-binding region comprise a charge mutation defined in any one of Embodiments 12 to 17, and the first antigen-binding region also comprises a disulfide bond reformation mutation defined in any one of Embodiments 2 to 8. The antibody or antigen-binding fragment thereof according to Embodiment 21. 23. The first antigen-binding region of the multispecific antibody is a combination of mutations selected from the following:

Table 5-1

Table 5-2

[0263] II. Preparation of Binding Molecules The present disclosure 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.

[0264] In one aspect, a method for preparing a binding molecule, such as an antibody, of the present disclosure is: a) Introducing nucleic acids encoding various chains of a binding molecule, such as an antibody, into a host cell; b) Expressing in the host cell and assembling the binding molecule, such as an antibody; Optionally, purifying the binding molecule, such as by protein A.

[0265] In yet another embodiment, a compound, such as L-arginine, is added to the medium during the production of the binding molecule, such as an antibody. In one embodiment, Arg is used to stabilize the protein.

[0266] In some embodiments, the products of the present disclosure maintain the same expression level and affinity as the binding molecule, such as an antibody, prior to mutation.

[0267] In some embodiments, the methods of the present disclosure obtain antibodies, such as stable binding molecules, such as multispecific antibodies (e.g., bispecific antibodies), in high yields.

[0268] In some embodiments, the methods of the present disclosure can express and assemble various chains of a binding molecule of an antibody, such as a multispecific antibody (e.g., bispecific antibody), in a cell line, preferably avoiding expression and in vitro recombination in two cell lines.

[0269] III Nucleic Acids and Host Cells The present disclosure provides nucleic acids encoding any chain or any monomer or domain of a binding molecule of the present disclosure, such as an antibody, such as a multispecific antibody, such as a bispecific antibody. Polynucleotide sequences encoding the various chains can be produced using methods well known in the art. In addition, the polynucleotides and nucleic acids of the present disclosure can include segments encoding a secretion signal peptide, operably linked to an antibody or protein encoding an antibody or binding molecule of the present disclosure, such as an antibody, such as a multispecific antibody, such as a bispecific antibody, thereby inducing the secretory expression of the binding molecules of the present disclosure, such as antibodies, such as multispecific antibodies, such as bispecific antibodies, and their various chains.

[0270] The present disclosure also provides a vector comprising the nucleic acid of the present disclosure. In one embodiment, the vector is an expression vector, for example, a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, and yeast artificial chromosomes (YACs). In a preferred embodiment, the expression vector of the present disclosure is a pCNDA vector, for example, a pCNDA3.1 expression vector.

[0271] The present disclosure also provides a host cell comprising the nucleic acid or vector. Host cells that are suitable for replication and support the expression of the binding molecules of the present disclosure, such as antibodies, for example, multispecific antibodies such as bispecific antibodies, are known in the art. Such cells may be transfected or transduced using a specific expression vector, and large quantities of cells containing the vector for seeding into large-scale fermentation tanks are grown, thereby obtaining an amount of multispecific antibody, for example, bispecific antibody sufficient for clinical applications.

[0272] In one embodiment, the host cell is a eukaryote. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells, e.g., Expi293 cells). Examples of mammalian host cell lines that can be used include the SV40-transformed monkey kidney CV1 line (COS-7), human embryonic kidney lines (293 or 293T cells), such as those described in Graham et al., J Gen Virol 36, 59 (1977), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, such as those described in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), chlorocebus kidney cells (VERO-76), human cervical cancer cells (HELA), dog kidney cells (MDCK), buffalo rat hepatocytes (BRL3A), human lung cells (W138), human hepatocytes (HepG2), mouse breast cancer cells (MMT060562), TRI cells (such as those described in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC5 cells, and FS4 cells. Other mammalian host cell lines that can be used include Chinese hamster ovary (CHO) cells, including dhfr-CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4, 216 (1980)), and myeloma cell lines such as Y0, NS0, P3X63, and Sp2 / 0. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell such as a CHO cell, a human embryonic kidney (HEK) cell, or a lymphocyte (e.g., Y0, NS0, Sp20 cells).

[0273] IV. Other molecules comprising the binding molecule of the present disclosure or a fragment thereof In some embodiments, the first and / or second target binding regions of the binding molecule of the present disclosure can conjugate with a drug, a precursor, or a toxin, or a receptor moiety comprising a drug, a precursor, or a toxin. Such a receptor moiety can be, for example, a non-natural amino acid.

[0274] Accordingly, the present disclosure also relates to immunoconjugates (ADCs) of the binding molecules of the present disclosure, such as antibodies, such as multispecific antibodies, such as bispecific antibodies.

[0275] In some embodiments, the binding molecule or fragment thereof of the present disclosure can also be constructed in a T cell receptor (TCR). In some embodiments, the variable region of the TCR comprises mutations or combinations of mutations according to the present disclosure, such as disulfide bond reformation mutations and / or charge mutations.

[0276] In some embodiments, the binding molecule or fragment thereof of the present disclosure can be fused with other proteins to form a fusion protein. 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), transforming growth factor (TGF, such as TGF-β), or their receptors, and fragments thereof.

[0277] V. Products and Uses In some embodiments, the present disclosure also relates to a composition (such as a pharmaceutical composition) comprising a binding molecule such as an antibody, such as a multispecific antibody, such as a bispecific antibody.

[0278] In one embodiment, the composition also comprises a pharmaceutical excipient. In one embodiment, the composition, such as a pharmaceutical composition, comprises a combination of the binding molecule of the present disclosure and one or more other types of therapeutic agents.

[0279] The compositions of the present disclosure may also contain pharmaceutical excipients suitable for pharmaceutical vectors and pharmaceutical vehicles known in the art, including buffers. As used herein, "pharmaceutical vector" includes any or all of physiologically compatible solvents, dispersion media, isotonic agents, absorption delaying agents, etc. For the use and application of pharmaceutical excipients, refer to "Handbook of Pharmaceutical Excipients", Version 8, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago. The compositions of the present disclosure may be in multiple forms. Such forms include liquid, semi-solid, and solid dosage forms such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomal agents, and suppositories. The preferred form depends on the expected mode of administration and therapeutic use. The binding molecules of the present disclosure having the desired purity can be mixed with one or more types of any pharmaceutical excipient to prepare a drug containing the binding molecules of the present disclosure, such as a lyophilized preparation or an aqueous solution.

[0280] In some embodiments, the present disclosure relates to a method of treating a disease, such as a tumor, such as cancer, using a binding molecule, such as an antibody, such as a multispecific antibody, such as a bispecific antibody, for therapeutic use or for preparing a therapeutic agent.

[0281] The binding molecules of the present disclosure, such as antibodies, such as multispecific antibodies, such as bispecific antibodies, can be combined with other therapeutic agents depending on their therapeutic use. For example, when using a binding molecule to treat a tumor, the therapeutic agent can be, for example, various therapeutic agents used to treat tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic drugs, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., immune checkpoint inhibitors or agonists).

[0282] In some embodiments, the present disclosure also provides a pharmaceutical combination or pharmaceutical combination product comprising the binding molecules of the present disclosure and one or more other types of therapeutic agents, etc.

[0283] Another object of the present disclosure is to provide a kit of parts comprising the pharmaceutical combination of the present disclosure, preferably, the kit is in a pharmaceutical dosage form. Accordingly, dosage units can be provided based on a dosing schedule or a drug dosing interval.

