Multi-specific antibody or antigen-binding fragment
Patent Information
- Application Number
- AU2025219353
- Authority / Receiving Office
- AU · AU
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-01-24
- Filing Date
- 2025-02-06
- Publication Date
- 2026-08-27
Smart Images
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Abstract
Description
RELATED APPLICATION 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 in their entirety for all purposes. TECHNICAL FIELD The present disclosure belongs to the field of biomedicine and relates to a multi-specific antibody or antigen-binding fragment, a composition, a method for preparing the same, and use thereof. BACKGROUND Bispecific antibodies are antibodies capable of simultaneously recognizing two different antigens or two different epitopes of the same antigen. This property enables bispecific antibodies to serve as targeting agents for both in vitro and in vivo immunodiagnosis and immunotherapy, holding great potential for a wide range of clinical applications. However, the production and development of bispecific antibodies are highly challenging. Traditionally, bispecific antibodies are prepared and obtained by hybridoma technology. Due to the random pairing of immunoglobulin heavy and light chains, a mixture of various antibody molecules is generated, such as heavy chain homodimerization and mismatched pairing of heavy and light chains, among which only one of the antibody molecules has the correct bispecific antibody structure. The presence of mismatched by-products significantly reduces the yield and necessitates complex purification methods to achieve product homogeneity. Therefore, it is essential to improve the efficiency of obtaining correct bispecific antibodies. The correct bispecific antibody molecules can be obtained by using several rational design strategies to reduce the mispairing of IgG heavy chains. One method is knobs-into-holes, which aims to modify the CH3-CH3 contact interface by introducing mutations into the CH3 domains of two different heavy chains, so that two different heavy chains are paired, thus avoiding heavy chain homodimerization. WO1998050431A2 discloses that an amino acid with a short side chain is used to replace the original amino acid in the CH3 domain of one heavy chain to produce a “hole”, and conversely, an amino acid with a large side chain is 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 having the “hole” structure and the “knob” structure preferentially pair with each other, and high-yield heterodimer formation is observed. However, in WO1998050431A2, two identical light chains are used to avoid heavy chain-light chain mispairing, and the use of a common light chain is impractical for the production of antibodies recognizing different antigens. Therefore, although this method resolves the problem of heavy chain homodimerization, it does not address the mispairing between the heavy and light chains from two antibodies. There are numerous methods to address the heavy chain / light chain mispairing in bispecific antibodies. WO2006106905A1 discloses enhancing the formation of bispecific antibodies by modulating the association at the VH-VL interface. Specifically, the binding between VH and VL is regulated by substituting amino acids present at the VH-VL interface with charged amino acids. For example, oppositely charged amino acid mutations are introduced at position Q39 in VH and position Q38 in VL. Nevertheless, this approach is still insufficient for the efficient production of the target bispecific antibodies. Appropriate optimization is still needed to further improve the specificity of heavy and light chain pairing, and reduce mismatched by-products to increase the yield of bispecific antibodies. SUMMARY OF THE INVENTION The present disclosure provides a multi-specific antibody or antigen-binding fragment thereof with improved heavy chain-light chain pairing specificity, as well as compositions, preparation methods, uses, and the like. By comprehensively considering various interactions among amino acids at the interaction interface of the heavy and light chains, such as electrostatic interaction, hydrophobic interaction, hydrogen bonding, and aromatic stacking, amino acid modifications are introduced at specific positions in the interaction interface of the heavy and light chains to improve the correct pairing of the heavy and light chains. The present disclosure provides a multi-specific 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; and B. a second antibody that specifically binds to a second antigen, comprising a second heavy chain H2 and a second light chain L2; wherein each of H1 and H2 comprises a CH1 domain, each of L1 and L2 comprises a CL domain, and amino acid mutations are present in the CH1 domains and the CL domains. In a preferred embodiment, mutated amino acid pairs are present in the CH1 and CL domains, respectively, of H1 and L1, and / or mutated amino acid pairs are present in the CH1 and CL domains, respectively, of H2 and L2. In a preferred embodiment, the mutated amino acid pairs are amino acid pairs that enhance the correct pairing of 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 by more than 50%, or more than 60%, or more than 70%, or more than 80%, or more than 90%, or 100%. In some embodiments, mutated amino acid pairs at one or more of the following positions are present in the CH1 and CL domains, respectively, of H1 and L1: CH1: G26, CL: L28; CH1: G26, CL: F11; CH1: V68, CL: L28; CH1: G49, CL: N31; CH1: H51, CL: N30; CH1: T70, CL: N31; CH1: K30, CL: S24; CH1: T22, CL: S7; CH1: P10, CL: S14; CH1: F53, CL: L28; CH1: T70, CL: S7; CH1: T70, CL: N30; CH1: H51, CL: N31; CH1: H51, CL: S67; CH1: S7, CL: T22; CH1: S19, CL: V98; CH1: A24, CL: S7; CH1: A12, CL: S14; CH1: F9, CL: T22; CH1: F9, CL: S24; CH1: L28, CL: T71; wherein the numbering is according to the IMGT exon numbering. In some embodiments, mutated amino acid pairs at one or more of the following positions are present in the CH1 and CL domains, respectively, of H2 and L2: CH1: G26, CL: L28; CH1: G26, CL: F11; CH1: V68, CL: L28; CH1: G49, CL: N31; CH1: H51, CL: N30; CH1: T70, CL: N31; CH1: K30, CL: S24; CH1: T22, CL: S7; CH1: P10, CL: S14; CH1: F53, CL: L28; CH1: T70, CL: S7; CH1: T70, CL: N30; CH1: H51, CL: N31; CH1: H51, CL: S67; CH1: S7, CL: T22; CH1: S19, CL: V98; CH1: A24, CL: S7; CH1: A12, CL: S14; CH1: F9, CL: T22; CH1: F9, CL: S24; CH1: L28, CL: T71; wherein the numbering is according to the IMGT exon numbering. Further, the mutated amino acid pairs present in the CH1 and CL domains, respectively, of H1 and L1, and / or of H2 and 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 a positively charged amino acid and a negatively charged amino acid; 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). Further, the mutated amino acid pairs present in the CH1 and CL domains, respectively, of H1 and L1, and / or of H2 and L2 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: P10K / 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, CL: N30E / K; CH1: T70R / D, CL: N31D / R; CH1: S7D / K, CL: T22K / D; CH1: S19R / E, CL: V98E / 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; wherein the numbering is according to the IMGT exon numbering. Further, the mutated amino acid pairs present in the CH1 and CL domains, respectively, of H1 and L1, and / or of H2 and L2 are those selected from any of the following groups: (1) G26R in the CH1 domain of H1, L28D in the CL domain of L1, G26D in the CH1 domain of H2, and L28R in the CL domain of L2; (2) P10K in the CH1 domain of H1, S14D in the CL domain of L1, P10D in the CH1 domain of H2, and S14K in the CL domain of L2; (3) F53R in the CH1 domain of H1, L28D in the CL domain of L1, F53D in the CH1 domain of H2, and L28R in the CL domain of L2; (4) T70D in the CH1 domain of H1, S7K in the CL domain of L1, T70K in the CH1 domain of H2, and S7D in the CL domain of L2; (5) F9D in the CH1 domain of H1, T22R in the CL domain of L1, F9R in the CH1 domain of H2, and T22D in the CL domain of L2; (6) T22K in the CH1 domain of H1, S7D in the CL domain of L1, T22D in the CH1 domain of H2, and S7K in the CL domain of L2; (7) T70E in the CH1 domain of H1, N30H in the CL domain of L1, T70H in the CH1 domain of H2, and N30E in the CL domain of L2; (8) H51D in the CH1 domain of H1, N30K in the CL domain of L1, H51K in the CH1 domain of H2, and N30D in the CL domain of L2; (9) T70K in the CH1 domain of H1, N31E in the CL domain of L1, T70E in the CH1 domain of H2, and N31K in the CL domain of L2; (10) H51D in the CH1 domain of H1, N31K in the CL domain of L1, H51K in the CH1 domain of H2, and N31D in the CL domain of L2; (11) H51K in the CH1 domain of H1, N30E in the CL domain of L1, H51E in the CH1 domain of H2, and N30K in the CL domain of L2; (12) T70R in the CH1 domain of H1, N31D in the CL domain of L1, T70D in the CH1 