WO2025167974A1 - Multi-specific antibody or antigen-binding fragment - Google Patents

Abstract

Provided in the present disclosure are a multi-specific antibody or antigen-binding fragment, a composition, a preparation method and the use. By means of comprehensively considering interfacial amino acid interactions, amino acid pair mutations are introduced into CH1 and CL domains of a antibody, thereby improving correct pairing between a heavy chain and a light chain in the multi-specific antibody or antigen-binding fragment.

Description

Multispecific antibodies or antigen-binding fragments

Related applications

This application claims priority to Chinese patent application No. 202410175872.5 filed on February 7, 2024 and Chinese patent application No. 202510121250.9 filed on January 24, 2025, the entire contents of which are incorporated herein by reference for all purposes.

Technical Field

The present disclosure belongs to the field of biomedicine and relates to multispecific antibodies or antigen-binding fragments, compositions, preparation methods, and uses thereof.

Background Art

Bispecific antibodies are antibodies that can simultaneously recognize two different antigens or two different epitopes of the same antigen. This property enables bispecific antibodies to be used as targeting agents for in vitro and in vivo immunodiagnosis and therapy, and they have great potential in a wide range of clinical applications. However, the production and development of bispecific antibodies is extremely challenging. Traditionally, bispecific antibodies are prepared using hybridoma technology. Due to the random pairing of immunoglobulin heavy and light chains, a mixture of various different antibody molecules is produced, such as heavy chain homodimerization and mispairing of heavy and light chains, of which only one has the correct bispecific antibody structure. The presence of mispaired byproducts significantly reduces the yield and requires complex purification methods to achieve product homogeneity. Therefore, it is necessary to improve the efficiency of obtaining correct bispecific antibodies.

The correct bispecific antibody molecule can be obtained by reducing the mispairing of IgG heavy chains using several rational design strategies. One of the methods is knobs-into-holes, which aims to modify the CH3-CH3 contact interface by introducing mutations in the CH3 domains of two different heavy chains, so that the two different heavy chains are paired, thereby avoiding heavy chain homodimerization. WO1998050431A2 replaces the original amino acids with amino acids with short side chains in the CH3 domain of one heavy chain to produce a "hole"; conversely, amino acids with large side chains are introduced into the CH3 domain of the other heavy chain to produce a "knob". By co-expressing the heavy chains of these two antibodies, the two different antibody heavy chains with "hole" and "knob" structures preferentially pair, and high-yield heterodimer formation is observed. However, WO1998050431A2 uses two identical light chains to avoid mispairing of heavy and light chains. Using a common light chain is unrealistic for producing antibodies that recognize different antigens. Therefore, while this approach solves the problem of heavy chain homodimerization, it does not solve the problem of mispairing of the heavy and light chains from the two antibodies with each other.

There are many approaches to addressing heavy/light chain disorder in bispecific antibodies. WO2006106905A1 improves bispecific antibody formation by modulating the association at the VH-VL interface. Specifically, the binding between VH and VL is modulated by substituting charged amino acids at the VH-VL interface, for example by mutating positions Q39 in VH and Q38 in VL with oppositely charged amino acids. However, this approach is insufficient for effectively generating the desired bispecific antibody.

Appropriate optimization is still needed to further improve the specificity of heavy and light chain pairing, reduce mispairing byproducts, and increase the yield of bispecific antibodies.

Summary of the Invention

The present disclosure provides a multispecific antibody or antigen-binding fragment with improved heavy-light chain specific pairing, as well as compositions, preparation methods, uses, etc. By comprehensively considering various interactions between amino acids at the heavy-light chain interaction interface, such as electrostatic interactions, hydrophobic interactions, hydrogen bonding, and aromatic stacking, amino acid modifications are introduced at specific positions in the heavy-light chain interaction interface to improve correct heavy-light chain pairing.

The present disclosure provides a multispecific antibody or antigen-binding fragment thereof, comprising:

A. a first antibody that specifically binds to a first antigen, comprising a first heavy chain H1 and a first light chain L1;

B. a second antibody that specifically binds to a second antigen, comprising a second heavy chain H2 and a second light chain L2;

Among them, H1 and H2 contain the CH1 domain, L1 and L2 contain the CL domain, and there are amino acid mutations in the CH1 domain and the CL domain.

In a preferred embodiment, H1/L1 has a mutated amino acid pair in the CH1 and CL domains respectively contained therein; and/or, H2/L2 has a mutated amino acid pair in the CH1 and CL domains respectively contained therein.

In a preferred embodiment, the amino acid pair that is mutated is one that improves proper pairing of the heavy and light chains.

In a more preferred embodiment, the mutated amino acid pairs can increase the correct pairing rate of heavy and light chains to more than 50%, or more than 60%, or more than 70%, or more than 80%, or more than 90%, or 100%.

In some embodiments, H1/L1 has one or more of the following amino acid pairs mutated in the CH1 and CL domains respectively:

CH1: G26, CL: L28; CH1: G26, CL: F11; CH1: V68, CL: L28; CH1: G49, CL: N31; CH1: H51, CL: N30; CH 1: T70, CL: N31; CH1: K30, CL: S24; CH1: T22, CL: S7; CH1: P10, CL: S14; CH1: F53, CL: L28; CH1: T 70. CL: S7; CH1: T70, CL: N30; CH1: H51, CL: N31; CH1: H51, CL: S67; CH1: S7, CL: T22; CH1: S19, C L: V98; CH1: A24, CL: S7; CH1: A12, CL: S14; CH1: F9, CL: T22; CH1: F9, CL: S24; CH1: L28, CL: T71;

The numbers are IMGT exon numbers.

In some embodiments, H2/L2 has one or more of the following amino acid pairs mutated in the CH1 and CL domains respectively:

CH1: G26, CL: L28; CH1: G26, CL: F11; CH1: V68, CL: L28; CH1: G49, CL: N31; CH1: H51, CL: N30; CH 1: T70, CL: N31; CH1: K30, CL: S24; CH1: T22, CL: S7; CH1: P10, CL: S14; CH1: F53, CL: L28; CH1: T 70. CL: S7; CH1: T70, CL: N30; CH1: H51, CL: N31; CH1: H51, CL: S67; CH1: S7, CL: T22; CH1: S19, C L: V98; CH1: A24, CL: S7; CH1: A12, CL: S14; CH1: F9, CL: T22; CH1: F9, CL: S24; CH1: L28, CL: T71;

The numbers are IMGT exon numbers.

