CN113121696A

CN113121696A - Bispecific antibody formed by Fab modification induction and preparation method and application thereof

Info

Publication number: CN113121696A
Application number: CN201911410305.9A
Authority: CN
Inventors: 周易
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2021-07-16
Also published as: WO2021136249A1

Abstract

The invention belongs to the field of antibody engineering, and provides a bispecific antibody formed by Fab modification induction, a preparation method and application thereof. According to the invention, various interactions between interface amino acids, such as electrostatic interaction, space interaction and the like are comprehensively considered, amino acid modification is introduced at a specific position of an interaction interface of the heavy and light chains of the antibody, and the connecting peptide between the heavy chain VH/CH1 and/or the connecting peptide between the light chain VL/CL is optimized, so that the correct pairing proportion of the heavy and light chains is increased to more than 99%, and the influence of the amino acid modification on the expression quantity of the mutant is remarkably reduced.

Description

Bispecific antibody formed by Fab modification induction and preparation method and application thereof

Technical Field

The invention belongs to the field of antibody engineering, and particularly relates to a bispecific antibody formed by Fab modification induction, a preparation method and application thereof.

Background

Bispecific antibodies have a variety of configurations, among which bispecific antibodies of the IgG class have similar structures, physicochemical properties, and Fc domain functions as normal antibodies. Typically, bispecific antibodies of the IgG type consist of two heavy chains of different amino acid sequence (i.e. heavy chain HC _ a against antigen a and heavy chain HC _ B against antigen B) and two light chains of different amino acid sequence (i.e. light chain LC _ a against antigen a and light chain LC _ B against antigen B). When 4 polypeptide chains are combined, homodimers and heterodimers are formed between the two heavy chains, and mismatches are formed between the light and heavy chains, thus resulting in 8 different combinations, only one of which is the desired target antibody molecule. The separation and purification of 8 molecules to obtain the target molecule is extremely inefficient and difficult.

Advances in the field of antibody engineering have led to significant advances in the production of bispecific antibodies of the IgG class. Most IgG type bispecific antibodies promote heterodimerization of the two heavy chains of the antibody by engineering the Fc region (Ridgway, Presta et al 1996; Carter 2001, US2010286374A1, CN106883297A, US20150307628A1), however, specific pairing of the light and heavy chains is more difficult due to the more complex interactions between the light and heavy chains. Specifically, it is desirable that, of the two heavy chains and the two light chains constituting the bispecific antibody, the light chain LC _ a specifically pairs with only the heavy chain HC _ a, but not with the heavy chain HC _ B, while the light chain LC _ B specifically pairs with only the heavy chain HC _ B, but not with the heavy chain HC _ a. CN 104968677A; WO2016172485a 2; WO 2014082179A; nat biotechnol.2014feb; 32(2) 191-8; protein Sci.2017Oct; 26(10) 2021-; protein Eng Des Sel.2017Sep 1; 30(9) 685-696; methods are disclosed how to correctly pair light and heavy chains, however there is still a need in the art to find suitable optimizations to further improve the specificity of light chain pairing, reduce mismatched by-products and the yield of bispecific antibodies.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a bispecific antibody formed by Fab modification induction and a preparation method and application thereof. The invention introduces amino acid modification at the specific position of the interaction interface of the light chain and the heavy chain of the antibody by comprehensively considering various interactions between the interface amino acids, such as electrostatic interaction, space interaction and the like, and introduces connecting peptide into the heavy chain and the light chain for optimization, so that the correct pairing proportion of the light chain and the heavy chain is improved to more than 99 percent, and the influence of the amino acid modification on the expression quantity of the mutant is obviously reduced.

A first aspect of the invention provides a bispecific antibody comprising: a heavy chain A capable of being combined with a specific antigen and a light chain a matched with the heavy chain A, and a heavy chain B capable of being combined with another specific antigen and a light chain B matched with the heavy chain B; heavy chain A and heavy chain B have antibody heavy chain variable region VH domain, antibody heavy chain constant region CH1 domain, CH2 domain, CH3 domain, light chain a and light chain B have antibody light chain variable region VL domain and light chain constant region CL domain; a connecting peptide is inserted between the VH domain of heavy chain A and the CH1 domain, and/or a connecting peptide is inserted between the VH domain of heavy chain B and the CH1 domain, and/or a connecting peptide is inserted between the VL domain of light chain a and the CL domain, and/or a connecting peptide is inserted between the VL domain of light chain B and the CL domain; the heavy chain a and light chain a, heavy chain B and light chain B have one or more of the following mutations compared to a wild-type human antibody: (a) q39 of the VH domain is mutated and Q38 of the VL domain is mutated; (b) l145 and/or L128 of the CH1 domain is mutated, and V133 of the CL domain is mutated; the positions of the above-mentioned amino acids are determined according to the EU index of KABAT numbering.

It will be appreciated that the linker peptide interposed between the VH domain of heavy chain a and the CH1 domain, the linker peptide interposed between the VH domain of heavy chain B and the CH1 domain, the linker peptide interposed between the VL domain of light chain a and the CL domain, and the linker peptide interposed between the VL domain of light chain B and the CL domain, if present, may all be the same, or partially the same, or different.