domain of H2, and N31R in the CL domain of L2; (13) S7D in the CH1 domain of H1, T22K in the CL domain of L1, S7K in the CH1 domain of H2, and T22D in the CL domain of L2; (14) S19R in the CH1 domain of H1, V98E in the CL domain of L1, S19E in the CH1 domain of H2, and V98R in the CL domain of L2; (15) A24D and G49K in the CH1 domain of H1, S7K and N31E in the CL domain of L1, A24K and G49E in the CH1 domain of H2, and S7D and N31K in the CL domain of L2; (16) A24D and H51T in the CH1 domain of H1, S7K and S67Q in the CL domain of L1, A24K and H51Q in the CH1 domain of H2, and S7D in the CL domain of L2; (17) A24D and K30R in the CH1 domain of H1, S7K and S24D in the CL domain of L1, A24K and K30D in the CH1 domain of H2, and S7D and S24R in the CL domain of L2; (18) A24D and G49E in the CH1 domain of H1, S7K and N31K in the CL domain of L1, A24K and G49K in the CH1 domain of H2, and S7D and N31E in the CL domain of L2; (19) F9E and T22K in the CH1 domain of H1, S7D and S24I in the CL domain of L1, F9T and T22D in the CH1 domain of H2, and S7K and S24F in the CL domain of L2; (20) T22K and K30R in the CH1 domain of H1, S7D and S24D in the CL domain of L1, T22D and K30D in the CH1 domain of H2, and S7K and S24R in the CL domain of L2; (21) K30R and G49K in the CH1 domain of H1, S24D and N31E in the CL domain of L1, K30D and G49E in the CH1 domain of H2, and S24R and N31K in the CL domain of L2; (22) K30R and H51T in the CH1 domain of H1, S24D and S67Q in the CL domain of L1, K30D and H51Q in the CH1 domain of H2, and S24R in the CL domain of L2; (23) K30R and H51D in the CH1 domain of H1, S24D and N30K in the CL domain of L1, K30D and H51K in the CH1 domain of H2, and S24R and N30D in the CL domain of L2; (24) K30R and T70K in the CH1 domain of H1, S24D and N31E in the CL domain of L1, K30D and T70E in the CH1 domain of H2, and S24R and N31K in the CL domain of L2; (25) K30R and H51K in the CH1 domain of H1, S24D and N30E in the CL domain of L1, K30D and H51E in the CH1 domain of H2, and S24R and N30K in the CL domain of L2; (26) L28E and H51D in the CH1 domain of H1, N30K and T71H in the CL domain of L1, L28R and H51K in the CH1 domain of H2, and N30D and T71F in the CL domain of L2; (27) P10Q, K30R and H51D in the CH1 domain of H1, S14E, S24D and N30K in the CL domain of L1, P10I, K30D and H51K in the CH1 domain of H2, and S14I, S24R and N30D in the CL domain of L2; (28) P10Q, T22K and K30R in the CH1 domain of H1, S7D, S14E and S24D in the CL domain of L1, P10I, T22D and K30D in the CH1 domain of H2, and S7K, S14I and S24R in the CL domain of L2; (29) A12D, T22K and K30R in the CH1 domain of H1, S7D, S14K and S24D in the CL domain of L1, A12F, T22D and K30D in the CH1 domain of H2, and S14G, S7K and S24R in the CL domain of L2; (30) A12D, H51D and K30R in the CH1 domain of H1, S14K, N30K and S24D in the CL domain of L1, A12F, K30D and H51K in the CH1 domain of H2, and S14G, S24R and N30D in the CL domain of L2; (31) T22K, K30R and G49K in the CH1 domain of H1, S7D, S24D and N31E in the CL domain of L1, T22D, K30D and G49E in the CH1 domain of H2, and S7K, S24R and N31K in the CL domain of L2; (32) T22K, K30R and H51T in the CH1 domain of H1, S7D, S24D and S67Q in the CL domain of L1, T22D, K30D and H51Q in the CH1 domain of H2, and S7K and S24R in the CL domain of L2; (33) T22K, G26A and K30R in the CH1 domain of H1, S7D, S24D and L28F in the CL domain of L1, T22D, G26Q and K30D in the CH1 domain of H2, and S7K, S24R and L28Q in the CL domain of L2; (34) G26A, K30R and T70K in the CH1 domain of H1, S24D, L28F and N31E in the CL domain of L1, G26Q, K30D and T70E in the CH1 domain of H2, and S24R, L28Q and N31K in the CL domain of L2; (35) G26A, K30R and G49K in the CH1 domain of H1, S24D, L28F and N31E in the CL domain of L1, G26Q, K30D and G49E in the CH1 domain of H2, and S24R, L28Q and N31K in the CL domain of L2; (36) G26A, K30R and H51D in the CH1 domain of H1, N30K, S24D and L28F in the CL domain of L1, G26Q, K30D and H51K in the CH1 domain of H2, and S24R, L28Q and N30D in the CL domain of L2; (37) G26A, K30R and H51T in the CH1 domain of H1, S24D, L28F and S67Q in the CL domain of L1, G26Q, K30D and H51Q in the CH1 domain of H2, and S24R and L28Q in the CL domain of L2; (38) G26L, K30R and H51D in the CH1 domain of H1, F11W, S24D and N30K in the CL domain of L1, G26W, K30D and H51K in the CH1 domain of H2, and F11L, S24R and N30D in the CL domain of L2; (39) G26L, K30R and T70K in the CH1 domain of H1, F11W, S24D and N31E in the CL domain of L1, G26W, K30D and T70E in the CH1 domain of H2, and F11L, S24R and N31K in the CL domain of L2; (40) G26L, K30R and G49K in the CH1 domain of H1, F11W, S24D and N31E in the CL domain of L1, G26W, K30D and G49E in the CH1 domain of H2, and F11L, S24R and N31K in the CL domain of L2; (41) G26L, K30R and H51T in the CH1 domain of H1, F11W, S24D and S67Q in the CL domain of L1, G26W, K30D and H51Q in the CH1 domain of H2, and F11L and S24R in the CL domain of L2; (42) T22K, G26L and K30R in the CH1 domain of H1, S7D, F11W and S24D in the CL domain of L1, T22D, G26W and K30D in the CH1 domain of H2, and S7K, F11L and S24R in the CL domain of L2; (43) T22K, K30R and V68S in the CH1 domain of H1, S7D, S24D and L28H in the CL domain of L1, T22D and K30D in the CH1 domain of H2, and S7K, S24R and L28T in the CL domain of L2; and (44) K30R, G49K and V68S in the CH1 domain of H1, S24D, N31E and L28H in the CL domain of L1, K30D and G49E in the CH1 domain of H2, and S24R, L28T and N31K in the CL domain of L2; wherein the numbering is according to the IMGT exon numbering. In some embodiments, the two heavy chains H1 and H2 in the multi-specific antibody or antigen-binding fragment thereof each further comprise a VH domain and an Fc domain (including a CH2 domain and a CH3 domain), wherein the VH domains each comprise an amino acid sequence targeting a different epitope. In some embodiments, the two light chains L1 and L2 in the multi-specific antibody or antigenbinding fragment thereof each further comprise a VL domain, wherein the VL domains each comprise an amino acid sequence targeting a different epitope. In some embodiments, the heavy chains H1 and H2 in the multi-specific antibody or antigenbinding fragment thereof comprise CH1 domains derived from IgG1, IgG2, IgG3, or IgG4, and the light chains L1 and L2 comprise CL domains derived from a kappa light chain or a lambda light chain. In some embodiments, the multi-specific antibody or antigen-binding fragment thereof is humanized. In some embodiments, the VH domain in H1 and the VL domain in L1 respectively comprise mutated amino acids bearing opposite charges to facilitate the preferential pairing of the heavy chain H1 with the light chain L1. In some embodiments, the VH domain in H2 and the VL domain in L2 respectively comprise mutated amino acids bearing opposite charges to facilitate the preferential pairing of the heavy chain H2 with the light chain L2. In a preferred embodiment, the VH domain in H1 and the VL domain in L1 respectively have substitution mutations of Q39E and Q38K, and / or the VH domain in H2 and the VL domain in L2 respectively have substitution mutations of Q39K and Q38E, wherein the numbering is according to the Kabat numbering. In some embodiments, 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 domains comprise a substitution of a naturally occurring non-cysteine residue to cysteine. Further preferably, H1 comprises the S354C mutation and H2 comprises the Y349C mutation. Preferably, the amino acid substitutions in the CH3 domains result in greater electrostatic complementarity. Preferably, the amino acid substitutions in the CH3 domains mean that the amino acid residues in the CH3 domain of H1 are replaced with one or more amino acid residues having a larger side chain volume, thereby producing a protrusion on the surface interacting with the CH3 domain of H2; and simultaneously, the amino acid residues in the CH3 domain of H2 are replaced with amino acid residues having a smaller side chain volume, thereby producing a cavity on the surface interacting with the CH3 domain of H1. Preferably, the protrusion is a knob mutation. Further preferably, the mutation producing the knob is T366W (wherein the numbering is according to the EU numbering). Preferably, the cavity is a hole mutation. Further preferably, the mutation producing the hole is at least one of T366S, L368A, and Y407V (wherein the numbering is according to the EU numbering). In a preferred embodiment, the CH3 domain of H1 comprises S354C and T366W substitutions (wherein the numbering is according to the EU numbering) and the CH3 domain of H2 comprises Y349C, T366S, L368A, and Y407V substitutions (wherein the numbering is according to the EU numbering). The present disclosure provides a nucleic acid, which comprises a nucleic acid molecule A encoding a first heavy chain H1 in the multi-specific