Furthermore, the mutated amino acid pairs present in the CH1 and CL domains respectively contained in H1/L1 and/or H2/L2 are amino acid pairs with opposite charges or amino acid pairs with non-electrostatic interactions; preferably, the amino acid pairs with opposite charges are positively charged amino acids and negatively charged amino acids; preferably, the positively charged amino acids are arginine (R), histidine (H) or lysine (K), and the negatively charged amino acids are aspartic acid (D) or glutamic acid (E).

Furthermore, the mutated amino acid pairs present in the CH1 and CL domains of H1/L1 and/or H2/L2, respectively, are selected from one or more of the following mutated amino acid pairs:

CH1: G26A/Q, CL: L28F/Q; CH1: G26L/W, CL: F11W/L; CH1: V68S/V, CL: L28H/T; CH1: G49K/E, CL: N31E/K; CH1: H51D/K, CL: N30K/D; CH1: T70K/E, CL: N31E/K; CH1 : K30R/D, CL: S24D/R; CH1: T22K/D, CL: S7D/K; CH1: G26R/D, CL: L28D/R; CH1: P1 0K/D, CL: S14D/K; CH1: F53R/D, CL: L28D/R; CH1: T70D/K, CL: S7K/D; CH1: F9D/R , CL: T22R/D; CH1: T70E/H, CL: N30H/E; CH1: H51D/K, CL: N31K/D; CH1: H51K/E, C L: N30E/K; CH1: T70R/D, CL: N31D/R; CH1: S7D/K, CL: T22K/D; CH1: S19R/E, CL: V 98E/R; CH1: A24D/K, CL: S7K/D; CH1: H51T/Q, CL: S67Q/S; CH1: A12D/F, CL: S14K /G; CH1: P10Q/I, CL: S14E/I; CH1: F9E/T, CL: S24I/F; CH1: L28E/R, CL: T71H/F;

The numbers are IMGT exon numbers.

Furthermore, the mutated amino acid pairs present in the CH1 and CL domains of H1/L1 and H2/L2, respectively, are selected from any one of the following groups:

(1) G26R of CH1 in H1, L28D of CL in L1, G26D of CH1 in H2, L28R of CL in L2;

(2) P10K of CH1 in H1, S14D of CL in L1, P10D of CH1 in H2, S14K of CL in L2;

(3) F53R of CH1 in H1, L28D of CL in L1, F53D of CH1 in H2, L28R of CL in L2;

(4) T70D of CH1 in H1, S7K of CL in L1, T70K of CH1 in H2, S7D of CL in L2;

(5) F9D of CH1 in H1, T22R of CL in L1, F9R of CH1 in H2, T22D of CL in L2;

(6) T22K of CH1 in H1, S7D of CL in L1, T22D of CH1 in H2, S7K of CL in L2;

(7) T70E of CH1 in H1, N30H of CL in L1, T70H of CH1 in H2, N30E of CL in L2;

(8) H51D of CH1 in H1, N30K of CL in L1, H51K of CH1 in H2, N30D of CL in L2;

(9) T70K of CH1 in H1, N31E of CL in L1, T70E of CH1 in H2, N31K of CL in L2;

(10) H51D of CH1 in H1, N31K of CL in L1, H51K of CH1 in H2, N31D of CL in L2;

(11) H51K of CH1 in H1, N30E of CL in L1, H51E of CH1 in H2, N30K of CL in L2;

(12) T70R of CH1 in H1, N31D of CL in L1, T70D of CH1 in H2, N31R of CL in L2;

(13) S7D of CH1 in H1, T22K of CL in L1, S7K of CH1 in H2, T22D of CL in L2;

(14) S19R of CH1 in H1, V98E of CL in L1, S19E of CH1 in H2, V98R of CL in L2;

(15) A24D and G49K of CH1 in H1, S7K and N31E of CL in L1, A24K and G49E of CH1 in H2, S7D and N31K of CL in L2;

(16) A24D and H51T of CH1 in H1, S7K and S67Q of CL in L1, A24K and H51Q of CH1 in H2, and S7D of CL in L2;

(17) A24D and K30R of CH1 in H1, S7K and S24D of CL in L1, A24K and K30D of CH1 in H2, S7D and S24R of CL in L2;

(18) A24D and G49E of CH1 in H1, S7K and N31K of CL in L1, A24K and G49K of CH1 in H2, S7D and N31E of CL in L2;

(19) F9E and T22K of CH1 in H1, S7D and S24I of CL in L1, F9T and T22D of CH1 in H2, S7K and S24F of CL in L2;

(20) T22K and K30R of CH1 in H1, S7D and S24D of CL in L1, T22D and K30D of CH1 in H2, S7K and S24R of CL in L2;

(21) K30R, G49K of CH1 in H1, S24D, N31E of CL in L1, K30D, G49E of CH1 in H2, S24R, N31K of CL in L2;

(22) K30R and H51T of CH1 in H1, S24D and S67Q of CL in L1, K30D and H51Q of CH1 in H2, and S24R of CL in L2;

(23) K30R and H51D of CH1 in H1, S24D and N30K of CL in L1, K30D and H51K of CH1 in H2, S24R and N30D of CL in L2;

(24) K30R, T70K of CH1 in H1, S24D, N31E of CL in L1, K30D, T70E of CH1 in H2, S24R, N31K of CL in L2;

(25) K30R and H51K of CH1 in H1, S24D and N30E of CL in L1, K30D and H51E of CH1 in H2, S24R and N30K of CL in L2;

(26) L28E and H51D of CH1 in H1, N30K and T71H of CL in L1, L28R and H51K of CH1 in H2, N30D and T71F of CL in L2;

(27) P10Q, K30R, H51D of CH1 in H1, S14E, S24D, N30K of CL in L1, P10I, K30D, H51K of CH1 in H2, S14I, S24R, N30D of CL in L2;

(28) P10Q, T22K, K30R of CH1 in H1, S7D, S14E, S24D of CL in L1, P10I, T22D, K30D of CH1 in H2, S7K, S14I, S24R of CL in L2;

(29) A12D, T22K, K30R of CH1 in H1, S7D, S14K, S24D of CL in L1, A12F, T22D, K30D of CH1 in H2, S14G, S7K, S24R of CL in L2;

(30) A12D, H51D, K30R of CH1 in H1, S14K, N30K, S24D of CL in L1, A12F, K30D, H51K of CH1 in H2, S14G, S24R, N30D of CL in L2;

(31) T22K, K30R, G49K of CH1 in H1, S7D, S24D, N31E of CL in L1, T22D, K30D, G49E of CH1 in H2, S7K, S24R, N31K of CL in L2;

(32) T22K, K30R, H51T of CH1 in H1, S7D, S24D, S67Q of CL in L1, T22D, K30D, H51Q of CH1 in H2, S7K, S24R of CL in L2;