Preferably, the linker peptide is 1-4 amino acids in length. I.e., the linker peptide inserted between the VH domain and the CH1 domain of heavy chain a, if present, is 1-4 amino acids in length; the linker peptide inserted between the VH domain and the CH1 domain of heavy chain B, if present, is 1-4 amino acids in length; the linker peptide inserted between the VL domain and the CL domain of light chain a, if present, is 1-4 amino acids in length; the linker peptide inserted between the VL domain and the CL domain of light chain b, if present, is 1-4 amino acids in length.

Preferably, the linking peptide is selected from: g, GG, GS, SG, SS, GGG, GGS, GSG, SGG, GSS, SGS, SSG, SSS, GGGG, GGGS, GGSG, GSGG, SGGG, GGSS, SSGG, GSSG, GSGS, SGSG, SGGS, GSSS, SGSS, SSGS, SSSG, A, AA, AS, SA, SS, AAA, AAS, ASA, SAA, ASS, SAS, SSA, SSS, AAAA, AAAS, AASA, ASAA, SAAA, AASS, SSAA, ASSA, ASAS, SASA, ASSS, SASS, SSAS, SSSA, GA, AG, GGA, GAG, GAA, AGA, AAG, GGGA, GGAG, GAGG, AGGG, AAAA, AAGG, GAAG, AGGA, AGAA, AAGA, AAAG, or any combination of other amino acids.

Preferably, the heavy chain a and heavy chain B, light chain a and light chain B in the bispecific antibody of the invention contain mutations selected from the group consisting of: (1) the L145 of the heavy chain A is mutated into an amino acid with positive charge, the V133 of the light chain a is mutated into an amino acid with negative charge, the L145 of the heavy chain B is mutated into an amino acid with negative charge, and the V133 of the light chain B is mutated into an amino acid with positive charge; (2) the L128 mutation of the heavy chain A is amino acid with positive charge, the V133 mutation of the light chain a is amino acid with negative charge, the L128 mutation of the heavy chain B is amino acid with negative charge, and the V133 mutation of the light chain B is amino acid with positive charge; (3) the L145 of the heavy chain A is mutated into an amino acid with positive charge, the V133 of the light chain a is mutated into an amino acid with negative charge, the L128 of the heavy chain B is mutated into an amino acid with negative charge, and the V133 of the light chain B is mutated into an amino acid with positive charge; (4) the L128 mutation of the heavy chain A is an amino acid with positive charge, the V133 mutation of the light chain a is an amino acid with negative charge, the L145 mutation of the heavy chain B is an amino acid with negative charge, and the V133 mutation of the light chain B is an amino acid with positive charge; (5) the Q39 and L145 of the heavy chain A are mutated into amino acids with positive charges, the Q38 and V133 of the light chain a are mutated into amino acids with negative charges, the Q39 and L145 of the heavy chain B are mutated into amino acids with negative charges, and the Q38 and V133 of the light chain B are mutated into amino acids with positive charges; (6) the Q39 and L128 of the heavy chain A are mutated into amino acids with positive charges, the Q38 and V133 of the light chain a are mutated into amino acids with negative charges, the Q39 and L128 of the heavy chain B are mutated into amino acids with negative charges, and the Q38 and V133 of the light chain B are mutated into amino acids with positive charges; (7) the Q39 and L145 of the heavy chain A are mutated into amino acids with positive charges, the Q38 and V133 of the light chain a are mutated into amino acids with negative charges, the Q39 and L128 of the heavy chain B are mutated into amino acids with negative charges, and the Q38 and V133 of the light chain B are mutated into amino acids with positive charges; (8) the Q39 and L128 of the heavy chain A are mutated into amino acids with positive charges, the Q38 and V133 of the light chain a are mutated into amino acids with negative charges, the Q39 and L145 of the heavy chain B are mutated into amino acids with negative charges, and the Q38 and V133 of the light chain B are mutated into amino acids with positive charges.

Further preferably, the positively charged amino acid refers to K (lysine) or R (arginine), the negatively charged amino acid refers to D (aspartic acid) or E (glutamic acid), and the heavy chain a and heavy chain B, light chain a and light chain B contain mutations selected from the group consisting of: 1) heavy chain A: L145K or L145R, light chain a: V133D or V133E, and heavy chain B: L145D or L145E, light chain b: V133K or V133R; 2) heavy chain A: L128K or L128R, light chain a: V133D or V133E, and heavy chain B: L128D or L128E, light chain b: V133K or V133R; 3) heavy chain A: L145K or L145R, light chain a: V133D or V133E, and heavy chain B: L128D or L128E, light chain b: V133K or V133R; 4) heavy chain A: L128K or L128R, light chain a: V133D or V133E, and heavy chain B: L145D or L145E, light chain b: V133K or V133R; 5) heavy chain A: (Q39K or Q39R) + (L145K or L145R), light chain a: (Q38D or Q38E) + (V133D or V133E), and heavy chain B: (Q39D or Q39E) + (L145D or L145E), light chain b: (Q38K or Q38R) + (V133K or V133R); 6) heavy chain A: (Q39K or Q39R) + (L128K or L128R), light chain a: (Q38D or Q38E) + (V133D or V133E), and heavy chain B: (Q39D or Q39E) + (L128D or L128E), light chain b: (Q38K or Q38R) + (V133K or V133R); 7) heavy chain A: (Q39K or Q39R) + (L145K or L145R), light chain a: (Q38D or Q38E) + (V133D or V133E), and heavy chain B: (Q39D or Q39E) + (L128D or L128E), light chain b: (Q38K or Q38R) + (V133K or V133R); 8) heavy chain A: (Q39K or Q39R) + (L128K or L128R), light chain a: (Q38D or Q38E) + (V133D or V133E), and heavy chain B: (Q39D or Q39E) + (L145D or L145E), light chain b: (Q38K or Q38R) + (V133K or V133R).