antibody or antigenbinding fragment thereof disclosed herein, a nucleic acid molecule B encoding a first light chain L1 in the multi-specific antibody or antigen-binding fragment thereof disclosed herein, a nucleic acid molecule C encoding a second heavy chain H2 in the multi-specific antibody or antigen-binding fragment thereof disclosed herein, and a nucleic acid molecule D encoding a second light chain L2 in the multi-specific antibody or antigen-binding fragment thereof disclosed herein. The present disclosure provides an expression vector or a host cell comprising a nucleic acid encoding the multi-specific antibody or antigen-binding fragment thereof disclosed herein. In some embodiment, the host cell is a eukaryotic cell or a prokaryotic cell; and preferably, the host cell is a eukaryotic cell; and further preferably, the host cell is a CHO cell or HEK293 cell. The present disclosure provides a composition comprising the multi-specific antibody or antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier and / or diluent and / or excipient. The present disclosure provides use of the multi-specific antibody or antigenbinding fragment thereof, the nucleic acid, the expression vector, the host cell or the composition in the preparation of a multi-specific antibody-fusion protein chimera. The present disclosure provides use of the above nucleic acid, expression vector, or host cell in the preparation of a multi-specific antibody or antigen-binding fragment thereof. The present disclosure provides a method for preparing a multi-specific antibody or antigen-binding fragment thereof, which comprises: (1) transforming a host cell with the expression vector disclosed herein; and (2) allowing the host cell to express the multi-specific antibody or antigenbinding fragment thereof. The present disclosure provides use of the multi-specific antibody or antigenbinding fragment thereof disclosed herein 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, which comprises administering to the subject an effective amount of the multi-specific antibody or antigen-binding fragment thereof disclosed herein. In the present disclosure, by analyzing the amino acids at the interaction interface between the CH1 domains and the CL domains of an antibody, the amino acids that affect the pairing of CH1 with CL domains are mutated and modified, so as to increase the possibility of correctly obtaining a heterologous multi-specific antibody or antigen-binding fragment thereof, and greatly improve the correct pairing rate of heavy and light chains while reducing the homologous dimerization of the heavy chains or the incorrect pairing of the heavy and light chains. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the CH1-CL structure from the crystal structure 6Y1L. DESCRIPTION OF THE SEQUENCES SEQ ID NO.1 IgG1_knob_WT ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK SEQ ID NO.2 IgG1_hole_WT ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK SEQ ID NO.3 Kappa_WT RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC SEQ ID NO.4 Zalutumumab_VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLE WVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RDGITMVRGVMKDYFDYWGQGTLVTVSS SEQ ID NO.5 Zalutumumab_VL AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIY DASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKV EIK SEQ ID NO.6 Onartuzumab_VH EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLE WVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCAT YRSYVTPLDYWGQGTLVTVSS SEQ ID NO.7 Onartuzumab_VL DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGK APKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPW TFGQGTKVEIK DETAILED DESCRIPTION All publications, patents and patent applications mentioned in this specification are hereby incorporated by reference into this disclosure to the same extent as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference into this disclosure. Before the present disclosure is described in detail below, it is to be understood that the present disclosure is not limited to the specific methodologies, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used in the present disclosure is for the purpose of describing particular embodiments only and is 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 one of ordinary skill in the art to which the present disclosure belongs. The three-letter codes and single-letter codes for amino acids used in the present disclosure are as described in J. Biol. Chem, 243, p3558(1968). Terminology The terms “including”, “comprising”, “having” or other similar words should be construed as encompassing a whole item or a whole group of items, and may also encompass other whole items or whole groups of items, and are non-exclusive or open-ended descriptions. The terms “and / or” should be understood to include both individual and combined options. For example, when two elements are connected 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 “multi-specific antibody” or “antigen-binding fragment” refers to a polypeptide or polypeptide complex capable of binding two or more epitopes. It may be an intact antibody or an antibody fragment that does not constitute an intact antibody structure, and it may also be a product with antigen-specific binding ability formed by engineering an intact antibody or antibody fragment or a single chain thereof (e.g., linking other peptide segments, rearranging functional units, etc.). Examples include (i) a Fab fragment, which is a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) a F(ab’)2 fragment, which is a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region, (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) dsFv, which is an antigen-binding fragment formed by VH and VL via interchain disulfide bonds; (vi) diabodies, bispecific antibodies, and multi-specific antibodies comprising scFv fragment, dsFv fragment, Fab fragment, and the like. In some embodiments, the multi-specific 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. In some embodiments, the bispecific antibody comprises an intact antibody structure. The term “first antibody” refers to an antibody in a multi-specific antibody or antigen-binding fragment thereof that binds to one antigen epitope, comprising a first heavy chain H1 and a first light chain L1, H1 and L1 being capable of binding to one antigen epitope. The term “second antibody” refers to an antibody in a multi-specific antibody or antigen-binding fragment thereof that binds to another antigen epitope, comprising a second heavy chain H2 and a second light chain L2, H2 and L2 being capable of binding to another antigen epitope. The heavy and 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 are generic designations referring to antibodies that bind to different antigen epitopes, and do not denote the order of the antibodies in the multi-specific antibody or antigen-binding fragment thereof. They shall not be construed as designating a specific or particular portion of the multi-specific antibody or antigen-binding fragment thereof provided in the present disclosure. The mutated amino acids in the first antibody and the second antibody may be reversed, i.e., any mutated amino acid present in the first antibody may alternatively be present in the second antibody, and any mutated amino acid present in the second antibody may alternatively be present in the first antibody. The first antibody and the second antibody respectively comprise heavy chains H1 and H2 as well as light chains L1 and L2, each of the heavy chains comprises a VH domain, a CH1 domain and an Fc domain (comprising a CH2 domain and a CH3 domain), wherein the VH domains each comprise an amino acid sequence targeting a different epitope; each of the light chains comprises a VL domain and a CL domain, wherein the VL domains each comprise an amino acid sequence targeting a different epitope. 