(33) T22K, G26A, K30R of CH1 in H1, S7D, S24D, L28F of CL in L1, T22D, G26Q, K30D of CH1 in H2, S7K, S24R, L28Q of CL in L2;

(34) G26A, K30R, T70K of CH1 in H1, S24D, L28F, N31E of CL in L1, G26Q, K30D, T70E of CH1 in H2, S24R, L28Q, N31K of CL in L2;

(35) G26A, K30R, G49K of CH1 in H1, S24D, L28F, N31E of CL in L1, G26Q, K30D, G49E of CH1 in H2, S24R, L28Q, N31K of CL in L2;

(36) G26A, K30R, H51D of CH1 in H1, N30K, S24D, L28F of CL in L1, G26Q, K30D, H51K of CH1 in H2, S24R, L28Q, N30D of CL in L2;

(37) G26A, K30R, H51T of CH1 in H1, S24D, L28F, S67Q of CL in L1, G26Q, K30D, H51Q of CH1 in H2, S24R, L28Q of CL in L2;

(38) G26L, K30R, H51D of CH1 in H1, F11W, S24D, N30K of CL in L1, G26W, K30D, H51K of CH1 in H2, F11L, S24R, N30D of CL in L2;

(39) G26L, K30R, T70K of CH1 in H1, F11W, S24D, N31E of CL in L1, G26W, K30D, T70E of CH1 in H2, F11L, S24R, N31K of CL in L2;

(40) G26L, K30R, G49K of CH1 in H1, F11W, S24D, N31E of CL in L1, G26W, K30D, G49E of CH1 in H2, F11L, S24R, N31K of CL in L2;

(41) G26L, K30R, H51T of CH1 in H1, F11W, S24D, S67Q of CL in L1, G26W, K30D, H51Q of CH1 in H2, F11L, S24R of CL in L2;

(42) T22K, G26L, K30R of CH1 in H1, S7D, F11W, S24D of CL in L1, T22D, G26W, K30D of CH1 in H2, S7K, F11L, S24R of CL in L2;

(43) T22K, K30R, V68S of CH1 in H1, S7D, S24D, L28H of CL in L1, T22D, K30D of CH1 in H2, S7K, S24R, L28T of CL in L2;

(44) K30R, G49K, V68S of CH1 in H1, S24D, N31E, L28H of CL in L1, K30D, G49E of CH1 in H2, S24R, L28T, N31K of CL in L2;

The numbers are IMGT exon numbers.

In some embodiments, the two heavy chains H1 and H2 in the multispecific antibody or its antigen-binding fragment further comprise a VH domain and an Fc domain (including a CH2 domain and a CH3 domain), wherein the VH domain contains an amino acid sequence that targets a different antigenic epitope. In some embodiments, the two light chains L1 and L2 in the multispecific antibody or its antigen-binding fragment further comprise a VL domain, wherein the VL domain contains an amino acid sequence that targets a different antigenic epitope. In some embodiments, the CH1 domain contained in the heavy chains H1 and H2 in the multispecific antibody or its antigen-binding fragment is derived from IgG1, IgG2, IgG3 or IgG4, and the CL domain contained in the light chains L1 and L2 is derived from a kappa light chain or a lambda light chain. In some embodiments, the multispecific antibody or its antigen-binding fragment is humanized.

In some embodiments, the VH domain in H1 and the VL domain in L1 contain oppositely charged mutant amino acids, respectively, to promote preferential pairing of the H1 and L1 heavy and light chains. In some embodiments, the VH domain in H2 and the VL domain in L2 contain oppositely charged mutant amino acids, respectively, to promote preferential pairing of the H2 and L2 heavy and light chains. In preferred embodiments, the VH domain in H1 and the VL domain in L1 contain Q39E and Q38K substitution mutations, respectively, and/or the VH domain in H2 and the VL domain in L2 contain Q39K and Q38E substitution mutations, respectively, where numbering is based on Kabat numbering.

In some embodiments, the CH3 domain in H1 and the CH3 domain in H2 comprise amino acid substitutions that preferentially pair the Fc domain of H1 with the Fc domain of H2. Preferably, the CH3 domain comprises a native non-cysteine to cysteine substitution. Further preferably, H1 comprises an S354C mutation and H2 comprises a Y349C mutation. Preferably, the amino acid substitutions in the CH3 domain result in greater electrostatic complementarity. Preferably, the amino acid substitutions in the CH3 domain involve replacing one or more amino acid residues in the CH3 domain of H1 with amino acid residues having a larger side chain volume, thereby creating a protrusion on the surface that interacts with the CH3 domain of H2; concurrently, replacing amino acid residues in the CH3 domain of H2 with amino acid residues having a smaller side chain volume, thereby creating a depression on the surface that interacts with the CH3 domain of H1. Preferably, the protrusion is a knob mutation. Further preferably, the knob-generating mutation is T366W (wherein numbering is EU numbering). Preferably, the depression is a hole mutation. Further preferably, the hole-generating mutation is at least one of T366S, L368A and Y407V (wherein numbering is EU numbering).

In a preferred embodiment, the CH3 domain of H1 comprises S354C, T366W substitutions (wherein the numbering is EU numbering), and the CH3 domain of H2 comprises Y349C, T366S, L368A, Y407V substitutions (wherein the numbering is EU numbering).

The present disclosure provides a nucleic acid comprising a nucleic acid molecule A encoding the first heavy chain H1 of the multispecific antibody or antigen-binding fragment thereof of the present disclosure, a nucleic acid molecule B encoding the first light chain L1 of the multispecific antibody or antigen-binding fragment thereof of the present disclosure, a nucleic acid molecule C encoding the second heavy chain H2 of the multispecific antibody or antigen-binding fragment thereof of the present disclosure, and a nucleic acid molecule D encoding the second light chain L2 of the multispecific antibody or antigen-binding fragment thereof of the present disclosure.

The present disclosure provides an expression vector or host cell comprising a nucleic acid encoding a multispecific antibody or antigen-binding fragment thereof of the present disclosure. In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell; preferably a eukaryotic cell, more preferably a CHO cell or HEK293 cell.

The present disclosure provides a composition comprising the multispecific antibody or antigen-binding fragment thereof of the present disclosure, and a pharmaceutically acceptable carrier and/or diluent and/or excipient.

The present disclosure provides uses of multispecific antibodies or antigen-binding fragments thereof, nucleic acids, expression vectors, host cells or compositions in preparing multispecific antibody-fusion protein chimeras.

The present disclosure provides use of the above-mentioned nucleic acid, expression vector, or host cell in preparing a multispecific antibody or an antigen-binding fragment thereof.