Wherein Q39R is Gln39 replaced with arginine (R), Q39K is Gln39 replaced with lysine (K), Q39E is Gln39 replaced with glutamic acid (E), Q39D is Gln39 replaced with aspartic acid (D), Q38R is Gln38 replaced with arginine (R), Q38K is Gln38 replaced with lysine (K), Q38E is Gln38 replaced with glutamic acid (E), Q38D is Gln38 replaced with aspartic acid (D), L145R is leucine 145 replaced with arginine (R), L145K is leucine 145 replaced with lysine (K), L145E is leucine 145 replaced with glutamic acid (E), L145D is leucine 145 replaced with aspartic acid (D), L128R is leucine 128 replaced with arginine (R), L128K is lysine (K), L128 is leucine 128 replaced with leucine (E), L128 is leucine E) replaced with leucine (E), L128 is leucine 128D) replaced with aspartic acid (128D), and L D is leucine (D), V133R indicates that valine 133 was replaced with arginine (R), V133K indicates that valine 133 was replaced with lysine (K), V133E indicates that valine 133 was replaced with glutamic acid (E), and V133D indicates that valine 133 was replaced with aspartic acid (D).

Preferably, the CH3 domains of heavy chain a and heavy chain B are designated CH3_ a domain and CH3_ B domain, respectively, said CH3_ a and CH3_ B domains containing mutations that facilitate the formation of bispecific antibodies, compared to the wild-type human antibody heavy chain constant region CH3 domain, and are not limited to, for example, WO9627011, CN101198698B, CN102459346B, CN105051069A, US2016177364a1, US2010286374a1, CN106883297A, US20150307628a1, CN104968677A, Nat biotechnol.2014feb; 32(2):191-8. In an embodiment of the invention, one of the mutations relating to the amino acids at the positions shown below is thus advantageously used to form bispecific antibodies: (a1) CH3_ a domain: F405E + K409F + K370D, CH3_ B domain: S364R + E357S; (a2) CH3_ a domain: F405E + K409F + K370D + S354C, CH3_ B domain: S364R + E357S + Y349C; (a3) CH3_ a domain: F405E + K409F + K370D + Y349C, CH3_ B domain: S364R + E357S + S354C (b1) CH3_ a domain: F405E + K409F + K392D, CH3_ B domain: D399K; (b2) CH3_ a domain: F405E + K409F + K392D + S354C, CH3_ B domain: D399K + Y349C; (b3) CH3_ a domain: F405E + K409F + K392D + Y349C, CH3_ B domain: D399K + S354C; (c1) CH3_ a domain: F405E + K409F + K439D, CH3_ B domain: E356K + E357K; (c2) CH3_ a domain: F405E + K409F + K439D + S354C, CH3_ B domain: E356K + E357K + Y349C; (c3) CH3_ a domain: F405E + K409F + K439D + Y349C, CH3_ B domain: E356K + E357K + S354C; (d1) CH3_ a domain: F405E + K409F + L368D, CH3_ B domain: S364R; (d2) CH3_ a domain: F405E + K409F + L368D + S354C, CH3_ B domain: S364R + Y349C; (d3) CH3_ a domain: F405E + K409F + L368D + Y349C, CH3_ B domain: S364R + S354C; (e1) CH3_ a domain: F405E + K409F + L368D, CH3_ B domain: S364K; (e2) CH3_ a domain: F405E + K409F + L368D + S354C, CH3_ B domain: S364K + Y349C; (e3) CH3_ a domain: F405E + K409F + L368D + Y349C, CH3_ B domain: S364K + S354C; (f1) CH3_ a domain: F405E + K409F + K360E, CH3_ B domain: Q347R; (f2) CH3_ a domain: F405E + K409F + K360E + S354C, CH3_ B domain: Q347R + Y349C; (f3) CH3_ a domain: F405E + K409F + K360E + Y349C, CH3_ B domain: Q347R + S354C; (g1) CH3_ a domain: F405E + K409F + K370D + K360E, CH3_ B domain: S364R + E357S + Q347R; (g2) CH3_ a domain: F405E + K409F + K370D + K360E + S354C, CH3_ B domain: S364R + E357S + Q347R + Y349C; (g3) CH3_ a domain: F405E + K409F + K370D + K360E + Y349C, CH3_ B domain: S364R + E357S + Q347R + S354C.