[0284] In one embodiment, the kit of parts of the present disclosure comprises, within the same package, - a first container comprising a pharmaceutical composition comprising the binding molecule of the present disclosure, - a second container comprising a pharmaceutical composition comprising another therapeutic agent.

[0285] Embodiments Embodiment 1. Design of mutant antibodies Disulfide bond reformation An effective strategy to enforce the pairing of identical light and heavy chains and avoid mismatches between the light and heavy chains of two antibodies is to reform the disulfide bond that links one light and heavy chain of the antibody and change the position of the disulfide bond so that mismatched light and heavy chains cannot effectively form disulfide bonds. We screened the amino acids at the contact surface of the heavy and light chains and selected two pairs of amino acids where the distance between the alpha carbon atoms is less than 8 Å, preferably less than 6 Å, and the side chains of the amino acids are facing each other. The selected amino acid pairs are as shown in Table 1 (EU number).

Table 8

[0286] Charge mutation Another effective strategy for forcing the pairing of identical light and heavy chains and avoiding mismatches between the light and heavy chains of two antibodies is to find amino acid pairs on the contact surfaces of the heavy and light chains, mutate them to amino acids carrying opposite charges, and promote homologous pairing based on the principle of opposite charge attraction. The selection criteria are conserved amino acid pairs at the interfaces of the heavy-chain variable region and the light-chain variable region, and the distance between the α-carbon atoms of the amino acid pairs is less than 12 Å with the side chains facing each other. The selected amino acid pairs are shown in Table 2 (Kabat number). [Table 9]

[0287] The final mutations used in the bispecific antibodies expressed in cell lines are a combination of disulfide bond reformation and charge mutations as shown in Table 3: [Table 10]

[0288] When antibody 1 is an anti-Her2 mAb and antibody 2 is an anti-Hel mAb, for the sequences of the full-length chains of exemplary bispecific antibodies, i.e., for the antibodies of Combinations 1 to 5 in Table 9, refer to Combinations 1 to 5 in Table 3 of the Sequence Listing.

[0289] Substitutions in the light-chain constant region To facilitate downstream purification and removal of minor mismatched antibodies, one of the light-chain constant regions of the antibodies can be selected for substitution from C kappa to C lambda (if one of the light-chain constant regions of the antibodies is a lambda light chain, no substitution is required), so that one of the two antibody Fabs is kappa and the other is lambda, and downstream purification is performed on kappa and lambda light chains using a selective affinity medium.

[0290] Embodiment 2. Expression of mutations and purification of mutations in HEK293 cells Various chains of the various antibodies of the present disclosure were sent to GENEWIZ for gene synthesis, constructed into a pCDNA3.1 template plasmid, and a certain amount of plasmid was prepared. See Table 4 for a summary of the sequences of the various antibody chains used in the embodiments.

Table 11-1

Table 11-2

Table 12

Table 13-1

Table 13-2

Table 14-1

Table 14-2

Table 15

[0291] The plasmid was filtered using a 0.22 μm filter head for transient expression of cells. Expi293 cells (Invitrogen) were passaged based on the desired transfection amount, and the cell density was adjusted to 3×10 6 cells / mL the day before transfection. The cell density was adjusted again to 3×10 6Adjusted to cells / mL. Opti-MEM culture medium (Gibco catalog number: 31985-070) at a final volume of 1 / 10 (v / v) was used as a transfection buffer and added to the expression plasmid at a ratio of antibody A heavy chain: antibody A light chain: antibody B heavy chain: antibody B light chain = 1:1:1:1, or other suitable ratio, and mixed well. An appropriate amount of polyethyleneimine (PEI) (Polysciences, 23966) was added to the plasmid in the previous step (the mass ratio of plasmid to PEI was 1:3), mixed well, and incubated at room temperature for 10 minutes to obtain a DNA / PEI mixture. The DNA / PEI mixture was gently poured into HEK293 cells, mixed well, and cultured at 37 °C and 8% CO2 for 24 hours. Then, additional VPA (Sigma, catalog number: P4543-100G) was added to a final concentration of 2 mM and 2% (v / v) feed (1 g / L Phytone Peptone + 1 g / L Difco Select Phytone), and the culture was continued for 6 days. The culture was collected, centrifuged at 4,000 revolutions per minute for 30 minutes, the cell supernatant was collected, filtered through a 0.45 μM membrane, and purified through protein A affinity chromatography. Samples with special requirements were subjected to further purification through ion exchange chromatography, KappaSelect affinity chromatography, and LambdaFabSelect affinity chromatography.

[0292] The specific operation steps for purification by protein A affinity chromatography were as follows: the supernatant was purified using a pre-packed column Hitrap Mabselect Sure (GE, 11-0034-95). Before the operation, the packed column was equilibrated with an equilibration solution (20 mM Tris, 150 mM NaCl, pH 7.2) five times the column volume. The collected supernatant was passed through the column, and then the packed column was washed with an equilibration solution ten times the column volume to remove non-specifically bound proteins. Elution was carried out by flowing a elution buffer (100 mM sodium citrate, pH 3.5) five times the column volume through the column, and the eluate was collected. 2 M Tris was added to neutralize the pH to 6.5. The proportion of correctly paired bispecific antibodies after purification was quantitatively measured using liquid chromatography-mass spectrometry (LC-MS).

[0293] The specific operation steps for purification by ion exchange chromatography were as follows: an ultrafiltration concentration tube (MILLIPORE, catalog number: UFC901096) was used to perform an exchange into a low-salt PB buffer at pH 6.0 (10 mM phosphate, pH 6.0), and Capto HiRes S10 / 100 (GE, catalog number 29275879) was used for precise purification. Before purification, the packed column was equilibrated with a low-salt PB at pH 6.0 five times the column volume. The exchanged sample was passed through the column, and then the packed column was washed with a low-salt PB buffer at pH 6.0 ten times the column volume to remove non-specifically bound proteins. During elution, the concentration of the low-salt PB buffer at pH 6.0 (10 mM phosphate, pH 6.0, 1 M NaCl) was linearly increased (linear gradient: within 30 column volumes, the proportion of the high-salt PB buffer at pH 6.0 was increased from 30% to 100%), and the main elution peak was collected. An ultrafiltration concentration tube (MILLIPORE, catalog number: UFC901096) was used to perform an exchange into a PBS buffer (Gibco, catalog number: 70011-044). The proportion of correctly paired bispecific antibodies after purification was quantitatively measured using liquid chromatography-mass spectrometry (LC-MS).

[0294] The specific operation steps for purification by KappaSelect affinity chromatography were to purify the supernatant using a pre-packed column HiTrap KappaSelect (GE, 17-5458-11). Before the operation, the packed column was equilibrated with an equilibration solution (PBS pH 7.4) five times the column volume, the collected supernatant was passed through the column, then the packed column was washed with an equilibration solution ten times the column volume to remove non-specifically bound proteins, and the packing was eluted with an elution buffer (0.1 M glycine buffer, pH 2.5) five times the column volume, and the eluate was collected. 2 M Tris was added to neutralize the pH to 6.5. The ratio of correctly paired bispecific antibodies after purification was quantitatively measured using liquid chromatography-mass spectrometry (LC-MS).