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 may be derived from the heavy chain of human IgG1, IgG2, IgG3, or IgG4. The term “CL domain” refers to the constant region of a light chain. The CL domain may be derived from a human kappa chain or a human lambda chain. The CH1 and CL domains can be determined by conventional methods in the art. Antibody numbering is applied according to well-known numbering systems. The term “IMGT exon numbering” is used for the amino acid position numbering of the CH1 domain of a heavy chain and the CL domain of a light chain (IMGT Scientific chart). For example, the positions of amino acid substitutions in the CH1 domain are numbered with reference to the CH1 of human IgG1 (positions 1-98 of SEQ ID NO: 1 or 2), and the positions of amino acid substitutions in the CL domain are numbered with reference to the k light chain (SEQ ID NO: 3). The term “Kabat numbering” is used for amino acid position numbering of antibody variable regions (Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition). The term “EU numbering” is widely used for amino acid position numbering of constant regions (including the Fc domain) (Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition). The correspondence among various antibody numbering systems is convertible according to numbering conventions well known in the art. The term “amino acid pair” refers to a pair of amino acids. An “oppositely charged amino acid pair” refers to an amino acid pair in which one amino acid is positively charged and the other negatively charged. A pair of positively charged and negatively charged amino acids can be introduced by respectively substituting native amino acid residues in the CH1 domain of a heavy chain and the CL domain of a light chain. Positively charged amino acids generally include arginine (R), histidine (H), and lysine (K), and negatively charged amino acids generally include aspartic acid (D) and glutamic acid (E). The charge distribution after introducing the oppositely charged amino acid pairs into an antibody is as follows: H1(CH1 positively charged) / L1(CL negatively charged) / H2(CH1 negatively charged) / L2(CL positively charged), or H1(CH1 negatively charged) / L1(CL positively charged) / H2(CH1 positively charged) / L2(CL negatively charged). One or more amino acid pairs may be introduced into the first antibody and the second antibody, and the amino acid pairs introduced into the first antibody and the second antibody may be the same or different. One or more amino acid pairs may also be introduced into the interface of VH and VL in combination with one or more charge pairs introduced into the interface of CH1 / CL domain, and amino acids introduced into the same chain (H1, L1, H2 or L2) generally carry the same charge. Amino acid pairs may also be introduced by substituting naturally occurring amino acid residues in the heavy chain CH1 domain and the light chain CL domain based on other non-electrostatic interactions, such as hydrophobic interactions, hydrogen bonding, aromatic stacking interactions, and the like, in order to improve the correct pairing of the heavy and light chains. When describing amino acid mutations, “CH1: V68S / V, CL: L28H / T” means that V at position 68 (IMGT exon numbering) in CH1 is replaced with S, and simultaneously L at position 28 (IMGT exon numbering) in CL is replaced with H; or V is retained at position 68 (IMGT exon numbering) in CH1, and simultaneously L at position 28 (IMGT exon numbering) in CL is replaced with T. All other substitutions follow the same naming conventions. The term “expression vector” refers to a replicon into which a nucleic acid molecule can be operably inserted for the replication or expression of the nucleic acid molecule. The term “host cell” refers to a cell comprising a nucleic acid molecule in the present disclosure. A “host cell” may be any type of cell, such as primary cells, cultured cells or cells derived from cell lines, or eukaryotic or prokaryotic cells. Examples include CHO cells, HEK293 cells, or Escherichia coli or the like. The term “composition” refers to a product comprising an antibody of the present disclosure and a pharmaceutically acceptable carrier. The antibodies of the present disclosure and compositions comprising the same may also be used in the manufacture of a medicament for treatment as mentioned in the present 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 materials well known in the art for pharmaceutical formulations. The properties of the carrier, excipient, or diluent will depend on the route of administration for the particular application. The term “effective amount” refers to a dose of a pharmaceutical formulation comprising an active ingredient of the present disclosure that produces a desired effect in a treated subject after administration to the subject in a single dose or in multiple doses. The term “individual” or “subject” refer 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 monkeys, rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind. The term “treatment” refers to clinical intervention in an attempt to alter the course of a disease in an individual or caused by treated cells, which may be performed for prophylaxis or intervention during the clinical pathologic process. Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and relieving or improving prognosis. The term “disease” refers to any alteration or disorder that impairs or interferes with the normal function of cells, tissues, or organs. EXAMPLES The present disclosure will be further illustrated below in conjunction with specific examples. It is to be understood that these examples are merely used to illustrate the present disclosure and are not intended to limit the scope of the disclosure. Experimental methods for which specific conditions are not indicated in the following examples are generally carried out according to conventional conditions such as those described in J. sambrook et al.(ed.), Molecular Cloning Experimental Guide, 3rd edition, 5 Science Press, 2002, or in accordance with the conditions recommended by the manufacturer. Example 1. Analysis of Interface Between CH1 and CL of IgG1 Antibody The IgG1 crystal structure (PDB ID: 6Y1L) was selected as a template, and 10 the CH1-CL portion therein was selected and used to fill in the missing structural portions, as shown in Fig. 1. The amino acids of the IgG1 CH1 (the sequence set forth in positions 1-98 of SEQ ID NO.1 or SEQ ID NO.2) and the kappa light chain CL (the sequence set forth in SEQ ID NO.3) were numbered according to the IMGT exon numbering. Amino acids with a distance range of 0-12 A centered on Ca atoms between 15 the CH1 and CL domains were scanned to obtain interaction sites amenable to amino acid mutation and modification. The summarized information was shown in Table 1. Table 1. Interaction sites at the interface between the IgG1 CH1 and kappa light chain CL Heavy chain position (IMGT EXON) Heavy chain position (Kabat numbering) Heavy chain amino acid Light chain position (IMGT EXON) Light chain position (Kabat numbering) Light chain amino acid Ca distance CP distance 7 120 S 20 127 S 9.06 6.61 7 120 S 22 129 T 10.48 7.95 7 120 S 24 131 S 12.35 10.69 7 120 S 21 128 G 11.70 10.76 9 122 F 14 121 S 7.05 5.42 9 122 F 17 124 Q 6.14 4.32 9 122 F 20 127 S 9.55 7.48 9 122 F 22 129 T 10.27 8.26 9 122 F 24 131 S 9.23 6.71 9 122 F 16 123 E 7.02 6.12 10 123 P 14 121 S 6.49 4.98 10 123 P 16 123 E 8.30 6.90 11 124 L 11 118 F 6.19 4.33 11 124 L 26 133 V 7.92 5.52 11 124 L 28 135 L 11.01 8.71 11 124 L 24 131 S 9.10 7.91 12 125 A 14 121 S 7.03 5.40 12 125 A 16 123 E 11.03 9.49 14 127 S 102 209 F 11.43 8.86 15 128 S 106 213 E 9.73 8.32 16 129 K 102 209 F 7.73 5.59 16 129 K 106 213 E 9.22 7.35 17 130 S 28 135 L 8.43 7.37 19 134 S 5 112 A 12.75 11.06 19 134 S 7 114 S 6.84 5.26 19 134 S 98 205 V 11.83 9.69 19 134 S 100 207 K 10.48 8.97 22 137 T 7 114 S 9.22 6.88 22 137 T 30 137 N 11.55 9.04 22 137 T 9 116 F 10.06 8.91 24 139 A 9 116 F 6.70 4.09 24 139 A 28 135 L 8.10 5.20 24 139 A 30 137 N 8.74 6.35 24 139 A 11 118 F 9.27 8.08 26 141 G 11 118 F 8.95 6.66 26 141 G 26 133 V 9.56 6.88 26 141 G 28 135 L 9.60 6.88 26 141 G 69 176 S 10.65 8.26 26 141 G 71 178 T 12.38 10.57 28 143 L 17 124 Q 9.97 7.78 28 143 L 24 131 S 8.60 5.56 28 143 L 26 133 V 8.43 6.10 28 143 L 71 178 T 9.06 6.88 28 143 L 69 176 S 10.32 9.25 28 143 L 14 121 S 10.17 8.34 30 145 K 17 124 Q 10.27 8.32 30 145 K 20 127 S 11.51 9.06 30 145 K 22 129 T 10.16 7.33 30 145 K 24 131 S 9.77 7.14 30 145 K 71 178 T 10.57 8.49 30 145 K 73 180 T 9.47 7.47 31 146 D 17 124 Q 11.92 10.90 47 167 T 62 169 K 12.65 11.21 48 168 S 62 169 K 10.85 9.93 49 169 G 30 137 N 8.85 7.57 49 169 G 31 138 N 8.76 7.66 51 172 H 30 137 N 8.42 6.68 51 172 H 57 164 T 7.89 5.76 51 172 H 67 174 S 7.55 4.62 51 172 H 31 138 N 9.54 8.47 51 172 H 60 167 D 9.84 8.80 51 172 H 65 172 T 11.42 9.94 53 174 F 55 162 S 6.62 4.52 53 174 F 69 176 S 7.19 4.53 53 174 F 28 135 L 10.55 9.12 53 174 F 67 174 S 8.42 7.48 56 177 V 55 162 S 7.10 5.49 56 177 V 71 178 T 10.26 8.13 56 177 V 73 180 T 12.20 10.09 56 177 V 69 176 S 10.77 9.28 58 179 Q 24 131 S 12.39 10.61 58 179 Q 73 180 T 8.66 6.12 58 179 Q 71 178 T 10.13 8.71 59 180 S 73 180 T 9.40 7.87 59 180 S 50 157 G 6.66 5.37 59 180 S 53 160 Q 9.35 8.16 60 182 S 22 129 T 11.85 9.27 60 182 S 73 180 T 9.71 7.51 62 184 L 73 180 T 11.91 10.03 64 186 S 53 160 Q 9.87 7.62 64 186 S 71 178 T 9.35 6.59 64 186 S 73 180 T 10.00 7.30 64 186 S 55 162 S 10.03 8.69 64 186 S 24 131 S 11.29 8.65 66 188 S 26 133 V 8.84 6.29 66 188 S 28 135 L 9.50 7.51 66 188 S 55 162 S 8.88 7.31 66 188 S 69 176 S 7.11 4.48 66 188 S 71 178 T 8.79 6.95 66 188 S 11 118 F 11.78 9.26 68 190 V 28 135 L 8.70 5.80 68 190 V 30 137 N 9.53 7.32 68 190 V 67 174 S 10.34 7.78 68 190 V --------1869---------- 176 S 9.22 6.99 68 190 V 11 118 F 11.52 9.51 70 192 T 7 114 S 9.22 6.57 70 192 