The present disclosure provides a method for preparing a multispecific antibody or an antigen-binding fragment thereof, comprising:

(1) transforming a host cell with the expression vector disclosed herein;

(2) causing the host cell to express the multispecific antibody or antigen-binding fragment thereof.

The present disclosure provides a use of the multispecific antibody or antigen-binding fragment thereof of the present disclosure in the preparation of a medicament for treating a disease in a subject in need thereof.

The present disclosure provides a method of treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the multispecific antibody or antigen-binding fragment thereof of the present disclosure.

The present invention analyzes the amino acids at the interaction interface of the antibody CH1-CL domain and mutates and modifies the amino acids that affect the pairing of CH1 and CL to improve the correct acquisition of heterologous multispecific antibodies or antigen-binding fragments thereof, thereby significantly improving the correct pairing rate of heavy and light chains while reducing heavy chain homodimerization or incorrect pairing of heavy and light chains.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Representation of the CH1-CL structure from the crystal structure of 6Y1L.

Sequence Description

SEQ ID NO.1IgG1_knob_WT

SEQ ID NO.2IgG1_hole_WT

SEQ ID NO. 3Kappa_WT

SEQ ID NO.4 Zalutumumab_VH

SEQ ID NO.5 Zalutumumab_VL

SEQ ID NO.6Onartuzumab_VH

SEQ ID NO.7Onartuzumab_VL

DETAILED DESCRIPTION

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference into the present disclosure to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference into the present disclosure.

Before describing the present disclosure in detail below, it should be understood that the present disclosure is not limited to the specific methodology, protocols and reagents described in the present disclosure, as these may vary. It should also be understood that the terms used in the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs.

The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

the term

The terms "include", "comprising", "having" or other similar words should be understood as including a whole or a whole group of things, and may also have other whole things or whole groups of things, and are non-exclusive or open descriptions.

The term "and/or" should be understood to include both individual and combined options. For example, when two elements are linked by "and/or," the first option refers to the applicability of only the first element without the second element, the second option refers to the applicability of only the second element without the first element, and the third option refers to the applicability of both the first and second elements.

The term "multispecific antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that can bind to two or more antigenic epitopes. It can be an intact antibody or an antibody fragment that does not constitute a complete antibody structure. It can also be a product with antigen-specific binding ability formed by modifying the intact antibody or antibody fragment or its single chain (e.g., by linking other peptide segments, rearranging functional units, etc.). For example, (i) Fab fragment, a monovalent fragment composed of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragment, a bivalent fragment composed of two Fab fragments connected by a disulfide bridge on the hinge region; (iii) Fd fragment composed of VH and CH1 domains; (iv) Fv fragment composed of the VH and VL domains of a single arm of an antibody; (v) dsFv, an antigen-binding fragment formed by VH and VL via an interchain disulfide bond; (vi) diabodies, bispecific antibodies and multispecific antibodies including fragments such as scFv, dsFv and Fab. In some embodiments, the multispecific antibody or antigen-binding fragment refers to a bispecific antibody, such as a human bispecific antibody, a humanized bispecific antibody, a chimeric bispecific antibody, or a murine bispecific antibody, etc. In some embodiments, the bispecific antibody comprises a complete antibody structure.

The term "first antibody" refers to an antibody that binds to one antigenic epitope in a multispecific antibody or an antigen-binding fragment thereof, and comprises a first heavy chain H1 and a first light chain L1, and H1 and L1 can bind to one antigenic epitope. The term "second antibody" refers to an antibody that binds to another antigenic epitope in a multispecific antibody or an antigen-binding fragment thereof, and comprises a second heavy chain H2 and a first light chain L2, and H2 and L2 can bind to another antigenic epitope. Heavy-light chain pairing refers to the pairing of the first heavy chain and the first light chain of the first antibody, or the pairing of the second heavy chain and the second light chain of the second antibody. In the present disclosure, the first antibody and the second antibody refer to universal labels, indicating antibodies that bind to different antigenic epitopes, and do not represent the order of antibodies in the multispecific antibody or its antigen-binding fragment, and should not be understood as marking specific or particular parts of the multispecific antibody or its antigen-binding fragment provided by the present disclosure. The mutated amino acids in the first antibody and the second antibody can be reversed, that is, any mutated amino acid in the first antibody can alternatively be in the second antibody, and any mutated amino acid in the second antibody can also alternatively be in the first antibody.

The first antibody and the second antibody respectively contain two heavy chains H1 and H2, which respectively contain a VH domain, a CH1 domain and an Fc domain (including a CH2 domain and a CH3 domain), wherein the VH domain contains an amino acid sequence targeting different antigenic epitopes; and two light chains L1 and L2, which respectively contain a VL domain and a CL domain, wherein the VL domain contains an amino acid sequence targeting different antigenic epitopes.

The term "CH1 domain" refers to the first constant region of a heavy chain and may also include a portion of the hinge region following the CH1 domain. The CH1 domain can be derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain. The term "CL domain" refers to the constant region of a light chain. The CL domain can be derived from a human kappa chain or a human lambda chain. The CH1 and CL domain structures can be determined by conventional methods in the art.

Antibody numbering is based on a well-known numbering system. The term "IMGT exon numbering" is used to number amino acid positions in the heavy chain CH1 domain and light chain CL domain (IMGT Scientific chart). For example, amino acid substitutions in CH1 are numbered relative to CH1 of human IgG1 (SEQ ID NO. 1 or 2, positions 1-98), and amino acid substitutions in CL are numbered relative to the kappa light chain (SEQ ID NO. 3). The term "Kabat numbering" is used to number amino acid positions in antibody variable regions (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition). The term "EU numbering" is widely used to number amino acid positions in constant regions (including Fc domains) (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition). The correlation between the antibody numbering systems is interchangeable according to the numbering method well known in the art.

The term "amino acid pair" refers to a pair of amino acids. An "oppositely charged amino acid pair" means one amino acid is positively charged and the other is negatively charged. A pair of positively and negatively charged amino acid pairs can be introduced by replacing the natural amino acid residues in the heavy chain CH1 domain and the light chain CL domain, respectively. Positively charged amino acids generally include arginine (R), histidine (H), and lysine (K), while negatively charged amino acids generally include aspartic acid (D) and glutamic acid (E). The charge distribution after introducing the oppositely charged amino acid pair is as follows: H1 (CH1 positive charge)/L1 (CL negative charge)/H2 (CH1 negative charge)/L2 (CL positive charge) or H1 (CH1 negative charge)/L1 (CL positive charge)/H2 (CH1 positive charge)/L2 (CL negative charge). One or more amino acid pairs can be introduced into the first antibody and the second antibody, and the amino acid pairs introduced into the first antibody and the second antibody can be the same or different. One or more amino acid pairs can also be introduced into the VH and VL interfaces, and in combination with one or more charge pairs introduced into the CH1/CL interface, amino acids introduced into the same chain (H1, L1, H2, or L2) generally have the same charge. Amino acid pairs can also be introduced by replacing native amino acid residues in the heavy chain CH1 domain and light chain CL domain based on other non-electrostatic interactions, such as hydrophobic interactions, hydrogen bonding, and aromatic stacking, to improve correct pairing of the heavy and light chains.