Q347R refers to glutamine Gln347 substituted by arginine (R), Y349C refers to tyrosine Tyr349 substituted by cysteine (C), S354C refers to serine Ser354 substituted by cysteine (C), E356K refers to glutamic Glu356 substituted by lysine (K), E357K refers to glutamic Glu357 substituted by lysine (K), E357S refers to glutamic Glu357 substituted by serine (S), K360E refers to lysine Lys360 substituted by lysine (K), S364R refers to serine Ser364 substituted by arginine (R), S364K refers to serine Ser364 substituted by lysine (K), L36368 refers to leucine Leu368 substituted by aspartic acid (D), K370D refers to lysine Lys370 substituted by aspartic acid (D), K392D refers to lysine Lys392 substituted by aspartic acid (D), D K refers to aspartic acid 399 substituted by lysine (K), K439E refers to glutamic acid (E) 439, F405E indicates that phenylalanine Phe405 was replaced by glutamic acid (E), K409F indicates that lysine Lys409 was replaced by phenylalanine (F).

In a second aspect of the invention, there is provided a composition comprising: (1) a heterodimer according to the first aspect of the invention, and (2) a pharmaceutically acceptable carrier and/or diluent and/or excipient.

In a third aspect, the present invention provides a polynucleotide comprising: the nucleotide molecule a encoding the heavy chain a of the bispecific antibody according to the first aspect of the invention, the nucleotide molecule a encoding the light chain a of the bispecific antibody according to the first aspect of the invention, the nucleotide molecule B encoding the heavy chain B of the bispecific antibody according to the first aspect of the invention, the nucleotide molecule B encoding the light chain B of the bispecific antibody according to the first aspect of the invention.

A fourth aspect of the present invention provides a vector combination comprising: the vector combination is selected from two or more of a recombinant vector containing a nucleotide molecule A, a recombinant vector containing a nucleotide molecule a, a recombinant vector containing the nucleotide molecule A and the nucleotide molecule a, a recombinant vector containing the nucleotide molecule B, a recombinant vector containing a nucleotide molecule B and a recombinant vector containing the nucleotide molecule B and the nucleotide molecule B, and the vector combination contains the nucleotide molecule A, the nucleotide molecule a, the nucleotide molecule B and the nucleotide molecule B.

The expression vector used in each of the above recombinant vectors is a conventional expression vector in the art, and means an expression vector containing appropriate regulatory sequences, such as a promoter sequence, a terminator sequence, a polyadenylation sequence, an enhancer sequence, a marker gene and/or sequence, and other appropriate sequences. The expression vector may be a virus or a plasmid, such as a suitable phage or phagemid, for more technical details see for example Sambrook et al, Molecular Cloning: a Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, 1989. Many known techniques and Protocols for nucleic acid manipulation are described in Current Protocols in Molecular Biology, second edition, Ausubel et al. The expression vector of the present invention is preferably pDR1, pcDNA3.1(+), pcDNA3.1/ZEO (+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, more preferably pTT 5.

In a fifth aspect, the invention provides a recombinant host cell comprising said vector combination.

The original host cell of the recombinant host cell of the present invention may be any host cell that is conventional in the art, as long as it is sufficient that the recombinant vector is stably self-replicating and the nucleotide is efficiently expressed. Wherein said primary host cell may be a prokaryotic or eukaryotic expression cell, said host cell preferably comprising: COS, CHO (Chinese hamster Ovary), NS0, sf9, sf21, DH5 α, BL21(DE3) or TG1, more preferably e.coli TG1, BL21(DE3) cells (expressing single chain or Fab antibodies) or CHO-K1 cells (expressing full length IgG antibodies). The recombinant host cell of the present invention can be obtained by transforming the expression vector into a host cell. Wherein the transformation method is a transformation method conventional in the art, preferably a chemical transformation method, a thermal shock method or an electric transformation method.

Preferably, the original host cell of the recombinant host cell is a eukaryotic cell, and more preferably a CHO cell or 293E cell.

In a sixth aspect, the invention provides the use of a bispecific antibody according to the first aspect of the invention, a composition according to the second aspect of the invention, a polynucleotide according to the third aspect of the invention, a vector combination according to the fourth aspect of the invention, or a recombinant host cell according to the fifth aspect of the invention for the preparation of a bispecific antibody, a bispecific fusion protein, and an antibody-fusion protein chimera.

In a seventh aspect, the present invention provides a method for producing the bispecific antibody of the first aspect of the present invention, wherein the bispecific antibody is expressed using the recombinant host cell of the fifth aspect of the present invention.

In the present invention, the recombinant host cell comprises the nucleotide molecule a encoding the heavy chain a of the bispecific antibody according to the first aspect of the present invention, the nucleotide molecule a encoding the light chain a of the bispecific antibody according to the first aspect of the present invention, the nucleotide molecule B encoding the heavy chain B of the bispecific antibody according to the first aspect of the present invention, and the nucleotide molecule B encoding the light chain B of the bispecific antibody according to the first aspect of the present invention, and the bispecific antibody is obtained by expression in the recombinant host cell and recovery.