T 9 116 F 9.90 8.16 70 192 T 28 135 L 10.17 8.35 70 192 T 30 137 N 8.03 5.19 70 192 T 31 138 N 9.79 7.45 96 221 K 16 123 E 11.88 9.52 Example 2. Exploration of Point Mutations for Enhancing Correct CH1-CL Pairing Using an Energy Calculation Method In a bispecific antibody, one heavy-light chain pair is designated as H1L1, and the other heavy-light chain pair is designated as H2L2. In the natural state, there are multiple heavy-light chain pairing patterns for a bispecific antibody. The correct pairing is H1L1H2L2, while incorrect pairing includes the following three patterns: H1L1H2L1, H1L2H2L2, and H1L2H2L1. Paired charge mutations were designed compatibly based on the distance between mutation sites and the opposite side, as well as the surrounding amino acid environment. By structural observation, certain CH1-CL interacting amino acids with a spacing of 7-12 A were selected as sites for generating polar amino acid mutations, with consideration given to introducing amino acids capable of electrostatic interactions, such as positively charged arginine (R), histidine (H), and lysine (K), and negatively charged aspartic acid (D) and glutamic acid (E). The energy difference generated by different heavy-light chain pairing on two respective sides in the presence of each mutation was obtained by the protein mutation energy calculation method of WeMol software, and then it was judged whether the mutation promoted the correct pairing of CH1 and CL. The energy calculation formula is as follows: △△G combination 1 = AG H1-L1- AG H1-L2 △△G combination 2 = AG H2-L2- AG H2-L1. The designed mutations were ranked by referring to the values of △G H1-L1, AG H2-L2, AAG combination 1, AAG combination 2, AAG combination 1 + AAG combination 2, AAG combination 1 - AAG combination 2 and the like, and the mutation pairs with strong energy were selected for experimental verification. Detailed design was shown in Table 2. Table 2. Single-point mutations promoting pairing of CH1 and CL obtained by energy calculation and structural observation Mol_ID CH1-CL Mutation H1 (Knob) L1 H2 (Hole) L2 1 G26R L28D G26D L28R 2 P10K S14D P10D S14K 3 F53R L28D F53D L28R 4 T70D S7K T70K S7D 5 F9D T22R F9R T22D 6 T22K S7D T22D S7K 7 T70E N30H T70H N30E 8 H51D N30K H51K N30D 9 T70K N31E T70E N31K 10 H51D N31K H51K N31D 11 H51K N30E H51E N30K 12 T70R N31D T70D N31R 13 S7D T22K S7K T22D 14 S19R V98E S19E V98R Example 3. Expression and Characterization of Bispecific Antibody Molecules Containing Single-Point Mutations in the CH1-CL Region Fourteen bispecific antibody constant region sequences with different CH1-CL mutations listed in Example 2 were combined with five pairs of bispecific antibody variable region sequences targeting different antigen combinations (A / B, C / D, E / F, G / H and I / J). The letter combinations each represent a bispecific antibody combination comprising the heavy and light chain variable regions of a first antibody directed against a first antigen and the heavy and light chain variable regions of a second antibody directed against a second antigen; and the heavy and light chain variable region sequences of each antibody are all different. For example, C / D represents a combination comprising the heavy and light chain variable regions of the anti-EGFR antibody from Zalutumumab (the sequences of which are as set forth in SEQ ID NO. 4 and SEQ ID NO. 5, respectively) and the heavy and light chain variable regions of the anti-c-Met antibody from Onartuzumab (the sequences of which are as set forth in SEQ ID NO. 6 and SEQ ID NO. 7, respectively). Meanwhile, the CH3 domains of heavy chains H1 and H2 of the bispecific antibodies were respectively subjected to S354C and T366W, and Y349C, T366S, L368A and Y407V mutations, to prevent heavy chain mispairing. Expression vectors were constructed and expressed in HEK293 cells. The products were purified and isolated by using a Protein A chromatography column, and antibody purity was analyzed by using SEC (Example 6), and the proportion of components in bispecific antibody molecules with different heavy and light chain pairing was analyzed by using mass spectrometry (Example 7). The proportion of the correctly paired pattern (i.e., H1L1H2L2) of the bispecific antibodies in the main peak of the mass spectrum was calculated (Table 3), where WT indicated that the bispecific antibodies had only S354C and T366W mutations in the CH3 domain of H1, and Y349C, T366S, L368A, and Y407V mutations in the CH3 domain of H2 (the heavy chain constant region sequences of WT were set forth in SEQ ID NOs.1-2, respectively, and the light chain constant region sequence of WT was set forth in SEQ ID NO.3), without amino acid mutations in the other domains. It can be seen that the mutations, such as Mol_ID 6 and 11 achieved a better effect in multiple bispecific antibody systems. Multiple paired mutations were combined to further optimize the correct pairing of bispecific antibodies. Table 3. Results of correct pairing of bispecific antibody molecules containing single-point pairing mutation in CH1-CL Mol_ID CH1-CL Mutation Percentage of correct pairing (H1+L1+H2+L2) H1 (Knob) L1 H2 (Hole) L2 A / B C / D E / F G / H I / J WT / / / / 73.3 93.7 21.3 30.5 23.4 1 G26R L28D G26D L28R / 100 / / / 2 P10K S14D P10D S14K 60 / / 49 23 3 F53R L28D F53D L28R 58.8 100 22.9 83.5 50.2 4 T70D S7K T70K S7D 75 93.2 21.8 47.7 / 5 F9D T22R F9R T22D 45.1 94.7 27.6 43.9 / 6 T22K S7D T22D S7K 73.2 94.5 35.9 52.3 40.5 7 T70E N30H T70H N30E 64.2 100 26.5 40.5 16.8 8 H51D N30K H51K N30D 86.5 100 26 56 16.6 9 T70K N31E T70E N31K 72.1 94.9 19.1 71.7 21.9 10 H51D N31K H51K N31D 70.3 100 29.2 52.5 15.3 11 H51K N30E H51E N30K 77.4 100 30.9 50.4 29.1 12 T70R N31D T70D N31R 58.1 100 26.4 57.6 42.4 13 S7D T22K S7K T22D 72.3 100 24.7 47.6 36.1 14 S19R V98E S19E V98R 63.4 / 18.1 / 38.4 Example 4. Combined Mutations for Enhancing the Correct Pairing of CH1 and CL The single-point pairing mutations that were experimentally validated in Example 3 to enhance the correct pairing of CH1-CL were combined, and some new amino acids with single-point pairing mutations in CH1-CL were introduced into the combinations. The added amino acids with single-point pairing mutations in CH1-CL can be selected from mutant residues introducing other non-electrostatic interactions (e.g. hydrophobic interactions, hydrogen bonding, and aromatic stacking), such as tryptophan (T), asparagine (Q), leucine (L) and the like. A detailed list of mutation combinations was shown in Table 4. The expression of bispecific antibodies was validated. Table 4. Combined mutations for enhancing the correct pairing of CH1 and CL Mol_ID CH1-CL Mutation H1 (Knob) L1 H2 (Hole) L2 15 A24D,G49K S7K,N31E A24K,G49E S7D,N31K 16 A24D,H51T S7K,S67Q A24K,H51Q S7D 17 A24D,K30R S7K,S24D A24K,K30D S7D,S24R 18 K30R,G49K S24D,N31E K30D,G49E S24R,N31K 19 K30R,H51T S24D,S67Q K30D,H51Q S24R 20 T22K,K30R S7D,S24D T22D,K30D S7K,S24R 21 K30R,H51D S24D,N30K K30D,H51K S24R,N30D 22 K30R,T70K S24D,N31E K30D,T70E S24R,N31K 23 K30R,H51K S24D,N30E K30D,H51E S24R,N30K 24 A24D,G49E S7K,N31K A24K,G49K S7D,N31E 25 T22K,K30R,G49K S7D,S24D,N31E T22D,K30D,G49E S7K,S24R,N31K 26 T22K,K30R,H51T S7D,S24D,S67Q T22D,K30D,H51Q S7K,S24R 27 A12D,T22K,K30R S7D,S14K,S24D A12F,T22D,K30D S14G,S7K,S24R 28 T22K,G26L,K30R S7D,F11W,S24D T22D,G26W,K30D S7K,F11L,S24R 29 P10Q,T22K,K30R S7D,S14E,S24D P10I,T22D,K30D S7K,S14I,S24R 30 T22K,G26A,K30R S7D,S24D,L28F T22D,G26Q,K30D S7K,S24R,L28Q 31 T22K,K30R,V68S S7D,S24D,L28H T22D,K30D S7K,S24R,L28T 32 F9E,T22K S7D,S24I F9T,T22D S7K,S24F 33 A12D,H51D,K30R S14K,N30K,S24D A12F,K30D,H51K S14G,S24R,N30D 34 G26L,K30R,H51D F11W,S24D,N30K G26W,K30D,H51K F11L,S24R,N30D 35 P10Q,K30R,H51D S14E,S24D,N30K P10I,K30D,H51K S14I,S24R,N30D 36 L28E,H51D N30K,T71H L28R,H51K N30D,T71F 37 G26A,K30R,H51D N30K,S24D,L28F G26Q,K30D,H51K S24R,L28Q,N30D 38 G26A,K30R,T70K S24D,L28F,N31E G26Q,K30D,T70E S24R,L28Q,N31K 39 G26A,K30R,G49K S24D,L28F,N31E G26Q,K30D,G49E S24R,L28Q,N31K 40 G26A,K30R,H51T S24D,L28F,S67Q G26Q,K30D,H51Q S24R,L28Q 41 G26L,K30R,T70K F11W,S24D,N31E G26W,K30D,T70E F11L,S24R,N31K 42 G26L,K30R,G49K F11W,S24D,N31E G26W,K30D,G49E F11L,S24R,N31K 43 G26L,K30R,H51T F11W,S24D,S67Q G26W,K30D,H51Q F11L,S24R 44 K30R,G49K,V68S S24D,N31E,L28H K30D,G49E S24R,L28T,N31K Example 5. Expression and Characterization of Bispecific Antibody Molecules Containing Combined Mutations Thirty bispecific antibody constant region sequences with different combinations of single-point pairing mutations in CH1-CL listed in Example 4 were combined with five pairs of bispecific antibody variable region sequences targeting different antigen combinations (A / B, C / D, E / F, G / H and I / J). The letter combinations each represent a bispecific antibody combination comprising the heavy and light chain variable regions of a first antibody directed against a first antigen and the heavy and light chain variable regions