[0295] The specific operation steps for purification by LambdaFabSelect affinity chromatography were to purify the supernatant using a pre-packed column Hitrap LambdaFabSelect (GE, 17-5482-11). Before the operation, the packed column was equilibrated with an equilibration solution (PBS pH 7.4) five times the column volume, the collected supernatant was passed through the column, then the packed column was washed with an equilibration solution ten times the column volume to remove non-specifically bound proteins, and the packing was eluted with an elution buffer (0.1 M acetate buffer, pH 3.5) five times the column volume, and the eluate was collected. 2 M Tris was added to neutralize the pH to 6.5. The ratio of correctly paired bispecific antibodies after purification was quantitatively measured using liquid chromatography-mass spectrometry (LC-MS).

[0296] Embodiment 3 Screening of mutant amino acids in the present disclosure Screening of mutation sites capable of directly forming disulfide bonds As a model antibody, trastuzumab was mutated to remove the native disulfide bond (HC220S, light chain C214S) between the heavy chain and the light chain, and then the amino acid pairs selected according to Table 1 were mutated to Cys. The mutated mAb was expressed in the HEK293 system, purified using Protein A, and then non-reducing polyacrylamide gel electrophoresis was performed as shown in FIGS. 9A to 9C. It can be seen that these mutated mAbs can be maintained as complete molecules in non-reducing polyacrylamide gel electrophoresis, and only minimal light chain components are removed. The selected disulfide bond mutation sites basically formed disulfide bonds in the normal way.

[0297] Furthermore, these antibodies that formed disulfide bonds were subjected to denaturing nrCE-SDS (FIGS. 9D to 9M), and quantitative measurements were performed on the integrity of the formed disulfide bonds. The antibody with re-formed disulfide bonds has more than 98% complete molecules, which can further prove that the re-formed disulfide bonds were correctly formed.

[0298] Effect of disulfide bond reformation mutation on the ratio of correct pairing Taking the F126 / Q124C disulfide bond reformation mutation in Table 1 as an example, the mutated antibody of the present disclosure was applied, and the wild-type antibody sequence was used as a control to test the effect of the disulfide bond reformation mutation on the ratio of correct pairing. After expression in HEK293 cells, only Protein A affinity chromatography purification was used to prevent the removal of mismatched molecules by other purification methods. The ratio of the anti-Her2 / anti-PD-1 antibody pair to the correct anti-Her2 / anti-Hel antibody pair when the bispecific antibody was formed was tested using LC-MS. Heterologous pairing between heavy chains was promoted through knob-into-hole.

Table 16

[0299] From the above table, it can be seen that the F126C / Q124C mutation can significantly increase the proportion of correct pairing of bispecific antibodies. For example, it can be found that the proportion of the correct Her2 / PD-1 antibody pair relative to the wild-type antibody pair reached 55.60%. After adding the F126C / Q124C mutation, the proportion of correct pairing increased to a maximum of 76.93%.

[0300] It can be seen that the proportion of correctly paired molecules formed by the F126C / Q124C mutation is the highest, significantly higher than that of the wild-type antibody sequence, and also somewhat higher than the mutant F126C / S121C disclosed in CN104011221B.

[0301] In addition, when the correct disulfide bond is not formed between the mutated Cys pairs, the Cys with a complete thiol often reacts with the free Cys in the cell or solution to form a disulfide bond and modify the protein molecule with Cys. This modification can be detected by LC-MS.

Table 17

[0302] Whether it is Her2 / Hel bsAb or Her2 / PD-1 bsAb, the modified components of free Cys are hardly detected after the F126C / Q124C mutation, which further indicates a very high proportion of correctly formed disulfide bonds. In contrast, the Her2 / PD-1 bispecific antibody introduced with the mutant F126C / S121C as disclosed in CN104011221B still has about 10% of the modified free Cys, which means that a part of the F126C / S121C disulfide bond could not be completely formed in the correct way.

[0303] Disulfide bond reformation mutation and V-region charge mutation In addition to the charge mutations (Q39K / Q38D) being added to the disulfide bond reformation mutations (F126C / Q124C), after expression in HEK293 cells, only protein A affinity chromatography purification was used to prevent the removal of mismatched molecules by other purification methods. The exact ratio of the anti-Her2 / anti-Hel antibody pair when the bispecific antibody was formed was tested using LC-MS (Figure 2). Hetero-dimerization between the heavy chains was promoted through knob-into-hole.

Table 18

[0304] It can be seen that after disulfide bond reformation, the ratio of correctly paired bispecific antibodies increased significantly. After removing the natural disulfide bonds of the antibody (combination 2 in Table 7 of Figure 2B), the correct pairing ratio increased compared to when only disulfide bonds were added (combination 1 in Table 7 of Figure 2A). Furthermore, when charge mutations were added to the V region (combination 3 in Figure 2C, Table 7), the correct pairing ratio increased further.

[0305] Four different antibody pair combinations of bsAb were used. After expression in HEk293 cells, only protein A affinity chromatography purification was used to prevent the removal of mismatched molecules by other purification methods. The exact ratio of correct pairing when the bispecific antibody was formed was tested using LC-MS (Figure 3). The universality of disulfide bond reformation and charge mutations on different antibodies was verified. At the same time, the expression levels of the antibodies were observed.

Table 19

[0306] The ratio of correct pairing was maintained at approximately 90% in the combinations of the four different antibodies, indicating that disulfide bond reformation and charge mutations are universal in different antibodies. The expression levels of the four different antibody compositions reached approximately 200 mg / L, which is a relatively high expression level and comparable to that of mAb.

[0307] Effect of Disulfide Bond Re - formation and Charge Mutations at Different Positions on the Ratio of Correct Pairing In the natural assembly and secretion process of intracellular IgG, after the heavy chain is expressed in the cell, CH1 binds to the molecular chaperone BiP and maintains an incomplete folding state before meeting the light chain. After meeting the light chain, the light chain first binds to VH via VL, then CL replaces BiP and binds to CH1. With the assistance of CL, CH1 is completely folded, and the assembly of the light chain and heavy chain is completed. (See 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 between VH and VL is the first process driving the assembly of the light chain and heavy chain. Therefore, the charge mutations of the present disclosure are mainly designed in the VH and VL regions. To further prove the superiority of our design, we also introduced charges between CH1 and CL to compare the effects of charge mutations in the V region and C region on the ratio of correct pairing. The only difference in the sequences between any of the mutant Her2 / Hel bispecific antibodies in Table 8 - 2 and the antibody of Combination 3 in Table 7 is the difference in the combination of the listed mutations, while the other sequences do not change.

Table 20

[0308] Using the same disulfide bond re - formation mutations, comparing the charge mutations Q39K / Q38D located in the V region, the combinations of H171K + T190K / N137D and K216E / E123R located in the C region, it can be seen that the charge mutations located in the V region resulted in a high proportion of correctly paired molecules. This proves the design superiority of placing charges in the V region.

[0309] Combinations of Other V - Region Charge Mutations and Disulfide Bond Re - formation Separate from Q39K and Q38D, other charge mutations and disulfide bond reformation mutations F126C / Q124C discovered in the V region were combined to form Combinations 1 - 5 in Table 3. After expression in HEK293 cells, only Protein A affinity chromatography purification was used to prevent the removal of mismatched molecules by other purification methods. The exact pairing ratio when the bispecific antibody was formed was tested using LC - MS (Figure 4). Heterologous pairing between heavy chains was promoted through knob - into - hole. At the same time, the antibody expression levels were observed.