When describing amino acid mutations, "CH1: V68S/V" and "CL: L28H/T" indicate that the V at position 68 (IMGT exon numbering) in CH1 is replaced with an S, while the L at position 28 (IMGT exon numbering) in CL is replaced with an H. Alternatively, "CH1: V68S/V" and "CL: L28H/T" indicate that the V at position 68 (IMGT exon numbering) in CH1 is retained, while the L at position 28 (IMGT exon numbering) in CL is replaced with a T. All other substitutions follow the same nomenclature.

The term "expression vector" is a replicon into which a nucleic acid molecule can be operably inserted so as to effect replication or expression of the nucleic acid molecule.

The term "host cell" refers to a cell that contains a nucleic acid molecule of the present disclosure. A "host cell" can be any type of cell, such as a primary cell, a cultured cell, or a cell from a cell line, or a eukaryotic cell or a prokaryotic cell. For example, a CHO cell, a HEK293 cell, or an E. coli cell, etc.

The term "composition" refers to a product comprising the disclosed antibodies and a pharmaceutically acceptable carrier. The disclosed antibodies and compositions comprising them can also be used to manufacture the medicaments for treatment mentioned in the disclosure.

The term "pharmaceutically acceptable carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation or other material known in the art for pharmaceutical formulation. The nature of the carrier, excipient or diluent will depend on the route of administration for a particular application.

The term "effective amount" refers to a dosage of a pharmaceutical formulation comprising an active ingredient of the present disclosure, which produces the desired effect in the treated subject after administration to a patient in single or multiple doses.

The term "individual" or "subject" refers to any animal, such as a mammal or marsupial. Individuals of the present disclosure include, but are not limited to, humans, non-human primates (e.g., cynomolgus or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any type of poultry.

The term "treatment" refers to clinical intervention intended to alter the disease process in an individual or cell, and can be either preventative or interventional in the clinical pathological process. Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, improving or relieving the condition, and alleviating or improving the prognosis.

The term "disease" refers to any change or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

Example

The present disclosure is further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present disclosure only and are not intended to limit the scope of the present disclosure. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed according to conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

Example 1 Analysis of the CH1 and CL Interface of IgG1 Antibody

The IgG1 crystal structure (PDB ID: 6Y1L) was selected as a template, and the CH1-CL portion was selected to fill in the missing structure, as shown in Figure 1. The amino acids of CH1 (sequence shown in SEQ ID NO.1 or SEQ ID NO.2, position 1-98) and kappa light chain CL (sequence shown in SEQ ID NO.3) of IgG1 were numbered using IMGT exon numbering. The spacing between CH1 and CL was centered with Cα as the core. The amino acids in the protein were identified to obtain the target sites for amino acid mutation modification. The summary information is shown in Table 1.

Table 1. Interaction sites at the interface between CH1 of IgG1 and the CL of kappa light chain

Example 2: Using energy calculation methods to explore point mutations that improve the correct pairing of CH1 and CL

In a bispecific antibody, one heavy and light chain is named H1L1, and the other heavy and light chain is named H2L2. In the natural state, there are many heavy and light chain pairing patterns. The correct pairing is H1L1H2L2, and the incorrect heavy and light chain pairing includes three forms: H1L1H2L1, H1L2H2L2, and H1L2H2L1. Based on the distance between the mutation site and the opposite side and the surrounding amino acid environment, the appropriate paired charged mutations are designed. Through structural observation, some pairs of The CH1-CL interacting amino acids are used as sites for generating polar amino acid mutations, and consideration is given to introducing electrostatically charged amino acids such as positively charged arginine (R), histidine (H), and lysine (K), as well as negatively charged aspartic acid (D) and glutamic acid (E). Using the protein mutation energy calculation method in WeMol software, the energy difference generated by different pairings of the heavy and light chains on both sides in the presence of each mutation is obtained, thereby determining whether the mutation promotes correct pairing between CH1 and CL. The energy calculation formula is:

ΔΔG combination 1=ΔG H1-L1-ΔG H1-L2

ΔΔG combination 2=ΔG H2-L2-ΔG H2-L1

Refer to the values of ΔG H1-L1, ΔG H2-L2, ΔΔG combination 1, ΔΔG combination 2, ΔΔG combination 1 + ΔΔG combination 2, and ΔΔG combination 1 - ΔΔG combination 2 to rank the mutation designs and select mutation pairs with strong energy for experimental verification. Detailed design information is available in Table 2.

Table 2. Single point mutations that promote CH1 and CL pairing, as determined by energy calculations and structural observations

Example 3 Expression and Characterization of Bispecific Antibody Molecules Containing Single-Point Mutations in the CH1-CL Region

The 14 bispecific antibody constant region sequences with different CH1-CL mutations listed in Example 2 were used in combination with 5 pairs of bispecific antibody variable region sequences with different antigen combinations (A/B, C/D, E/F, G/H, I/J, where the letter combinations represent bispecific antibody combinations of the first antibody heavy and light chain variable regions against the first antigen and the second antibody heavy and light chain variable regions against the second antigen, respectively. The heavy and light chain variable region sequences of each antibody were different. For example, C/D represents the heavy and light chain variable regions of the anti-EGFR antigen from Zalutumumab (wherein The sequences of the bispecific antibodies are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively) and the heavy and light chain variable regions of Onartuzumab (whose sequences are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively) are combined, and the CH3 domains of the bispecific antibodies H1 and H2 are mutated with S354C, T366W and Y349C, T366S, L368A, Y407V, respectively, to prevent mispairing between the heavy chains. An expression vector was constructed, expressed in HEK293 cells, purified and separated using a Protein A column, the purity of the antibody was analyzed by SEC (Example 6), and the proportion of bispecific antibody molecules with different heavy and light chain pairings was analyzed by mass spectrometry (Example 7). The proportion of correctly paired bispecific antibodies, H1L1H2L2, within the main mass spectrometry peaks was calculated (Table 3). WT indicates that the bispecific antibody harbors only the S354C and T366W mutations in the CH3 domain of H1 and the Y349C, T366S, L368A, and Y407V mutations in the CH3 domain of H2 (the WT heavy chain constant region sequences are shown in SEQ ID NOs. 1-2, and the light chain constant region sequence is shown in SEQ ID NO. 3), with no amino acid mutations in other domains. Mutations 6 and 11 demonstrate good results across multiple bispecific antibody systems. Multiple paired mutations were combined to further optimize the effectiveness of correctly paired bispecific antibodies.