Wherein the bispecific antibody can be purified from the recombinant host cell by standard experimental means. For example, when the heterodimeric protein comprises an antibody Fc fragment, protein a may be used for purification. Purification methods include, but are not limited to, chromatographic techniques such as size exclusion, ion exchange, affinity chromatography, and ultrafiltration, or suitable combinations of the foregoing.

In the present invention, the molar ratio of the nucleotide molecule A, the nucleotide molecule a, the nucleotide molecule B and the nucleotide molecule B in the recombinant host cell is (1-3): (1-3): 1-3, such as 1:1:1, 1:1:1.5:1.5, 1:1:2:2, 1:1:2.5:2.5, 1:1:3:3, 3:3:1:1, 2.5:2.5:1:1, 2:2:1:1, or 1.5:1.5:1: 1.

In an embodiment of the invention wherein the light chain is selected from a kappa chain or a lambda chain, wherein the constant region is derived from IgG (e.g. IgG1, IgG2, IgG3, IgG4), IgA (e.g. IgA1, IgA2), IgD, IgE or IgM.

In the present invention, the CH1 and CL are derived from an antibody Fab fragment, preferably from a human antibody Fab fragment. In general, the CH1 and CL domains of a human antibody Fab fragment are derived from a wild-type human antibody Fab fragment. The human antibody Fab fragments of the invention also include individual amino acid changes to the wild-type human antibody Fab sequence, including, for example, certain amino acids mutated at glycosylation sites, or other nonsense mutations. In addition to the mutations mentioned in the present invention, it is also possible to include other mutations which do not affect the function of the Fab fragment of the antibody.

In the present invention, the CH3 is derived from an antibody Fc fragment, preferably from a human antibody Fc fragment. In general, the CH3 domain of a human antibody Fc fragment is derived from a wild-type human antibody Fc fragment. The wild-type human antibody Fc refers to an amino acid sequence present in the human population, although Fc fragments may have some slight differences among individuals. The human antibody Fc fragments of the invention also include individual amino acid changes to the wild-type human antibody Fc sequence, including, for example, certain amino acids mutated at glycosylation sites, or other nonsense mutations. For the CH3 and CH2 domains, it is possible to include, in addition to the mutations mentioned in the present invention, other mutations which do not affect the function of the antibody, in particular the Fc region.

In the present invention, the numbering of the amino acid positions is determined according to the position indexed by Kabat EU numbering. Those skilled in the art know that even if the amino acid sequence is changed in the above regions due to insertion or deletion of amino acids or other mutation, the position numbering of each amino acid corresponding to the standard sequence as determined by the Kabat EU numbering index remains unchanged. The EU index is described in Kabat et al, Sequences of Proteins of Immunological Interest, published Health Service 5 th edition, National Institutes of Health, Bethesda, Md. (1991).

The invention has the beneficial effects that:

the invention introduces amino acid modification at the specific position of the interaction interface of the light chain and the heavy chain of the antibody by comprehensively considering various interactions between interface amino acids, such as electrostatic interaction, space interaction and the like, optimizes the connecting peptide between the heavy chain VH/CH1 and/or the connecting peptide between the light chain VL/CL, improves the correct pairing proportion of the light chain and the heavy chain to more than 99 percent, and obviously reduces the influence of the amino acid modification on the expression quantity of the mutant, thereby improving the yield of the antibody and reducing the production cost.

Drawings

FIG. 1 shows the results of electrophoretic analysis of light and heavy chain pairs. 4-12% SDS-PAGE protein gel electrophoresis. Lanes from left to right are: molecular weight Standard of protein, A₂b、A₆b、B₁₂a、B₁₆a。

FIG. 2 shows that the binding activity of EGFR x HER2 bispecific antibody and antigen EGFR-ECD-Fc is detected by ELISA.

FIG. 3 shows that the binding activity of EGFR x HER2 bispecific antibody and antigen HER2-ECD-Fc is detected by ELISA.

FIG. 4 shows that the EGFR x HER2 bispecific antibody was detected by ELISA to bind to both antigens HER2-ECD-Fc and EGFR-ECD-Fc.

FIG. 5 shows the detection of the binding activity of EGFR x cMet bispecific antibody to the antigen EGFR-ECD-Fc by ELISA.

FIG. 6 shows the detection of the binding activity of EGFR x cMet bispecific antibody to antigen cMet-ECD-Fc by ELISA.

FIG. 7 shows ELISA detection of the binding activity of EGFR x cMet bispecific antibody with the antigens cMet-ECD-Fc and EGFR-ECD-Fc.

Detailed Description

The following examples and experimental examples are intended to further illustrate the present invention and should not be construed as limiting the present inventionAnd (5) preparing. The examples do not include a detailed description of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells^ndedition，Cold spring Harbor Laboratory Press.