Table 21

[0310] By the mutation methods of Combinations 1 - 5, an accurate pairing ratio exceeding 90% was achieved. Expression levels close to those of mAb (200 - 300 mg / L) were achieved in Combinations 1 - 3. The expression levels of Combinations 4 and 5 were slightly lower but also up to approximately 100 mg / L at most. Therefore, the bispecific antibodies containing the combination of mutations of the present disclosure can achieve relatively good expression levels.

[0311] Combination of V - region charge mutations and other disulfide bond reformation mutations Separate from F126C / Q124C, other discovered disulfide bond reformation mutations were combined with the V - region charge mutations Q39K and Q38D to form Combinations 6 - 14 in Table 3. The only sequence difference between any mutant Her2 / Hel bispecific antibody containing Combinations 6 - 14 in Table 3 and any of Combinations 1 - 5 in Table 3 is the difference in the listed combination of mutations, while other sequences remain unchanged.

[0312] After expression in HEK293 cells, only protein A affinity chromatography purification was used to prevent the removal of mismatched molecules by other purification methods. The exact pairing ratio when the bispecific antibody was formed was tested using LC-MS. Heterologous pairing between the heavy chains was promoted through knob-into-hole. At the same time, the antibody expression levels were observed.

Table 22

[0313] The mutation methods of combinations 6 to 14 can achieve an exact pairing ratio of up to 79.46% to 98.90% for bsAb, and the expression levels reached the same level as that of mAb (200 - 300 mg / L). Therefore, the bispecific antibodies containing the mutation combinations of the present disclosure can achieve relatively good exact pairing ratios and expression levels.

[0314] Embodiment 4. Affinity test experiment of the antibodies in the present disclosure To test the equilibrium dissociation constant (KD) of the bispecific antibody binding with each antigen in the present disclosure, the Biolayer Interference (BLI) technology was used. The BLI method affinity test was carried out using an existing method (Estep, P et al., High throughput solution Based measurement of antibody - antigen affinity and epitope binning. MAbs, 2013.5(2):270 - 278).

[0315] Thirty minutes before the start of the experiment, an appropriate number of ProA (ForteBio, 18-5010) sensors based on the sample size were immersed in SD buffer (PBS 1×, BSA 0.1%, Tween®-20 0.05%). 100 μL of SD buffer and the antigen fusion protein were obtained and added to a 96-well black polystyrene half-area microplate (Greiner, 675076), respectively. The sensor positions were selected based on the sample positions and the layout. The instrument setting parameters were as follows: execution steps: baseline, loading about 1 nm, baseline, association and dissociation; the execution time of each step depended on the sample binding and dissociation rates, the rotation speed was 400 rpm, and the temperature was 30 degrees. The KD value was analyzed using ForteBio analysis software.

[0316] Using the anti-Her2 antibody as a model, after introducing the mutations of Combinations 1 to 5 in Table 3, the affinity data with its antigen Her2 are shown in the following table.

Table 23

[0317] From the results in the above table, it was found that there was no significant difference in affinity between various mutations and the wild-type antibody. Therefore, the introduction of mutations and disulfide bonds at the binding surfaces of the light and heavy chains in the V region according to the present disclosure does not affect antibody affinity.

[0318] Embodiment 5. Expansion of the multivalent configuration The bispecific antibodies of the present disclosure can be applied not only in the monovalent 1+1 configuration (Figure 1), but also expanded to multivalent configurations such as the 2+1 N-terminal configuration (Figure 5A), the 2+1 C-terminal configuration (Figure 5B), the 2+2 C-terminal configuration (Figure 5C), and other forms. Apart from the tandem Fab configuration that does not require heterologous pairing of heavy chains, heterologous pairing between heavy chains was promoted through the company's Innobody mutations.

[0319] The multivalent 2+1 N-terminal construct, 2+1 C-terminal construct, and 2+2 C-terminal construct were expressed and purified according to the method of Embodiment 2, and the ratio of correct pairing was tested using mass spectrometry (Figure 6). See Table 11 for an overview of the results.

Table 24

[0320] From the above table, it can be seen that even when the mutations specified in this patent are applied to molecules with a multivalent construct, a very high percentage of correct pairing can still be maintained. Furthermore, when applied to molecules with a multivalent construct, it was also found that disulfide bond reformation and charge mutations had a much higher percentage of correct pairing than disulfide bond reformation mutations alone.

[0321] Embodiment 6. Cell killing experiment The BCMA / GPRC5D / CD3 trispecific antibody (see Figure 7 for the wild type and the introduced mutations specified in this patent, and see the Sequence Listing for the sequence) binds simultaneously to BCMA and GPRC5D on the surface of H929 cells and CD3 on the surface of T cells in primary PBMC, activates T cells, and mediates the killing of BCMA- or GPRC5D-positive tumor cells. This study used the lactate dehydrogenase (LDH) method to test the LDH levels released by dead cells in the supernatant collected 24 hours after the addition of exemplary antibodies under co-culture conditions of PBMC, BCMA-positive H929 cells, and GPRC5D-positive H929 cells, thereby evaluating the killing ability of antibody-mediated T cells against BCMA tumor cells and GPRC5D tumor cells. The effect of the mutations specified in this patent on the trispecific antibody was evaluated based on the BCMA / GPRC5D / CD3 trispecific antibody without the charge mutations and disulfide bond reformation mutations disclosed in this disclosure (Figure 7A) and the BCMA / GPRC5D / CD3 trispecific antibody with the mutations disclosed in this disclosure introduced (Figure 7B).

[0322] PBMC (SallyBio, SLB-HP100A) was taken out from the liquid nitrogen tank and thawed rapidly at 37°C. Pre-warmed 1640 medium (Gibco, 22400-071) (containing 0.1% DNase) was added dropwise to obtain a 10 mL mixture. The mixture was centrifuged at 400 g for 5 minutes, resuspended using 10 mL of 1640 medium, 10 μL of DNase was added, and monolayer culture was carried out overnight at 37°C and 5% CO2. The suspended cells were pipetted, centrifuged at 400 g for 5 minutes, resuspended using 1640 medium, counted, and the cell density was adjusted to 4×10 6 cells / mL and used as effector cells. BCMA- or GPRC5D-positive H929 cells (ATCC, CRL-9068) were used as target cells, centrifuged at 400 g for 5 minutes, resuspended using 1640 medium, counted, and the cell density was adjusted to 2×10 5 cells / mL. Target cells with the adjusted cell density were added to a 96-well plate at 100 μL per well, and the serially diluted antibody was added to the 96-well plate at 50 μL per well, and then effector cell PBMC was added to the 96-well plate at 50 μL per well. The final effector-target ratio was 10:1. The above 96-well plate was placed in a 37°C, 5% CO2 incubator, and after 24 hours of culture, the supernatant was collected. The level of LDH released into the supernatant by dead cells was tested according to the instructions of the LDH test kit (CytoTox96 non-radioactive cytotoxicity kit, Promega), and the percentage of MM cells killed by the exemplary antibody was calculated.

[0323] In the experiment conducted according to the above test method, the exemplary antibody can induce a killing effect on H929 cells of human PBMC in a dose-dependent manner (see Figure 8).