Table 3. Correct pairing results of bispecific antibodies containing CH1-CL single-point pairing mutations

Example 4 Combination mutations to improve correct pairing of CH1 and CL

The single-point pairing mutations experimentally verified to improve the correct pairing of CH1-CL in Example 3 were combined, and some new CH1-CL single-point pairing mutation amino acids were added to participate in the combination. The added CH1-CL single-point pairing mutation amino acids can be selected to introduce other non-electrostatic interactions (such as hydrophobic interactions, hydrogen bonds, aromatic stacking interactions) Mutation residues, such as tryptophan (T), asparagine (Q), leucine (L), etc. A detailed list of mutation combinations is shown in Table 4. Expression verification was performed.

Table 4. Combination mutations for improving correct pairing of CH1 and CL

Example 5 Expression and characterization of bispecific antibodies containing combined mutations

The 30 bispecific antibody constant region sequences with different CH1-CL single-point paired mutation combinations listed in Example 4 were used together with 5 pairs of bispecific antibody variable region sequences for different antigen combinations (A/B, C/D, E/F, G/H, I/J, where the letter combination represents the bispecific antibody combination of the heavy and light chain variable regions of the first antibody for the first antigen and the heavy and light chain variable regions of the second antibody for the second antigen, respectively. The heavy and light chain variable region sequences of each antibody are different. For example, C/D represents the heavy and light chain of Zalutumumab against EGFR antigen. The variable regions (whose sequences are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively) and the heavy and light chain variable regions of Onartuzumab (whose sequences are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively) of the anti-c-Met antigen were combined, and the CH3 domains of the H1 and H2 of the bispecific antibodies were mutated with S354C, T366W and Y349C, T366S, L368A, Y407V, respectively, to prevent mispairing between the heavy chains. An expression vector was constructed, expressed in HEK293 cells, purified and separated using a Protein A column, the purity of the antibody was analyzed by SEC (Example 6), and the proportion of bispecific antibody molecules with different heavy and light chain pairings was analyzed by mass spectrometry (Example 7). The percentage of correctly paired bispecific antibodies (H1L1H2L2) within the main mass spectrometry peaks was calculated. The results are shown in Table 5. WT indicates that only the CH3 domain of H1 harbors the S354C and T366W mutations, and the CH3 domain of H2 harbors the Y349C, T366S, L368A, and Y407V mutations, with no other amino acid mutations. Mutations 25, 30, 31, 34, 37, 38, 39, and 44 demonstrated excellent performance across multiple bispecific antibody systems. Mutation 30 achieved a correct pairing rate exceeding 90% across all bispecific antibody systems. Each bispecific antibody system had a mutation design that achieved 100% correct pairing. Swapping the constant regions on either side can also be employed to achieve even better pairing.

In addition to mutations in the CH1-CL interface residues, there are also sites between the antibody VH and VL that affect heavy-light chain binding. The variable region amino acid numbering follows the Kabat rule. For example, mutating Q39 of VH to E or K corresponds to mutating Q38 of VL to K or E. By combining bispecific antibodies containing 6, 8, 9, 12, 17, 18, and 19 mutation types with different variable regions carrying the above mutations, the mispairing of heavy and light chains can be significantly improved.

Table 5. Correct pairing results of bispecific antibodies containing CH1-CL combination mutations

Example 6. Construction of bispecific antibodies and their transient transfection expression in eukaryotic cells

The target gene fragments of the heavy chain and light chain sequences of the present invention verified by sequencing were cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid.

Expi293F ^™ cells (Thermo Fisher Scientific) were cultured in serum-free medium, seeded in shake flasks (Corning Inc.), and cultured on a shaker at 37°C in an 8% _CO2 environment. The cell density was adjusted, and the recombinant expression vector containing the target gene fragment and the PEI transfection reagent were mixed in appropriate proportions and added to the cell culture shake flask. After 6 days of cell culture, the expression supernatant was collected, the cell debris was removed by high-speed centrifugation, and affinity purification was performed using a Protein A column. The column was rinsed with PBS until the A280 reading dropped to the baseline. The target protein was eluted with an acidic eluent of pH 3.0-pH 3.5 and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample was appropriately concentrated, the solution was exchanged into PBS for aliquoting. The final purified antibody was subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination.

Example 7. Mass spectrometry analysis

The molecular weight of the antibody sample was determined using a Thermo Vanquish ultra-high performance liquid chromatograph in series with a Thermo QE Plus mass spectrometer. The bispecific antibody sample was diluted with Tris-HCl solution, and PNGase F enzyme (Merck, 11365177001) was added to remove N-sugars, and then the N-sugar-free intact molecular weight was determined. Data analysis was performed using Biopharma Finder software, and the measured molecular weight was compared with the theoretical molecular weight to match and confirm the peak components. The theoretical molecular weight was calculated by GPMAW software. If the difference between the measured molecular weight and the theoretical molecular weight was within 50 ppm, it was considered a match. This analysis confirmed the pairing results of the bispecific antibody molecules described in Examples 3 and 5.

The embodiments of the present disclosure described above are merely exemplary, and any person skilled in the art will recognize or be able to determine the equivalents of a variety of different specific compounds, materials, and operations without requiring undue experimentation. All such equivalents are within the scope of the present disclosure and are included in the appended claims.

Claims (16)