The experimental materials and sources used in the following examples and the methods of formulating the experimental reagents are specifically described below.

1. Experimental materials:

293E cells: from the NRC biotechnology Research Institute.

2. Experimental reagent:

PBS: purchased from Biotechnology (Shanghai) Inc., cat # B548117.

Citric acid: purchased from the national pharmaceutical group chemical agents limited.

Prime star HS DNA polymerase: available from Takara corporation under the trade designation R010A.

Endotoxin-free plasmid macroextraction kit: purchased from TIANGEN, cat # DP 117.

3. An experimental instrument:

HiTrap MabSelectSuRe column: purchased from GE company.

AKTA-FPLC fast protein liquid chromatography system: purchased from GE company.

C1000 Touch Thermal Cycler PCR instrument: purchased from Bio-Rad.

Chemidoc MP gel imager: purchased from Bio-Rad.

A centrifuge: purchased from Eppendorf company.

G1600AX capillary electrophoresis apparatus: purchased from agilent.

MicroCal PEAQ-DSC microcalorimetry differential scanning calorimeter: purchased from marvens corporation.

Octet molecular interaction system: purchased from ForteBio corporation.

Xevo G2-XS Tof time-of-flight mass spectrum: purchased from Waters corporation.

Example 1 CH1/CL Point mutation design

When two different antibody molecules are co-expressed in a cell, the 4 polypeptide chains heavy chain a, light chain a, heavy chain B and light chain B will pair randomly to produce a bispecific antibody and a mismatch antibody. To reduce the occurrence of mismatches, it is desirable that the two heavy and two light chains that make up the bispecific antibody are those in which light chain a is specifically paired only with heavy chain a and not with heavy chain B, while light chain B is specifically paired only with heavy chain B and not with heavy chain a. There is therefore a need to engineer the antibody light and heavy chains. Table 1 lists the interacting amino acids at the CH1/CL interface of the antibody, and by mutating these amino acids it is possible to promote the correct pairing of the light and heavy chains of the bispecific antibody. The light chain of an antibody is selected from the group consisting of kappa and lambda chains, and the amino acids located at the interaction interface are highly conserved across the constant regions C κ and C λ of the light chain. Thus, while all mutations in the light chain of the antibody of the invention are made in the kappa chain, the same applies to the lambda chain.

TABLE 1 amino acids interacting at the CH1/CL interface

1.1 construction of recombinant antibodies with CH 1/Ck mutations

Heavy chain HC (SEQ ID NO: 1) and light chain LC (SEQ ID NO: 2) of anti-HER 2 antibody Trastuzumab were subcloned into mammalian cell expression vector pTT5 to obtain a recombinant expression vector for mammalian cell expression of Trastuzumab. The heavy chain (SEQ ID NO: 3) and light chain (SEQ ID NO: 4) of the anti-EGFR antibody Cetuximab were subcloned into the mammalian cell expression vector pTT5 to obtain a recombinant expression vector for mammalian cell expression of Cetuximab. Heavy chain (SEQ ID NO: 5) and light chain (SEQ ID NO: 6) of anti-IL17 antibody anti-IL17Ab were subcloned into mammalian cell expression vector pTT5 to obtain a recombinant expression vector for mammalian cell expression of anti-IL17 Ab. According to Table 1 of example 1, there was an interaction between Val (or called V) at position 133 in the EU numbering system of the antibody light chain and Leu (or called L) at position 128 in the EU numbering system and Leu at position 145 in the antibody heavy chain. The heavy chain L128 or L145 of Trastuzumab, Cetuximab and anti-IL17Ab and the light chain V133 were mutated by overlap PCR method, and finally mutant vectors for expression in mammalian cells corresponding to the mutants shown in Table 2 were obtained, respectively.

1.2 transient expression of Trastuzumab, Cetuximab and anti-IL17Ab wild type and mutant, and examination of the influence of different mutation combinations on antibody expression level

The expression vectors corresponding to the mutation combinations of step 1 were transfected into 293E cells in suspension culture with PEI, and the cotransformation ratio of the recombinant expression vectors for the heavy and light chains was 1:1. After culturing for 5-6 days, collecting transient expression culture supernatant, and detecting the expression quantity of the antibody by a Fortebio method by using an Fc capture method. As shown in Table 2, 1) all the wild-type and mutant antibodies were normally expressed, indicating that the introduction of oppositely charged amino acids into the heavy (L128 or L145) and light chain V133 did not affect the pairing of the light and heavy chains of the antibodies. 2) The expression level of the mutant Trastuzumab is obviously reduced compared with that of the wild type, and similar results are shown in Figure 1A and 1B in WO2016172485A 2. Specifically, WO2016172485a2 reports that when the heavy chain L128 or L145 of the anti-VEGF antibody Ranibizumab is mutated to a negatively charged amino acid such as D or E, while the light chain V133 is mutated to a positively charged amino acid such as R or K, the expression level of the mutant protein is significantly reduced. 3) However, this example shows that the expression level of the mutant is not reduced after the introduction of amino acids with opposite charges into the heavy chain (L128 or L145) and the light chain V133 of Cetuximab and anti-IL17 Ab. Thus, the introduction of oppositely charged amino acids in the heavy chain (L128 or L145) and light chain V133 of an antibody does not necessarily affect the expression of the antibody, and the results shown in Figure 1A and 1B in WO2016172485A2 are not generally regular.