[0324] In the experiment conducted according to the above test method, the killing effect of PBMC from the same donor on BCMA or GPRC5D-positive H929 cell lines induced by exemplary antibodies was compared, and the mutations specified by this patent did not affect the killing effect of the exemplary antibodies even when introduced. However, after introducing the mutations specified in this patent, the exemplary antibodies can be expressed and prepared in one cell line, saving the effort of expression, preparation, and in vitro recombination in two cell lines.

Table 25-1

Table 25-2

Table 25-3

Table 25-4

Table 25-5

Table 25-6

Table 25-7

Table 25-8

Table 25-9

Table 25-10

Table 25-11

Table 25-12

Table 25-13

Table 25-14

Table 25-15

Table 25-16

Table 25-17

Table 25-18

Table 25-19

Table 25-20

Table 25-21

Table 25-22

Table 25-23

Table 25-24

Table 25-25

Table 25-26

Table 25-27

Table 25-28

Table 25-29

Table 25-30

Claims

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 comprise a heavy chain CH1 and a light chain CL, and comprises F126C in CH1 and Q124C (EU number) in CL, optionally further comprising C220S / A / V in CH1 and C214S / A / V (EU number) in CL, preferably one antigen-binding region comprises F126C and Q124C (EU number) in CL, and the first antigen-binding region comprises a heavy chain variable region VH containing Q39K and a light chain variable region VL containing Q38D, and the second antigen-binding region comprises a heavy chain variable region VH containing Q39D and a light chain variable region VL containing Q38K. The first antigen and the second antigen may be the same or different. An antibody or its antigen-binding fragment.

2. The antibody is a multispecific antibody comprising 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 and the second antigen-binding region of the multispecific antibody are, respectively: Table 1 The antibody or antigen-binding fragment thereof according to claim 1, comprising a combination of mutations.

3. 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, At least one or more antigen-binding regions include a heavy chain CH1 and a light chain CL, and include a disulfide bond reformation mutation at the CH1-CL interface, preferably, only one antigen-binding region includes a disulfide bond reformation mutation at the CH1-CL interface. An antibody or antigen-binding fragment thereof, wherein the first antigen and the second antigen may be the same or different.

4. The disulfide bond reformation mutation described above is (i) Substitution of a non-Cys amino acid at position 126 of the heavy chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 124 of the light chain CL with a Cys amino acid (EU number), (ii) Substitution of a non-Cys amino acid at position 168 of the heavy chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 164 of the light chain CL with a Cys amino acid (EU number), (iii) Substitution of a non-Cys amino acid at position 170 of the heavy chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 162 or 164 of the light chain CL with a Cys amino acid (EU number), (iv) Substitution of a non-Cys amino acid at position 173 of the heavy chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 160 or 162 of the light chain CL with a Cys amino acid (EU number), (v) Substitution of a non-Cys amino acid at position 128 of the heavy chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 118 of the light chain CL with a Cys amino acid (EU number), (vi) Substitution of a non-Cys amino acid at position 134 of the heavy chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 116 of the light chain CL with a Cys amino acid (EU number), (vii) Substitution of a non-Cys amino acid at position 136 of the heavy chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 114 of the light chain CL with a Cys amino acid (EU number), (viiii) Substitution of a non-Cys amino acid at position 171 of the heavy chain CH1 with a Cys amino acid (EU number), and substitution of a non-Cys amino acid at position 162 of the light chain CL with a Cys amino acid (EU number), or (ix) The antibody or antigen-binding fragment according to claim 3, comprising the substitution of a non-Cys amino acid at position 139 of the heavy chain CH1 with a Cys amino acid (EU number), and the substitution of a non-Cys amino acid at position 116 of the light chain CL with a Cys amino acid (EU number).

5. The disulfide bond reformation mutation described above is (i) F126C in CH1 and Q124C in CL (EU number), (ii) H168C in CH1 and T164C in CL (EU number), (iii) F170C in CH1 and T164C in CL (EU number), (iv) F170C in CH1 and S162C in CL (EU number), (v) V173C in CH1 and S162C in CL (EU number), (vi) V173C in CH1 and Q160C in CL (EU number), (vii) L128C in CH1 and F118C in CL (EU number), (viiii) S134C in CH1 and F116C in CL (EU number), (ix) S136C in CH1 and S114C in CL (EU number), (x) P171C in CH1 and S162C in CL (EU number), The antibody or antigen-binding fragment thereof according to claim 2, comprising (xi) T139C in CH1 and F116C (EU number) in CL.

6. The antibody or antigen-binding fragment according to claim 3, wherein the disulfide bond reformation mutation also includes the substitution of an interchain Cys at the heavy chain constant region-light chain constant region interface with a non-Cys.

7. The antibody or antigen-binding fragment according to claim 6, wherein the interchain Cys substitution with non-Cys includes a non-Cys substitution of Cys at position 220 in CH1 and a non-Cys substitution of Cys at position 214 in CL, or the interchain Cys substitution with non-Cys includes C220S / A / V in CH1 and C214S / A / V (EU number) in CL.

8. The disulfide bond reformation mutation described above is (i) F126C and C220S in CH1, and Q124C and C214S (EU numbers) in CL, (ii) H168C and C220S in CH1, and T164C and C214S (EU number) in CL, (iii) F170C and C220S in CH1, and T164C and C214S (EU number) in CL, (iv) F170C and C220S in CH1, and S162C and C214S (EU number) in CL, (v) V173C and C220S in CH1, and S162C and C214S (EU number) in CL, (vi) V173C and C220S in CH1, and Q160C and C214S (EU numbers) in CL, (vii) L128C and C220S in CH1, and F118C and C214S (EU number) in CL, (viiii) S134C and C220S in CH1, and F116C and C214S (EU number) in CL, (ix) S136C and C220S in CH1, and S114C and C214S (EU number) in CL, (x) P171C and C220S in CH1, and S162C and C214S (EU number) in CL, or The antibody or antigen-binding fragment thereof according to claim 3, comprising T139C and C220S in (xi)CH1, and F116C and C214S (EU number) in CL.

9. The CH1 region is human IgG1 CH1, for example, human IgG1 CH1, human IgG2 CH1, human IgG3 CH1, or human IgG4 CH1, and for example, the CH1 region includes the amino acid sequence shown in SEQ ID NO:

116. The antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the CL region is a human kappa light chain CL region or a human lambda light chain CL region, and for example, the CL region includes the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO:

55.