A multispecific antibody or antigen-binding fragment thereof, comprising: A. a first antibody that specifically binds to a first antigen, comprising a first heavy chain H1 and a first light chain L1; B. a second antibody that specifically binds to a second antigen, comprising a second heavy chain H2 and a second light chain L2; Among them, H1 and H2 contain the CH1 domain, L1 and L2 contain the CL domain, and there are amino acid mutations in the CH1 domain and the CL domain; Preferably, H1/L1 and/or H2/L2 contain mutated amino acid pairs in the CH1 and CL domains respectively contained therein; preferably, the mutated amino acid pairs are amino acid pairs that improve the correct pairing of the heavy and light chains; further preferably, the mutated amino acid pairs can increase the correct pairing rate of the heavy and light chains to more than 50%, or more than 60%, or more than 70%, or more than 80%, or more than 90%, or 100%. The multispecific antibody or antigen-binding fragment thereof according to claim 1, wherein H1/L1 and/or H2/L2 contain one or more of the following amino acid pairs mutated in their respective CH1 and CL domains: CH1: G26, CL: L28; CH1: G26, CL: F11; CH1: V68, CL: L28; CH1: G49, CL: N31; CH1: H51, CL: N30; CH 1: T70, CL: N31; CH1: K30, CL: S24; CH1: T22, CL: S7; CH1: P10, CL: S14; CH1: F53, CL: L28; CH1: T 70. CL: S7; CH1: T70, CL: N30; CH1: H51, CL: N31; CH1: H51, CL: S67; CH1: S7, CL: T22; CH1: S19, C L: V98; CH1: A24, CL: S7; CH1: A12, CL: S14; CH1: F9, CL: T22; CH1: F9, CL: S24; CH1: L28, CL: T71; The numbers are IMGT exon numbers. The multispecific antibody or antigen-binding fragment thereof according to claim 2, wherein the mutated amino acid pairs present in the CH1 and CL domains of H1/L1 and/or H2/L2, respectively, are amino acid pairs with opposite charges or amino acid pairs with non-electrostatic interactions; Preferably, the pair of amino acids with opposite charges is a positively charged amino acid and a negatively charged amino acid; further preferably, the positively charged amino acid is arginine (R), histidine (H) or lysine (K), and the negatively charged amino acid is aspartic acid (D) or glutamic acid (E). The multispecific antibody or antigen-binding fragment thereof according to claim 2 or 3, wherein the mutated amino acid pairs present in the CH1 and CL domains of H1/L1 and/or H2/L2, respectively, are selected from one or more of the following mutated amino acid pairs: CH1: G26A/Q, CL: L28F/Q; CH1: G26L/W, CL: F11W/L; CH1: V68S/V, CL: L28H/T; CH1: G49K/E, CL: N31E/K; CH1: H51D/K, CL: N30K/D; CH1: T70K/E, CL: N31E/K; CH1 : K30R/D, CL: S24D/R; CH1: T22K/D, CL: S7D/K; CH1: G26R/D, CL: L28D/R; CH1: P1 0K/D, CL: S14D/K; CH1: F53R/D, CL: L28D/R; CH1: T70D/K, CL: S7K/D; CH1: F9D/R , CL: T22R/D; CH1: T70E/H, CL: N30H/E; CH1: H51D/K, CL: N31K/D; CH1: H51K/E, C L: N30E/K; CH1: T70R/D, CL: N31D/R; CH1: S7D/K, CL: T22K/D; CH1: S19R/E, CL: V 98E/R; CH1: A24D/K, CL: S7K/D; CH1: H51T/Q, CL: S67Q/S; CH1: A12D/F, CL: S14K /G; CH1: P10Q/I, CL: S14E/I; CH1: F9E/T, CL: S24I/F; CH1: L28E/R, CL: T71H/F; The numbers are IMGT exon numbers. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 2 to 4, wherein the mutated amino acid pairs present in the CH1 and CL domains of H1/L1 and H2/L2, respectively, are selected from any one of the following groups: (1) G26R of CH1 in H1, L28D of CL in L1, G26D of CH1 in H2, L28R of CL in L2; (2) P10K of CH1 in H1, S14D of CL in L1, P10D of CH1 in H2, S14K of CL in L2; (3) F53R of CH1 in H1, L28D of CL in L1, F53D of CH1 in H2, L28R of CL in L2; (4) T70D of CH1 in H1, S7K of CL in L1, T70K of CH1 in H2, S7D of CL in L2; (5) F9D of CH1 in H1, T22R of CL in L1, F9R of CH1 in H2, T22D of CL in L2; (6) T22K of CH1 in H1, S7D of CL in L1, T22D of CH1 in H2, S7K of CL in L2; (7) T70E of CH1 in H1, N30H of CL in L1, T70H of CH1 in H2, N30E of CL in L2; (8) H51D of CH1 in H1, N30K of CL in L1, H51K of CH1 in H2, N30D of CL in L2; (9) T70K of CH1 in H1, N31E of CL in L1, T70E of CH1 in H2, N31K of CL in L2; (10) H51D of CH1 in H1, N31K of CL in L1, H51K of CH1 in H2, N31D of CL in L2; (11) H51K of CH1 in H1, N30E of CL in L1, H51E of CH1 in H2, N30K of CL in L2; (12) T70R of CH1 in H1, N31D of CL in L1, T70D of CH1 in H2, N31R of CL in L2; (13) S7D of CH1 in H1, T22K of CL in L1, S7K of CH1 in H2, T22D of CL in L2; (14) S19R of CH1 in H1, V98E of CL in L1, S19E of CH1 in H2, V98R of CL in L2; (15) A24D and G49K of CH1 in H1, S7K and N31E of CL in L1, A24K and G49E of CH1 in H2, S7D and N31K of CL in L2; (16) A24D and H51T of CH1 in H1, S7K and S67Q of CL in L1, A24K and H51Q of CH1 in H2, and S7D of CL in L2; (17) A24D and K30R of CH1 in H1, S7K and S24D of CL in L1, A24K and K30D of CH1 in H2, S7D and S24R of CL in L2; (18) A24D and G49E of CH1 in H1, S7K and N31K of CL in L1, A24K and G49K of CH1 in H2, S7D and N31E of CL in L2; (19) F9E and T22K of CH1 in H1, S7D and S24I of CL in L1, F9T and T22D of CH1 in H2, S7K and S24F of CL in L2; (20) T22K and K30R of CH1 in H1, S7D and S24D of CL in L1, T22D and K30D of CH1 in H2, S7K and S24R of CL in L2; (21) K30R, G49K of CH1 in H1, S24D, N31E of CL in L1, K30D, G49E of CH1 in H2, S24R, N31K of CL in L2; (22) K30R and H51T of CH1 in H1, S24D and S67Q of CL in L1, K30D and H51Q of CH1 in H2, and S24R of CL in L2; (23) K30R and H51D of CH1 in H1, S24D and N30K of CL in L1, K30D and H51K of CH1 in H2, S24R and N30D of CL in L2; (24) K30R, T70K of CH1 in H1, S24D, N31E of CL in L1, K30D, T70E of CH1 in H2, S24R, N31K of CL in L2; (25) K30R and H51K of CH1 in H1, S24D and N30E of CL in L1, K30D and H51E of CH1 in H2, S24R and N30K of CL in L2; (26) L28E and H51D of CH1 in H1, N30K and T71H of CL in L1, L28R and H51K of CH1 in H2, N30D and T71F of CL in L2; (27) P10Q, K30R, H51D of CH1 in H1, S14E, S24D, N30K of CL in L1, P10I, K30D, H51K of CH1 in H2, S14I, S24R, N30D of CL in L2; (28) P10Q, T22K, K30R of CH1 in H1, S7D, S14E, S24D of CL in L1, P10I, T22D, K30D of CH1 in H2, S7K, S14I, S24R of CL in L2; (29) A12D, T22K, K30R of CH1 