TABLE 2 influence of CH1/CL mutations on antibody expression levels

Example 2 light and heavy chain pairing experiment

2.1VH/VL and CH1/CL combination of mutations

The introduction of mutations at the antibody VH/VL interface amino acids can facilitate the correct pairing of the light and heavy chains of the bispecific antibody. CN104968677A, WO2016172485a2 disclose that the introduction of oppositely charged amino acids into Q39 of the heavy chain and Q38 of the light chain of an antibody facilitates the correct pairing of the light and heavy chains of a bispecific antibody. In addition to example 1, this example further designed the combination of mutations shown in table 3 by selecting as templates the anti-EGFR antibody Panitumumab, the anti-HER 2 antibody Trastuzumab and the anti-cMet antibody Onartuzumab. Heavy chain HC (SEQ ID NO: 1) and light chain LC (SEQ ID NO: 2) of Trastuzumab, heavy chain HC (SEQ ID NO: 7) and light chain LC (SEQ ID NO: 8) of Onartuzumab, heavy chain HC (SEQ ID NO: 9) and light chain LC (SEQ ID NO: 10) of Panitumumab were subcloned into mammalian cell expression vector pTT5 to obtain a recombinant expression vector for mammalian cell expression. The heavy chain HC and light chain LC coding genes of Trastuzumab, Panitumumab and Onartuzumab are subjected to combined mutation by using an overlapping PCR method, and finally, recombinant expression vectors for expressing the mutants in mammalian cells are obtained respectively.

TABLE 3

2.2 introduction of linker peptide between antibodies VH/CH1 and or VL/CL, it is beneficial to increase expression level of mutant

Example 1 shows that antibody heavy (L128 or L145) and light chain V133 mutations introduce oppositely charged amino acids and do not necessarily affect antibody expression. The invention further creatively discovers that when the mutation of the heavy chain (L128 or L145) and the light chain V133 of the antibody introduces amino acids with opposite charges to cause the expression quantity to be obviously reduced, the introduction of the connecting peptide between the antibodies VH/CH1 and/or VL/CL is favorable for improving the expression level of the mutant. Specifically, a linker1 is inserted before Ala at position 118 in EU numbering of an antibody heavy chain, a linker2 is inserted before Arg at position 108 in EU numbering of an antibody light chain, and the

linkers

1 and 2 are linker peptides with a length of 0-4 amino acids, and linker peptide sequences of 2 amino acids and 4 amino acids, preferably GG and GGGS. Among them, linker1 is a2 amino acid linker peptide such as "GG" with the most obvious effect. Accordingly, mutants as shown in Table 4 were constructed. The expression vectors corresponding to the combination of mutations were transfected into 293E cells in suspension culture with PEI, and the recombinant expression vectors for the heavy and light chains were cotransferred in a ratio of 1:1. After culturing for 5-6 days, collecting transient expression culture supernatant, detecting the expression quantity of the antibody by Fortebio by using an Fc capture method, purifying the Protein by Protein A affinity chromatography, and measuring a Tm value by using a differential scanning calorimeter MicroCal PEAQ-DSC. The results are shown in table 4, 1) there was no significant difference in expression levels of combination P0 and combination P1, indicating that the linker peptide did not affect antibody expression; 2) connecting peptides are inserted between antibodies VH/CH1 and/or VL/CL, particularly connecting peptide GG is inserted between VH/CH1, so that the expression quantity of the mutant can be obviously improved; 3) the point mutation does not affect the thermal stability of Fab, and the insertion of the connecting peptide only affects the melting peak coupling of Fab and CH3, but does not affect the thermal stability of Fab.

TABLE 4 influence of VH/VL and CH1/CL mutation combinations on expression level, thermostability

Note: melting peak coupling of Fab and CH3

2.3 orthogonal experiments

In Table 3 of example 2, mutants incorporating the linker peptide "GG" between the antibodies VH/CH1 were all normally expressed, indicating that the light and heavy chains were correctly paired. Therefore, light and heavy chains of the combination P2, the combination P6, the combination P12 and the combination P16 are further selected for orthogonal experiments. Specifically, the heavy chains of the combination P2 and the combination P6 are positively charged by mutation, the light chains of the combination P12 and the combination P16 are negatively charged by mutation, and the light chains of the combination P12 and the combination P16 are positively charged, so that theoretically, repulsion of the same charge between the light chains of the combination P2 and the combination P6 and the heavy chains of the combination P12 and the combination P16 cannot be paired, and repulsion of the same charge between the light chains of the combination 12 and the combination P16 and the heavy chains of the combination P2 and the combination P6 cannot be paired.