10. CH1 is (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 129, and containing F126C and C220S, or containing or being composed of the amino acid sequence shown in SEQ ID NO: 129, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 133, or containing H168C and C220S, or containing or being composed of the amino acid sequence shown in SEQ ID NO: 133 (iii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 135, and containing F170C and C220S, or containing or being composed of the amino acid sequence shown in SEQ ID NO: 135, (iv) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 137, and containing V173C and C220S, or containing or being composed of the amino acid sequence shown in SEQ ID NO: 137, (v) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 139, and containing L128C and C220S, or containing or being composed of the amino acid sequence shown in SEQ ID NO: 139 (vi) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 141, and containing S134C and C220S, or containing or being composed of the amino acid sequence shown in SEQ ID NO: 141, (vii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 143, and containing S136C and C220S, or containing or being composed of the amino acid sequence shown in SEQ ID NO: 143, (viiii) A molecule comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 145, and comprising P171C and C220S, or comprising or composed of the amino acid sequence shown in SEQ ID NO:

145. (ix) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 147, and including T139C and C220S, or including or consisting of the amino acid sequence shown in SEQ ID NO: 147, and / or CL (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 128, and including Q124C and C124S, or including or being composed of the amino acid sequence shown in SEQ ID NO: 128, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 149, and containing T164C and C124S, or containing or being composed of the amino acid sequence shown in SEQ ID NO: 149, (iii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 151, and containing S162C and C124S, or containing or being composed of the amino acid sequence shown in SEQ ID NO: 151, (iv) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 35, and containing Q160C and C124S, or containing or being composed of the amino acid sequence shown in SEQ ID NO: 35 (v) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 37, and containing F118C and C124S, or containing or being composed of the amino acid sequence shown in SEQ ID NO: 37 (vii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in Sequence ID No. 98, or containing F116C and C124S, or containing or being composed of the amino acid sequence shown in Sequence ID No.

98. (viiii) The antibody or antigen-binding fragment according to claim 3, comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 100, or comprising S114C and C124S, or comprising or composed of the amino acid sequence shown in SEQ ID NO:

100.

11. 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, At least one or more antigen-binding regions include 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 includes a charge mutation that causes two amino acids on the amino acid pair to be changed to amino acids having opposite charges. An antibody or antigen-binding fragment thereof, wherein the first antigen and the second antigen may be the same or different.

12. The position of the amino acid pair at the interface between the heavy chain variable region and the light chain variable region is selected from the following: 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 number). More preferably, the position of the amino acid pair at the interface between 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 in at least one second antigen-binding region, or The antibody or antigen-binding fragment according to claim 11, wherein the mutated amino acid at the position of the amino acid pair in the first antigen-binding region has the opposite charge to the mutated amino acid at the same position in the second antigen-binding region.

13. (1) In the first antigen-binding region, the amino acid at position 39 of the heavy chain variable region is replaced with a positively charged amino acid, and the amino acid at position 38 of the light chain variable region is replaced with a negatively charged amino acid; In the second antigen-binding region, the amino acid at position 39 of the heavy chain variable region is replaced with a negatively charged amino acid, and the amino acid at position 38 of the light chain variable region is replaced 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 replaced with a positively charged amino acid, and the amino acid at position 87 of the light chain variable region is replaced with a negatively charged amino acid; In the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is replaced with a negatively charged amino acid, and the amino acid at position 87 of the light chain variable region is replaced 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 replaced with a positively charged amino acid, and the amino acid at position 44 of the light chain variable region is replaced with a negatively charged amino acid; In the second antigen-binding region, the amino acid at position 45 of the heavy chain variable region is replaced with a negatively charged amino acid, and the amino acid at position 44 of the light chain variable region is replaced 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 replaced with a positively charged amino acid, and the amino acid at position 43 of the light chain variable region is replaced with a negatively charged amino acid; In the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is replaced with a negatively charged amino acid, and the amino acid at position 43 of the light chain variable region is replaced with a positively charged amino acid; (5) In the first antigen-binding region, the amino acid at position 91 of the heavy chain variable region is replaced with a positively charged amino acid, and the amino acid at position 44 of the light chain variable region is replaced with a negatively charged amino acid; In the second antigen-binding region, the amino acid at position 91 of the heavy chain variable region is replaced with a negatively charged amino acid, and the amino acid at position 44 of the light chain variable region is replaced with a positively charged amino acid; For example, the antibody or antigen-binding fragment according to claim 12, 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.

14. (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; 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. (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; 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; (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; 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; (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; 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; (v) The antibody or antigen-binding fragment thereof according to claim 13, wherein 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 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.

15. The antibody or antigen-binding fragment thereof according to any one of claims 11 to 14, wherein the antibody further comprises a disulfide bond reformation mutation as defined in any one of claims 3 to 8.

16. An antibody or antigen-binding fragment according to any one of claims 1 to 8 and 10 to 14, wherein one, more, or all antigen-binding regions that bind to the same antigen contain the same mutation.

17. 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 the heavy chain variable region VH and the light chain variable region VL, for example, the first antigen-binding region is the Fab fragment of the antibody and / or the second antigen-binding region is the Fab fragment of the antibody. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and 10 to 14.

18. The first antigen-binding region of the multispecific antibody is a combination of mutations selected from the following: Table 1-1 Table 1-2 The following combinations of mutations are included, in which the second antigen-binding region is optionally selected from the following: Table 2 The antibody or antigen-binding fragment thereof according to claim 17, including the antibody or antigen-binding fragment thereof.

19. The first antigen-binding region and the second antigen-binding region of the multispecific antibody are, respectively, the following combinations of mutations: Table 3-1 Table 3-2 Table 3-3 The antibody or antigen-binding fragment thereof according to claim 18, comprising:

20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and 10 to 14, wherein the light chain constant region included in 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 included in one or more or all of the second antigen-binding regions is a lambda light chain constant region, or the light chain constant region included in one or more of all of the first antigen-binding regions is a lambda light chain constant region, and the light chain constant region included in one or more or all of the second antigen-binding regions is a kappa light chain constant region.

21. The antibody also includes a first Fc region and a second Fc region, and the first Fc region and the second Fc region are the same or different. For example, the first Fc region and the second Fc region are each human IgG Fc, for example human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, and each contains or is composed of the amino acid sequence of SEQ ID NO: 122 or 123, or has at least 90% identity thereto, for example 95%, 96%, 97%, 99%, or more identity thereto, and optionally the first and / or second Fc region contains the L234A / L235A mutation, the antibody or antigen-binding fragment according to claims 1 to 8 and 10 to 14.

22. Preferably, based on knob-into-hole technology, the corresponding knob mutation and hole mutation are introduced into the first Fc region and the second Fc region. a) The polypeptide in one Fc region contains mutant T366W, while the polypeptide in the other Fc region contains T366S, L368A, and Y407V (numbered based on the EU index), or b) One Fc region includes amino acid substitutions S354C and T366W, and the other Fc region includes amino acid substitutions Y349C, T366S, L368A, and Y407V (numbered based on the EU index), more preferably a) One Fc region polypeptide contains or is composed of the amino acid sequence shown in SEQ ID NO: 125, and the other Fc region polypeptide contains or is composed of the amino acid sequence shown in SEQ ID NO: 124, b) The polypeptide in one Fc region contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 125, while the polypeptide in the other Fc region contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

124. c) The polypeptide in one Fc region contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 125, and includes mutations S354C and T366W, while the polypeptide in the other Fc region contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 124, and includes mutations Y349C, T366S, L368A, and Y407V, d) The polypeptide in one Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO: 20 or 131, and the polypeptide in the other Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO: 101 or 130, e) The polypeptide in one Fc region contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 20 or 131, while the polypeptide in the other Fc region contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 101 or 130, or f) The multispecific antibody according to claim 21, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 20 or 131, and comprises mutant T366W, while the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 101 or 130, and comprises T366S, L368A, and Y407V.