of a second antibody directed against a second antigen; and the heavy and light chain variable region sequences of each antibody are all different. For example, C / D represents a combination comprising the heavy and light chain variable regions of the anti-EGFR antibody from Zalutumumab (the sequences of which are as set forth in SEQ ID NO. 4 and SEQ ID NO. 5, respectively) and the heavy and light chain variable regions of the anti-c-Met antibody from Onartuzumab (the sequences of which are as set forth in SEQ ID NO. 6 and SEQ ID NO. 7, respectively). Meanwhile, the CH3 domains of heavy chains H1 and H2 of the bispecific antibodies were respectively subjected to S354C and T366W, and Y349C, T366S, L368A and Y407V mutations, to prevent heavy chain mispairing. Expression vectors were constructed and expressed in HEK293 cells. The products were purified and isolated by using a Protein A chromatography column, and antibody purity was analyzed by using SEC (Example 6), and the proportion of components in bispecific antibody molecules with different heavy and light chain pairing was analyzed by using mass spectrometry (Example 7). The proportion of the correctly paired pattern (i.e., H1L1H2L2) of the bispecific antibodies in the main peak of the mass spectrum was calculated, and the results were shown in Table 5. WT indicated that the bispecific antibodies had only S354C and T366W mutations in the CH3 domain of H1, and Y349C, T366S, L368A, and Y407V mutations in the CH3 domain of H2, without amino acid mutations in the other domains. It can be seen that the combined mutations, such as Mol_ID 25, 30, 31, 34, 37, 38, 39 and 44 achieved a better effect in multiple bispecific antibody systems, among which the correct pairing rate of Mol_ID30 in each bispecific antibody system was more than 90%. Each bispecific antibody system had a mutation design that achieved 100% correct pairing. The constant regions on both sides can also be exchanged to achieve better pairing effects. In addition to the interfacial residue mutations between CH1 and CL, there were also sites in VH and VL of an antibody that affect the association of heavy and light chains. The amino acid numbering of variable regions follows the Kabat rule. For example, Q39 in VH was mutated to E or K, and Q38 in the corresponding VL was mutated to K or E. The mismatching of heavy and light chains can be significantly improved by combining bispecific antibody molecules containing the mutation types of Mol_ID 6, 8, 9, 12, 17, 18 and 19 with different variable regions having the above mutations. Table 5. Results of correct pairing of bispecific antibody molecules containing CH1-CL combined mutations Mol_ ID Fv Mutation CH1-CL Mutation Str ategy Percentage of correct pairing (H1+L1+H2+L2) H1 L1 H2 L2 H1 (Knob) L1 H2 (Hole) L2 A / B C / D E / F G / H I / J 15 A24D,G49K S7K,N31E A24K,G49E S7D,N31K Combo Mutation 62.8 91.7 12.4 50.9 44.8 16 A24D,H51T S7K,S67Q A24K,H51Q S7D Combo Mutation 72.1 84.4 21.1 51.1 24.6 17 A24D,K30R S7K,S24D A24K,K30D S7D,S24R Combo Mutation 61.4 100 0 72.4 62.9 18 K30R,G49K S24D,N31E K30D,G49E S24R,N31K Combo Mutation 77.9 92.4 35.6 60.4 66.2 19 K30R,H51T S24D,S67Q K30D,H51Q S24R Combo Mutation 86.4 91.7 47.4 61.4 58.5 20 T22K,K30R S7D,S24D T22D,K30D S7K,S24R Combo Mutation 81.1 100 62 53.6 65.9 21 K30R,H51D S24D,N30K K30D,H51K S24R,N30D Combo Mutation 92.8 89.5 71.7 71.6 49.4 22 K30R,T70K S24D,N31E K30D,T70E S24R,N31K Combo Mutation 72.7 79.8 83.1 65.6 48.6 23 K30R,H51K S24D,N30E K30D,H51E S24R,N30K Combo Mutation 75.6 89.3 50.7 58.1 / 24 A24D,G49E S7K,N31K A24K,G49K S7D,N31E Combo Mutation 68.2 100 28.7 54.3 37.5 25 T22K,K30R,G49K S7D,S24D,N31E T22D,K30D,G49E S7K,S24R,N31K Combo Mutation 71.7 100 82.5 64 64.5 26 T22K,K30R,H51T S7D,S24D,S67Q T22D,K30D,H51Q S7K,S24R Combo Mutation 77.4 100 100 56.1 47.9 27 A12D,T22K,K30R S7D,S14K,S24D A12F,T22D,K30D S14G,S7K,S24R Combo Mutation 53.3 / 7.1 44.7 / 28 T22K,G26L,K30R S7D,F11W,S24D T22D,G26W,K30D S7K,F11L,S24R Combo Mutation 67.9 100 47.6 53.8 39.9 29 P10Q,T22K,K30R S7D,S14E,S24D P10I,T22D,K30D S7K,S14I,S24R Combo Mutation 86.1 100 28.1 68.3 47.6 30 T22K,G26A,K30R S7D,S24D,L28F T22D,G26Q,K30D S7K,S24R,L28Q Combo Mutation 90.3 100 94.5 93.6 100 31 T22K,K30R,V68S S7D,S24D,L28H T22D,K30D S7K,S24R,L28T Combo Mutation 69.8 100 72.1 76.8 78.7 32 F9E,T22K S7D,S24I F9T,T22D S7K,S24F Combo Mutation 41.7 100 7.7 54.1 45.9 33 A12D,H51D,K30R S14K,N30K,S24D A12F,K30D,H51K S14G,S24R,N30D Combo Mutation 78.6 92.6 24.2 62.5 34.9 34 G26L,K30R,H51D F11W,S24D,N30K G26W,K30D,H51K F11L,S24R,N30D Combo Mutation 100 100 64.6 84 51.7 35 P10Q,K30R,H51D S14E,S24D,N30K P10I,K30D,H51K S14I,S24R,N30D Combo Mutation 75.7 93.8 32.5 / 40.4 36 L28E,H51D N30K,T71H L28R,H51K N30D,T71F Combo Mutation 51.4 100 10.9 48.4 20.7 37 G26A,K30R,H51D N30K,S24D,L28F G26Q,K30D,H51K S24R,L28Q,N30D Combo Mutation 48.2 94.5 100 100 100 38 G26A,K30R,T70K S24D,L28F,N31E G26Q,K30D,T70E S24R,L28Q,N31K Combo Mutation 100 83.5 87.4 100 100 39 G26A,K30R,G49K S24D,L28F,N31E G26Q,K30D,G49E S24R,L28Q,N31K Combo Mutation 94.3 79.3 84.4 100 100 40 G26A,K30R,H51T S24D,L28F,S67Q G26Q,K30D,H51Q S24R,L28Q Combo Mutation 65.8 70.6 88.9 75.5 52 41 G26L,K30R,T70K F11W,S24D,N31E G26W,K30D,T70E F11L,S24R,N31K Combo Mutation 87.9 93.5 100 / 33.4 42 G26L,K30R,G49K F11W,S24D,N31E G26W,K30D,G49E F11L,S24R,N31K Combo Mutation 79.1 100 54.6 75.8 29.5 43 G26L,K30R,H51T F11W,S24D,S67Q G26W,K30D,H51Q F11L,S24R Combo Mutation 100 100 38.4 77 31.7 44 K30R,G49K,V68S S24D,N31E,L28H K30D,G49E S24R,L28T,N31K Combo Mutation 100 91.2 89.2 94.2 71.9 8 39E 38K 39K 38E H51D N30K H51K N30D Site Mutation + V region Mutation 100 19 39E 38K 39K 38E K30R,H51T S24D,S67Q K30D,H51Q S24R Site Mutation + V region Mutation 82.8 6 39K 38E 39E 38K T22K S7D T22D S7K Site Mutation + V region Mutation 60.7 19 39K 38E 39E 38K K30R,H51T S24D,S67Q K30D,H51Q S24R Site Mutation + V region Mutation 49.9 9 39E 38K 39K 38E T70K N31E T70E N31K Site Mutation + V region Mutation 69.7 17 39E 38K 39K 38E A24D,K30R S7K,S24D A24K,K30D S7D,S24R Site Mutation + V region Mutation 100 12 39K 38E 39E 38K T70R N31D T70D N31R Site Mutation + V region Mutation 65.3 18 39K 38E 39E 38K K30R,G49K S24D,N31E K30D,G49E S24R,N31K Site Mutation + V region Mutation 96 Example 6. Construction and Transient Transient Expression of Bispecific Antibodies in Eukaryotic Cells The gene fragments of interest of the heavy chain and light chain sequences of the present disclosure verified by sequencing were cloned into the pTT5 expression 10 vector to prepare transfection-grade expression plasmids. Expi293FTM cells (Thermo Fisher Scientific) were cultured in serum-free medium, inoculated in shake flasks (Corning Inc) and cultured on a shaker at 37°C in an 8% CO2 atmosphere. The cell density was adjusted, and the recombinant expression vectors containing the gene fragments of interest and PEI transfection reagents were 15 mixed in a proper ratio and added into a cell culture shake flask. After 6 days of cell culture, the expression supernatant was collected, and cell debris was removed by highspeed centrifugation. Affinity purification was performed using a Protein A column. The column was rinsed with PBS until the A280 reading dropped to the baseline level. The protein of interest was eluted with an acidic eluent (pH 3.0 to pH 3.5) and neutralized with 1M Tris-HCl (pH 8.0 to 9.0). After the eluted samples were appropriately concentrated, buffer-exchanged into PBS and aliquoted for later use. The final purified antibodies were subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination. Example 7. Mass Spectrometry Detection and Analysis The molecular weight of antibody samples was determined using a Thermo Vanquish ultra-high-performance liquid chromatograph in tandem with a Thermo QE Plus mass spectrometer. The bispecific antibody samples were diluted with Tris-HCl solution, treated with PNGase F (Merck, 11365177001) to remove N-glycans from the bispecific antibody samples, and then were subjected to intact molecular weight measurement after removing N-glycans. Data analysis was performed using Biopharma Finder software. The measured molecular weights were compared with the theoretical molecular weights to match and confirm the peak components. The theoretical molecular weights were calculated by GPMAW software. A difference within 50 ppm between the measured and theoretical molecular weights was regarded as a match. Through this analysis, the pairing results of the bispecific antibody molecules described in Examples 3 and 5 were confirmed. The embodiments of the present disclosure described above are exemplary only, and any person skilled in the art may recognize or ascertain numerous equivalents of the specific compounds, materials, and procedures described herein without undue experimentation. All such equivalents are within the scope of the present disclosure and are encompassed by the appended claims.