in H1, S7D, S14K, S24D of CL in L1, A12F, T22D, K30D of CH1 in H2, S14G, S7K, S24R of CL in L2; (30) A12D, H51D, K30R of CH1 in H1, S14K, N30K, S24D of CL in L1, A12F, K30D, H51K of CH1 in H2, S14G, S24R, N30D of CL in L2; (31) T22K, K30R, G49K of CH1 in H1, S7D, S24D, N31E of CL in L1, T22D, K30D, G49E of CH1 in H2, S7K, S24R, N31K of CL in L2; (32) T22K, K30R, H51T of CH1 in H1, S7D, S24D, S67Q of CL in L1, T22D, K30D, H51Q of CH1 in H2, S7K, S24R of CL in L2; (33) T22K, G26A, K30R of CH1 in H1, S7D, S24D, L28F of CL in L1, T22D, G26Q, K30D of CH1 in H2, S7K, S24R, L28Q of CL in L2; (34) G26A, K30R, T70K of CH1 in H1, S24D, L28F, N31E of CL in L1, G26Q, K30D, T70E of CH1 in H2, S24R, L28Q, N31K of CL in L2; (35) G26A, K30R, G49K of CH1 in H1, S24D, L28F, N31E of CL in L1, G26Q, K30D, G49E of CH1 in H2, S24R, L28Q, N31K of CL in L2; (36) G26A, K30R, H51D of CH1 in H1, N30K, S24D, L28F of CL in L1, G26Q, K30D, H51K of CH1 in H2, S24R, L28Q, N30D of CL in L2; (37) G26A, K30R, H51T of CH1 in H1, S24D, L28F, S67Q of CL in L1, G26Q, K30D, H51Q of CH1 in H2, S24R, L28Q of CL in L2; (38) G26L, K30R, H51D of CH1 in H1, F11W, S24D, N30K of CL in L1, G26W, K30D, H51K of CH1 in H2, F11L, S24R, N30D of CL in L2; (39) G26L, K30R, T70K of CH1 in H1, F11W, S24D, N31E of CL in L1, G26W, K30D, T70E of CH1 in H2, F11L, S24R, N31K of CL in L2; (40) G26L, K30R, G49K of CH1 in H1, F11W, S24D, N31E of CL in L1, G26W, K30D, G49E of CH1 in H2, F11L, S24R, N31K of CL in L2; (41) G26L, K30R, H51T of CH1 in H1, F11W, S24D, S67Q of CL in L1, G26W, K30D, H51Q of CH1 in H2, F11L, S24R of CL in L2; (42) T22K, G26L, K30R of CH1 in H1, S7D, F11W, S24D of CL in L1, T22D, G26W, K30D of CH1 in H2, S7K, F11L, S24R of CL in L2; (43) T22K, K30R, V68S of CH1 in H1, S7D, S24D, L28H of CL in L1, T22D, K30D of CH1 in H2, S7K, S24R, L28T of CL in L2; (44) K30R, G49K, V68S of CH1 in H1, S24D, N31E, L28H of CL in L1, K30D, G49E of CH1 in H2, S24R, L28T, N31K of CL in L2; The numbers are IMGT exon numbers. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that The two heavy chains H1 and H2 further comprise a VH domain and an Fc domain, respectively. Preferably, the Fc domain comprises a CH2 domain and a CH3 domain, wherein the VH domains respectively contain amino acid sequences targeting different antigenic epitopes; The two light chains L1 and L2 also contain VL domains, wherein the VL domains contain amino acid sequences that target different antigen epitopes; Preferably, the CH1 domain contained in the heavy chains H1 and H2 of the multispecific antibody or antigen-binding fragment thereof is derived from human IgG1, IgG2, IgG3 or IgG4, and the CL domain contained in the light chains L1 and L2 is derived from a κ light chain or a λ light chain; Preferably, the multispecific antibody or antigen-binding fragment thereof is humanized. The multispecific antibody or antigen-binding fragment thereof according to claim 6, characterized in that the VH domain in H1 and the VL domain in L1 respectively contain one or more mutant amino acids with opposite charges to promote preferential pairing of the H1 and L1 heavy and light chains, and/or the VH domain in H2 and the VL domain in L2 respectively contain one or more mutant amino acids with opposite charges to promote preferential pairing of the H2 and L2 heavy and light chains; Preferably, the VH domain in H1 and the VL domain in L1 have Q39E and Q38K substitution mutations, respectively, and the VH domain in H2 and the VL domain in L2 have Q39K and Q38E substitution mutations, respectively; The number is the Kabat number. The multispecific antibody or antigen-binding fragment thereof according to claim 6 or 7, wherein the CH3 domain in H1 and the CH3 domain in H2 comprise amino acid substitutions such that the Fc domain of H1 preferentially pairs with the Fc domain of H2; Preferably, the CH3 domain comprises a native non-cysteine to cysteine substitution; further preferably, H1 comprises an S354C mutation and H2 comprises a Y349C mutation; Preferably, the CH3 domain in H1 contains a knob mutation, and the CH3 domain in H2 contains a hole mutation; further preferably, the knob mutation is T366W, and the hole mutation is at least one of T366S, L368A, and Y407V; Preferably, the CH3 domain in H1 comprises S354C, T366W substitutions, and the CH3 domain in H2 comprises Y349C, T366S, L368A, Y407V substitutions; The number is the EU number. A nucleic acid comprising a nucleic acid molecule A encoding the first heavy chain H1 of the multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, a nucleic acid molecule B encoding the first light chain L1, a nucleic acid molecule C encoding the second heavy chain H2, and a nucleic acid molecule D encoding the second light chain L2. An expression vector or host cell comprising the nucleic acid of claim 9; The host cell is a eukaryotic cell or a prokaryotic cell; preferably a eukaryotic cell, more preferably a CHO cell or a HEK293 cell. A composition comprising the multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier and/or diluent and/or excipient. Use of the multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the nucleic acid according to claim 9, the expression vector or host cell according to claim 10, or the composition according to claim 11 in preparing a multispecific antibody-fusion protein chimera. Use of the nucleic acid according to claim 9, the expression vector or the host cell according to claim 10 in preparing a multispecific antibody or an antigen-binding fragment thereof. A method for preparing the multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, comprising: (1) transforming a host cell with the expression vector according to claim 10; (2) causing the host cell to express the multispecific antibody or antigen-binding fragment thereof. Use of the multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in the preparation of a medicament for treating a disease in a subject in need thereof. A method for treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.