23. The mutation is 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, preferably One Fc region's CH3 contains S364R and D399K mutations, and the other Fc region's CH3 mutations contain Y349T, K370S, and K409D mutations, more preferably a) One Fc region polypeptide contains or is composed of the amino acid sequence shown in SEQ ID NO: 126, and the other Fc region polypeptide contains or is composed of the amino acid sequence shown in SEQ ID NO: 127, b) The polypeptide in one Fc region contains 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: 126, while the polypeptide in the other Fc region contains 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: 127, or c) The antibody or antigen-binding fragment according to claim 21, wherein one Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 126, and comprises mutations S364R and D399K, while the polypeptide of the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 127, and comprises mutations Y349T, K370S, and K409D.

24. The antigen bound to the antibody or its 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, e.g., PD-1, PD-L1, CD47, immune activation molecules, e.g., 4-1BB, CD40, and OX40, and can be optionally selected. The antibody or antigen-binding fragment thereof according to claims 1 to 8 and 10 to 14, wherein the first antigen and the second antigen are different, the antibody is a bispecific antibody, and preferably the bispecific antibody binds to two antigens selected from the following: 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.

25. The bispecific antibody of this disclosure 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. (A) The first Fab is connected to the N end of the first Fc region at the C end of the CH1 (by or without a linker, e.g., a hinge region), and the second Fab is connected to the N end of the second Fc region at the C end of the CH1 (by or without a linker, e.g., a hinge region), preferably The bispecific antibody is an IgG-like antibody having the configuration shown in Figure 1, more preferably The aforementioned antibody Heavy chain 1: From the N-terminus to the C-terminus, it includes or is composed of the heavy chain variable region of the first Fab, the heavy chain steady region CH1, and the heavy chain steady region CH1 is connected to the N-terminus of the first Fc region at its C-terminus, with or without a linker (e.g., a hinge region). Light chain 1: From the N-terminus to the C-terminus, it includes or is composed of the light chain variable region and the light chain constant region of the first Fab. Heavy chain 2: From the N-terminus to the C-terminus, the heavy chain variable region of the second Fab, the heavy chain steady region CH1, and the second Fc region are included or comprised thereof, and the heavy chain steady region CH1 is connected to the N-terminus of the second Fc region at its C-terminus, with or without a linker (e.g., a hinge region). Light chain 2: From the N-terminus to the C-terminus, it includes or is composed of the light chain variable region - light chain steady region of the second Fab, and optionally (1) The first antigen-binding region specifically binds to Her2, and the heavy chain 1 and light chain 1 each contain the amino acid sequence shown in the following sequence number, or contain 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 sequence number: i) Sequence ID 43 and Sequence ID 45, ii) Sequence ID 48 and Sequence ID 49, iii) Sequence ID 21 and Sequence ID 22, iv) Sequence IDs 25 and 26, v) Sequence IDs 25 and 29, vi) Sequence ID 31 and Sequence ID 32, or vii) Sequence ID 31 and Sequence ID 29, and / or (2-1) The second antigen-binding region specifically binds to Hel, and the heavy chain 2 and light chain 2 each contain an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing or shown in the following sequence numbers, or consist of the sequence shown in the following sequence numbers: i) Sequence ID 41 and Sequence ID 42, ii) Sequence IDs 23 and 24, iii) Sequence ID 27 and Sequence ID 28, iv) Sequence IDs 27 and 30, v) Sequence ID 33 and Sequence ID 34, or vi) Sequence ID 33 and Sequence ID 30, (2-2) The second antigen-binding region specifically binds to PD-1, and the heavy chain 2 and light chain 2 each contain an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing or shown below, or consist of the sequences shown in the following SEQ ID NOs: (2-3) The second antigen-binding region specifically binds to CD47, and the heavy chain 2 and light chain 2 each contain an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing or shown below, or consist of the sequence shown in the following SEQ ID NOs: (2-4) The second antigen-binding region specifically binds to cMet, and the heavy chain 2 and light chain 2 each contain an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence containing or shown below, or are composed of the sequences shown in the following SEQ ID NOs: (B) 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, with one second Fab linked to the N-terminus of the first Fc region by the C-terminus of its CH1 (by or without a linker, e.g., a hinge region), and another second Fab linked to the N-terminus of the heavy chain variable region of the first Fab by the C-terminus of its CH1 (by or without a linker), preferably The bispecific antibody has a 2+1 N-terminal configuration, the configuration of which is shown in Figure 5A, and more preferably The aforementioned bispecific antibody Heavy chain 1: From the N-terminus to the C-terminus, it includes or is composed of the following: heavy chain variable region of the second Fab - heavy chain steady region CH1 - heavy chain variable region of the first Fab - heavy chain steady region CH1 - second Fc region, wherein the heavy chain steady region CH1 of the first Fab is connected to the N-terminus of the second Fc region at its C-terminus, with or without a linker (e.g., hinge region), and the second Fab is connected to the N-terminus of the heavy chain variable region of the first Fab at its C-terminus of CH1 (with or without a linker). Light chain 1: From the N-terminus to the C-terminus, it includes or is composed of the light chain variable region and the light chain constant region of the first Fab. Heavy chain 2: From the N-terminus to the C-terminus, the heavy chain variable region of the second Fab, the heavy chain steady region CH1, and the first Fc region are included or comprised thereof, and the heavy chain steady region CH1 is connected to the N-terminus of the first Fc region at its C-terminus, with or without a linker (e.g., a hinge region). Light chain 2: From the N-terminus to the C-terminus, it includes or is composed of the light chain variable region - light chain steady region of the second Fab, The aforementioned bispecific antibody comprises two light chains 2, and optionally The first antigen-binding region binds to CD3, and the second antigen-binding region binds to GPRC5D, for example, 1) Heavy chain 1 contains the amino acid sequence shown in SEQ ID NO: 58, or contains or is composed of 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:

58. Light chain 1 contains the amino acid sequence shown in Sequence ID No. 59, or contains or is composed of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in Sequence ID No.

59. Heavy chain 2 contains the amino acid sequence shown in SEQ ID NO: 57, or contains or is composed of 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: 57, and / or Light chain 2 contains the amino acid sequence shown in SEQ ID NO: 56, or contains or is composed of 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: 56, and / or 2) Heavy chain 1 contains the amino acid sequence shown in SEQ ID NO: 62, or contains or is composed of 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:

62. Light chain 1 contains the amino acid sequence shown in SEQ ID NO: 63, or contains or is composed of 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:

63. Heavy chain 2 contains the amino acid sequence shown in SEQ ID NO: 61, or contains or is composed of 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: 61, and / or Light chain 2 contains the amino acid sequence shown in SEQ ID NO: 60, or contains or is composed of 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, (C) The bispecific antibody of the present disclosure 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 one first Fab, and the second antigen-binding regions comprise one second Fab, and the two second Fabs are linked to the N-terminus of the first Fc region and the N-terminus of the second Fc region, respectively, at the C-terminus of their CH1 (by or without linker, e.g., hinge region), and the N-terminus of the heavy chain variable region of the first Fab is linked to the C-terminus of the second Fc region (by or without linker), preferably The bispecific antibody has a 2+1 C-terminal configuration, which is shown in Figure 5B, and more preferably... The aforementioned bispecific antibody Heavy chain 1: From the N-terminus to the C-terminus, it includes or is composed of the heavy chain variable region of the second Fab - heavy chain steady region CH1 - second Fc region - heavy chain variable region of the first Fab - heavy chain steady region CH1, and the second Fab is connected to the N-terminus of the second Fc region at the C-terminus of its CH1 (by linker, e.g., hinge region, or not), and the N-terminus of the heavy chain variable region of the first Fab is connected to the C-terminus of the second Fc region (by linker or not). Light chain 1: From the N-terminus to the C-terminus, it includes or is composed of the light chain variable region and the light chain constant region of the first Fab. Heavy chain 2: From the N-terminus to the C-terminus, the heavy chain variable region of the second Fab, the heavy chain steady region CH1, and the first Fc region are included or comprised thereof, and the heavy chain steady region CH1 is connected to the N-terminus of the first Fc region at its C-terminus, with or without a linker (e.g., a hinge region). Light chain 2: From the N-terminus to the C-terminus, it includes or is composed of the light chain variable region - light chain steady region of the second Fab, The aforementioned bispecific antibody comprises two light chains 2, and optionally The first antigen-binding region binds to CD3, and the second antigen-binding region binds to GPRC5D, for example, 1) Heavy chain 1 contains the amino acid sequence shown in SEQ ID NO: 64, or contains or is composed of 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:

64. Light chain 1 contains the amino acid sequence shown in Sequence ID No. 59, or contains or is composed of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in Sequence ID No.