Claims
1. A multi-specific antibody or antigen-binding fragment thereof, which comprises:A. a first antibody that specifically binds to a first antigen, comprising a first heavy chain H1 and a first light chain L1; andB. a second antibody that specifically binds to a second antigen, comprising a second heavy chain H2 and a second light chain L2;wherein each of H1 and H2 comprises a CH1 domain, each of L1 and L2 comprises a CL domain, and amino acid mutations are present in the CH1 domains and the CL domains;preferably, mutated amino acid pairs are present in the CH1 and CL domains, respectively, of H1 and L1, and / or of H2 and L2;preferably, the mutated amino acid pairs are amino acid pairs that enhance the correct pairing of heavy and light chains; andfurther preferably, the mutated amino acid pairs increase the correct pairing rate of heavy and light chains by more than 50%, or more than 60%, or more than 70%, or more than 80%, or more than 90%, or 100%.
2. The multi-specific antibody or antigen-binding fragment thereof of claim 1, wherein mutated amino acid pairs at one or more of the following positions are present in the CH1 and CL domains, respectively, of H1 and L1, and / or of H2 and L2:CH1: G26, CL: L28; CH1: G26, CL: F11; CH1: V68, CL: L28; CH1: G49, CL: N31; CH1: H51, CL: N30; CH1: T70, CL: N31; CH1: K30, CL: S24; CH1: T22, CL: S7; CH1: P10, CL: S14; CH1: F53, CL: L28; CH1: T70, CL: S7; CH1: T70, CL: N30; CH1: H51, CL: N31; CH1: H51, CL: S67; CH1: S7, CL: T22; CH1: S19, CL: V98; CH1: A24, CL: S7; CH1: A12, CL: S14; CH1: F9, CL: T22; CH1: F9, CL: S24; CH1: L28, CL: T71;wherein the numbering is according to the IMGT exon numbering.
3. The multi-specific antibody or antigen-binding fragment thereof of claim 2, wherein the mutated amino acid pairs present in the CH1 and CL domains, respectively, of H1 and L1, and / or of H2 and L2 are amino acid pairs with opposite charges or aminoacid pairs with non-electrostatic interactions;preferably, the amino acid pairs with opposite charges are 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).
4. The multi-specific antibody or antigen-binding fragment thereof of claim 2 or 3, wherein the mutated amino acid pairs present in the CH1 and CL domains, respectively, of H1 and L1, and / or of H2 and L2 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: P10K / 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, CL: N30E / K; CH1: T70R / D, CL: N31D / R; CH1: S7D / K, CL: T22K / D; CH1: S19R / E, CL: V98E / 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;wherein the numbering is according to the IMGT exon numbering.
5. The multi-specific antibody or antigen-binding fragment thereof of any one of claims 2-4, wherein the mutated amino acid pairs present in the CH1 and CL domains, respectively, of H1 and L1, and / or of H2 and L2 are those selected from any of the following groups:(1) G26R in the CH1 domain of H1, L28D in the CL domain of L1, G26D in the CH1 domain of H2, and L28R in the CL domain of L2;(2) P10K in the CH1 domain of H1, S14D in the CL domain of L1, P10D in the CH1 domain of H2, and S14K in the CL domain of L2;(3) F53R in the CH1 domain of H1, L28D in the CL domain of L1, F53D in the CH1 domain of H2, and L28R in the CL domain of L2;(4) T70D in the CH1 domain of H1, S7K in the CL domain of L1, T70K in the CH1 domain of H2, and S7D in the CL domain of L2;(5) F9D in the CH1 domain of H1, T22R in the CL domain of L1, F9R in the CH1 domain of H2, and T22D in the CL domain of L2;(6) T22K in the CH1 domain of H1, S7D in the CL domain of L1, T22D in the CH1 domain of H2, and S7K in the CL domain of L2;(7) T70E in the CH1 domain of H1, N30H in the CL domain of L1, T70H in the CH1 domain of H2, and N30E in the CL domain of L2;(8) H51D in the CH1 domain of H1, N30K in the CL domain of L1, H51K in the CH1 domain of H2, and N30D in the CL domain of L2;(9) T70K in the CH1 domain of H1, N31E in the CL domain of L1, T70E in the CH1 domain of H2, and N31K in the CL domain of L2;(10) H51D in the CH1 domain of H1, N31K in the CL domain of L1, H51K in the CH1 domain of H2, and N31D in the CL domain of L2;(11) H51K in the CH1 domain of H1, N30E in the CL domain of L1, H51E in the CH1 domain of H2, and N30K in the CL domain of L2;(12) T70R in the CH1 domain of H1, N31D in the CL domain of L1, T70D in the CH1 domain of H2, and N31R in the CL domain of L2;(13) S7D in the CH1 domain of H1, T22K in the CL domain of L1, S7K in the CH1 domain of H2, and T22D in the CL domain of L2;(14) S19R in the CH1 domain of H1, V98E in the CL domain of L1, S19E in the CH1 domain of H2, and V98R in the CL domain of L2;(15) A24D and G49K in the CH1 domain of H1, S7K and N31E in the CL domain of L1, A24K and G49E in the CH1 domain of H2, and S7D and N31K in the CL domain of L2;(16) A24D and H51T in the CH1 domain of H1, S7K and S67Q in the CL domain of L1, A24K and H51Q in the CH1 domain of H2, and S7D in the CL domain of L2;(17) A24D and K30R in the CH1 domain of H1, S7K and S24D in the CL domain of L1, A24K and K30D in the CH1 domain of H2, and S7D and S24R in the CL domain of L2;(18) A24D and G49E in the CH1 domain of H1, S7K and N31K in the CL domain of L1, A24K and G49K in the CH1 domain of H2, and S7D and N31E in the CL domain of L2;(19) F9E and T22K in the CH1 domain of H1, S7D and S24I in the CL domainof L1, F9T and T22D in the CH1 domain of H2, and S7K and S24F in the CL domain of L2;(20) T22K and K30R in the CH1 domain of H1, S7D and S24D in the CL domain of L1, T22D and K30D in the CH1 domain of H2, and S7K and S24R in the CL domain of L2;(21) K30R and G49K in the CH1 domain of H1, S24D and N31E in the CL domain of L1, K30D and G49E in the CH1 domain of H2, and S24R and N31K in the CL domain of L2;(22) K30R and H51T in the CH1 domain of H1, S24D and S67Q in the CL domain of L1, K30D and H51Q in the CH1 domain of H2, and S24R in the CL domain of L2;(23) K30R and H51D in the CH1 domain of H1, S24D and N30K in the CL domain of L1, K30D and H51K in the CH1 domain of H2, and S24R and N30D in the CL domain of L2;(24) K30R and T70K in the CH1 domain of H1, S24D and N31E in the CL domain of L1, K30D and T70E in the CH1 domain of H2, and S24R and N31K in the CL domain of L2;(25) K30R and H51K in the CH1 domain of H1, S24D and N30E in the CL domain of L1, K30D and H51E in the CH1 domain of H2, and S24R and N30K in the CL domain of L2;(26) L28E and H51D in the CH1 domain of H1, N30K and T71H in the CL domain of L1, L28R and H51K in the CH1 domain of H2, and N30D and T71F in the CL domain of L2;(27) P10Q, K30R and H51D in the CH1 domain of H1, S14E, S24D and N30K in the CL domain of L1, P10I, K30D and H51K in the CH1 domain of H2, and S14I, S24R and N30D in the CL domain of L2;(28) P10Q, T22K and K30R in the CH1 domain of H1, S7D, S14E and S24D in the CL domain of L1, P10I, T22D and K30D in the CH1 domain of H2, and S7K, S14I and S24R in the CL domain of L2;(29) A12D, T22K and K30R in the CH1 domain of H1, S7D, S14K and S24D in the CL domain of L1, A12F, T22D and K30D in the CH1 domain of H2, and S14G, S7K and S24R in the CL domain of L2;(30) A12D, H51D and K30R in the CH1 domain of H1, S14K, N30K andS24D in the CL domain of L1, A12F, K30D and H51K in the CH1 domain of H2, and S14G, S24R and N30D in the CL domain of L2;(31) T22K, K30R and G49K in the CH1 domain of H1, S7D, S24D and N31E in the CL domain of L1, T22D, K30D and G49E in the CH1 domain of H2, and S7K, S24R and N31K in the CL domain of L2;(32) T22K, K30R and H51T in the CH1 domain of H1, S7D, S24D and S67Q in the CL domain of L1, T22D, K30D and H51Q in the CH1 domain of H2, and S7K and S24R in the CL domain of L2;(33) T22K, G26A and K30R in the CH1 domain of H1, S7D, S24D and L28F in the CL domain of L1, T22D, G26Q and K30D in the CH1 domain of H2, and S7K, S24R and L28Q in the CL domain of L2;(34) G26A, K30R and T70K in the CH1 domain of H1, S24D, L28F and N31E in the CL domain of L1, G26Q, K30D and T70E in the CH1 domain of H2, and S24R, L28Q and N31K in the CL domain of L2;(35) G26A, K30R and G49K in the CH1 domain of H1, S24D, L28F and N31E in the CL domain of L1, G26Q, K30D and G49E in the CH1 domain of H2, and S24R, L28Q and N31K in the CL domain of L2;(36) G26A, K30R and H51D in the CH1 domain of H1, N30K, S24D and L28F in the CL domain of L1, G26Q, K30D and H51K in the CH1 domain of H2, and S24R, L28Q and N30D in the CL domain of L2;(37) G26A, K30R and H51T in the CH1 domain of H1, S24D, L28F and S67Q in the CL domain of L1, G26Q, K30D and H51Q in the CH1 domain of H2, and S24R and L28Q in the CL domain of L2;(38) G26L, K30R and H51D in the CH1 domain of H1, F11W, S24D and N30K in the CL domain of L1, G26W, K30D and H51K in the CH1 domain of H2, and F11L, S24R and N30D in the CL domain of L2;(39) G26L, K30R and T70K in the CH1 domain of H1, F11W, S24D and N31E in the CL domain of L1, G26W, K30D and T70E in the CH1 domain of H2, and F11L, S24R and N31K in the CL domain of L2;(40) G26L, K30R and G49K in the CH1 domain of H1, F11W, S24D and N31E in the CL domain of L1, G26W, K30D and G49E in the CH1 domain of H2, and F11L, S24R and N31K in the CL domain of L2;(41) G26L, K30R and H51T in the CH1 domain of H1, F11W, S24D and S67Qin the CL domain of L1, G26W, K30D and H51Q in the CH1 domain of H2, and F11L and S24R in the CL domain of L2;(42) T22K, G26L and K30R in the CH1 domain of H1, S7D, F11W and S24D in the CL domain of L1, T22D, G26W and K30D in the CH1 domain of H2, and S7K, F11L and S24R in the CL domain of L2;(43) T22K, K30R and V68S in the CH1 domain of H1, S7D, S24D and L28H in the CL domain of L1, T22D and K30D in the CH1 domain of H2, and S7K, S24R and L28T in the CL domain of L2; and(44) K30R, G49K and V68S in the CH1 domain of H1, S24D, N31E and L28H in the CL domain of L1, K30D and G49E in the CH1 domain of H2, and S24R, L28T and N31K in the CL domain of L2;wherein the numbering is according to the IMGT exon numbering.
6. The multi-specific antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the two heavy chains H1 and H2 each further comprise a VH domain and an Fc domain; and preferably, the Fc domain comprises a CH2 domain and a CH3 domain, wherein the VH domains each comprise an amino acid sequence targeting a different epitope;the two light chains L1 and L2 each further comprise a VL domain, wherein the VL domains each comprise an amino acid sequence targeting a different epitope;preferably, the heavy chains H1 and H2 in the multi-specific antibody or antigen-binding fragment thereof comprise CH1 domains derived from IgG1, IgG2, IgG3, or IgG4, and the light chains L1 and L2 comprise CL domains derived from a kappa light chain or a lambda light chain;preferably, the multi-specific antibody or antigen-binding fragment thereof is humanized.
7. The multi-specific antibody or antigen-binding fragment thereof of claim 6, wherein the VH domain in H1 and the VL domain in L1 respectively comprise one or more mutated amino acids with opposite charges to facilitate the preferential pairing of the heavy chain H1 and the light chain L1, and / or the VH domain in H2 and the VL domain in L2 respectively comprise one or more mutated amino acids with opposite charges to facilitate the preferential pairing of the heavy chain H2 and the light chainL2;preferably, the VH domain in H1 and the VL domain in L1 have Q39E and Q38K substitutions, respectively, and the VH domain in H2 and the VL domain in L2 have Q39K and Q38E substitutions, respectively;wherein the numbering is according to the Kabat numbering.
8. The multi-specific antibody or antigen-binding fragment thereof of 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 domains comprise a substitution of a naturally occurring non-cysteine residue to cysteine residue; further preferably, H1 comprises the S354C mutation and H2 comprises the Y349C mutation;preferably, the CH3 domain in H1 comprises a knob mutation, and the CH3 domain in H2 comprises 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 and T366W substitutions, and the CH3 domain in H2 comprises Y349C, T366S, L368A, and Y407V substitutions;wherein the numbering is according to the EU numbering.
9. A nucleic acid, which comprises a nucleic acid molecule A encoding a first heavy chain H1, a nucleic acid molecule B encoding a first light chain L1, a nucleic acid molecule C encoding a second heavy chain H2 and a nucleic acid molecule D encoding a second light chain L2, of the multi-specific antibody or antigen-binding fragment thereof of any one of claims 1-8.
10. An expression vector or a host cell comprising the nucleic acid of claim 9, wherein the host cell is a eukaryotic cell or a prokaryotic cell; and preferably, the host cell is a eukaryotic cell; and further preferably, the host cell is a CHO cell or HEK293 cell.
11. A composition comprising the multi-specific antibody or antigen-binding fragment thereof of any one of claims 1-8, and a pharmaceutically acceptable carrierand / or diluent and / or excipient.
12. Use of the multi-specific antibody or antigen-binding fragment thereof of any one of claims 1-8, the nucleic acid of claim 9, the expression vector or host cell of claim 10 or a composition of claim 11 in the preparation of a multi-specific antibodyfusion protein chimera.
13. Use of the nucleic acid of claim 9, the expression vector or host cell of claim 10 in the preparation of a multi-specific antibody or antigen-binding fragment thereof.
14. A method for preparing the multi-specific antibody or antigen-binding fragment thereof of any one of claims 1-8, comprising:(1) transforming a host cell with the expression vector of claim 10; and(2) allowing the host cell to express the multi-specific antibody or antigenbinding fragment thereof.
15. Use of the multi-specific antibody or antigen-binding fragment thereof of any one of claims 1-8 in the preparation of a medicament for treating a disease in a subject in need thereof.
16. A method of treating a disease in a subject in need thereof comprising administering to the subject an effective amount of the multi-specific antibody or antigen-binding fragment thereof of any one of claims 1-8.