59. Heavy chain 2 contains the amino acid sequence shown in SEQ ID NO: 57, or contains or is composed of 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: 57, and / or Light chain 2 contains the amino acid sequence shown in SEQ ID NO: 56, or contains or is composed of 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: 56, and / or 2) Heavy chain 1 contains the amino acid sequence shown in SEQ ID NO: 65, or contains or is composed of 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:

65. Light chain 1 contains the amino acid sequence shown in SEQ ID NO: 63, or contains or is composed of 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:

63. Heavy chain 2 contains the amino acid sequence shown in SEQ ID NO: 61, or contains or is composed of 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: 61, and / or Light chain 2 contains the amino acid sequence shown in SEQ ID NO: 60, or contains or is composed of 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, (D) The bispecific antibody of the present disclosure 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 regions comprise a first Fab, the second antigen-binding regions comprise a second Fab, the two second Fabs are linked to the N-terminuses of two Fc regions at their CH1 C-terminuses (by or without linkers, e.g., hinge regions) (the Fc regions may be the same or different), and the N-terminuses of the heavy chain variable regions of the two first Fabs are linked to the C-terminuses of the two Fc regions (by or without linkers), preferably The bispecific antibody has a 2+2 C-terminal configuration, and the configuration is shown in Figure 5C, more preferably The aforementioned bispecific antibody Heavy chain: Hereinafter, from the N-terminus to the C-terminus, it includes or is composed of the heavy chain variable region of the second Fab - heavy chain steady region CH1 - Fc region - heavy chain variable region of the first Fab - heavy chain steady region CH1, the second Fab is connected to the N-terminus of the Fc region at the C-terminus of its CH1 (by linker, e.g., hinge region, or not), and the N-terminus of the heavy chain variable region of the first Fab is connected to the C-terminus of the Fc region (by linker or not), Light chain 1: From the N-terminus to the C-terminus, it includes or is composed of the light chain variable region and the light chain constant region of the first Fab. Light chain 2: From the N-terminus to the C-terminus, it includes or is composed of the light chain variable region - light chain steady region of the second Fab, The aforementioned bispecific antibody contains two heavy chains, and optionally The first antigen-binding region binds to CD3, and the second antigen-binding region binds to GPRC5D, for example, 1) The heavy chain contains the amino acid sequence shown in SEQ ID NO: 66, or contains or is composed of 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:

66. Light chain 1 contains the amino acid sequence shown in SEQ ID NO: 59, or contains or is composed of 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: 59, and / or Light chain 2 contains the amino acid sequence shown in SEQ ID NO: 56, or contains or is composed of 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: 56, and / or 2) The heavy chain contains the amino acid sequence shown in SEQ ID NO: 67, or contains or is composed of 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:

67. Light chain 1 contains the amino acid sequence shown in SEQ ID NO: 63, or contains or is composed of 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: 63, and / or The antibody or antigen-binding fragment thereof according to claim 24, wherein the light chain 2 contains the amino acid sequence shown in SEQ ID NO: 60, or contains or is composed of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

60.

26. The first antigen and the second antigen are different, and the antibody also includes an antigen-binding region that specifically binds to one or more other antigens, preferably The antibody is a trispecific antibody, more preferably The triplicate antibody of this disclosure 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, and the first Fab is linked to the N-terminus of the first Fc region at the C-terminus of its CH1 (by a linker, e.g., a hinge region). The second Fab is linked to the N-terminus of the second Fc region at the C-terminus of its CH1 (by or without a linker, e.g., a hinge region), and the third antigen-binding region includes an scFv (e.g., VH-VL or VL-VH) linked to the C-terminus of the second Fc region at the N-terminus of its VH or VL (e.g., scFv is VL-VH, e.g., the N-terminus of VL), by or without a linker, for example, The configuration of the triple-specific antibody is a 2+1 C-terminal configuration, as shown in Figure 7A or B, and more preferably The multispecific antibody described above Heavy chain 1: From the N-terminus to the C-terminus, it includes or is composed of the heavy chain variable region of the first Fab - heavy chain steady region CH1 - first Fc region, and the first Fab is connected to the N-terminus of the first Fc region at the C-terminus of its CH1 (by a linker, e.g., a hinge region, or not). Light chain 1: From the N-terminus to the C-terminus, it includes or is composed of the light chain variable region and the light chain constant region of the first Fab. Heavy chain 2: Hereinafter, from the N-terminus to the C-terminus, the heavy chain variable region of the second Fab - heavy chain steady region CH1 - second Fc region - scFv is included or composed thereof, wherein the heavy chain steady region CH1 is connected to the N-terminus of the second Fc region at its C-terminus by a linker (e.g., hinge region) or not, and the scFv is connected to the C-terminus of the second Fc region at its VH or VL N-terminus or C-terminus (e.g., scFv is VL-VH, e.g., the N-terminus of VL), with or without a linker. Light chain 2: From the N-terminus to the C-terminus, including the constant region of the second Fab light chain - which includes or is composed of the constant region of the light chain, optionally The first antigen-binding region specifically binds to GPRC5D, the second antigen-binding region specifically binds to CD3, and / or the third antigen-binding region specifically binds to BCMA, for example, Heavy chain 1 contains the amino acid sequence shown in SEQ ID NO: 71, or contains or is composed of 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:

71. Light chain 1 contains the amino acid sequence shown in SEQ ID NO: 72, or contains or is composed of 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:

72. Heavy chain 2 contains the amino acid sequence shown in SEQ ID NO: 73, or contains or is composed of 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: 73, and / or The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and 10 to 14, wherein the light chain 2 comprises the amino acid sequence shown in SEQ ID NO: 74, or comprises or is composed of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

74.

27. A nucleic acid encoding an antibody or an antigen-binding fragment thereof, or any or more of its chains, as described in any one of claims 1 to 26.

28. An expression vector comprising the nucleic acid described in claim 27.

29. A host cell comprising the nucleic acid described in claim 27 or the expression vector described in claim 28.

30. A method for preparing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 26, a) Introducing the nucleic acid encoding various chains of the antibody or its antigen-binding fragment into the host cell, b) The antibody or its antigen-binding fragment is expressed and assembled within the host cell, A method for optionally purifying the antibody or its antigen-binding fragment with, for example, protein A.

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