As shown in Table 5, the heavy chain A of P2 and P6 was named heavy chain A₂And A₆The light chain is named as light chain a, and the heavy chains B of P12 and P16 are named as heavy chains B₁₂And B₁₆And the light chain is designated light chain b. To further investigate the effect of newly introduced mutations on the correct pairing of light and heavy chains, we transiently expressed A₂b and A₆B, and B₁₂a and B₁₆a, by comparing the Ab and Ba expression under the same transient condition, the tendency of correct pairing of light and heavy chains is examined. The expression vectors were transfected into 293E cells in suspension culture with PEI, and the recombinant expression vectors for the heavy and light chains were cotransferred in a ratio of 1:1. After culturing for 5-6 days, collecting transient expression culture supernatant, and purifying Protein by Protein A affinity chromatography. The results of SDS-PAGE are shown in FIG. 1. Judged from the molecular weight, A₂b、A₆b、B₁₂a、B₁₆a is about 100kD, and all are heavy chain dimers. Indicating that heavy chain a cannot pair with light chain B and heavy chain B cannot pair with light chain a. Thus, the light and heavy chains of combination P2, combination P6 and combination P12, combination P16 are completely orthogonal.

TABLE 5

Example 3 preparation of EGFR x HER2 bispecific antibody

3.1 light and heavy chain pairing experiment

To construct EGFR × HER2 bispecific antibodies, the anti-EGFR antibody combination P2, P3, P5, the anti-HER 2 antibody combination T4 of example 2 were randomly selected for light and heavy chain pairing experiments. To verify whether the light and heavy chains of the EGFR × HER2 bispecific antibody were perfectly matched correctly, the present example co-transfects the light chain of the EGFR antibody and the HER2 antibody with the heavy chain of the EGFR antibody as shown in table 7, and observes whether the light chain of the HER2 antibody would interfere with the correct matching of the EGFR antibody. The EGFR antibody and the HER2 antibody light chain were additionally co-transfected with the HER2 antibody heavy chain to see if the EGFR antibody light chain would interfere with the correct pairing of the HER2 antibody. Expression vectors were transfected with PEI into 293E cells in suspension culture at a: b ═ 1:1:1. After culturing for 5-6 days, collecting transient expression culture supernatant, and purifying Protein by Protein A affinity chromatography. The ratio of the two light chains was analyzed by reductive capillary gel electrophoresis. The results are shown in table 7, the light chain of the HER2 antibody T4 was able to pair properly with the heavy chain of T4 and was not interfered by the light chains of EGFR antibodies P2, P3, P5. The EGFR antibodies P2, P3 and P5 light chain can be correctly paired with the EGFR antibodies P2, P3 and P5 heavy chain, wherein the EGFR antibody P2 is minimally affected, and when the HER2 antibody light chain and the P2 antibody light chain are transfected in equal proportion, the pairing of the P2 antibody is completely undisturbed.

TABLE 7 EGFR × HER2 bispecific antibody light and heavy chain pairing experiments

Construction, expression and purification of EGFR x HER2 bispecific antibody molecules

EGFR antibody P2 and HER2 antibody T4 were selected to construct an EGFR × HER2 bispecific antibody. The introduction of a S364R + E357S + Y349C + I253N point mutation in the heavy chain CH3 domain of P2 and a F405E + K409F + K370D + S354C point mutation in the heavy chain CH3 domain of T4 promoted preferential heterodimer formation of the heavy chains of P2 and T4, as shown in table 8. Expression vectors were transfected into suspension cultured 293E cells with PEI in the ratio heavy chain a: light chain a: heavy chain B: and (3) culturing the light chain b-3: 3:1:1 for 5-6 days, collecting transient expression culture supernatant, and purifying the Protein by Protein A affinity chromatography. LC-MS analysis showed that no light heavy chain mismatches and heavy chain homodimers were detected and the bispecific antibody was about 100% pure.

TABLE 8 construction method of EGFR x HER2 bispecific antibody molecule

3.3 detection of EGFR x HER2 bispecific antibody binding antigen Activity by ELISA method

The recombinant EGFR-ECD-Fc protein was diluted to 3. mu.g/ml with the coating solution, and 50. mu.l/well was added to the microplate at 4 ℃ overnight. The plates were washed 3 times with PBST, 200. mu.l/well blocking solution was added, and after 1 hour at 37 ℃ the plates were washed 1 time with PBST for future use. The anti-EGFR XHER 2 bispecific antibody and the control antibody (EGFR antibody P2) were diluted to 100. mu.g/ml with a diluent, diluted 4-fold to form 12 concentration gradients (maximum concentration of 100000ng/ml and minimum concentration of 0.02ng/ml), and the blocked ELISA plates were added in sequence at 100. mu.l/well and left at 37 ℃ for 1 hour. The plates were washed 3 times with PBST, and HRP-labeled mouse anti-human Fab antibody was added and left at 37 ℃ for 30 minutes. After PBST washing for 3 times, the residual liquid drops are patted dry on absorbent paper, 100 mu l of TMB is added into each hole, and the plate is placed for 5 minutes in a dark place at room temperature (20 +/-5 ℃); adding stop solution into each hole to stop the reaction of the substrate, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism6, mapping and calculating EC₅₀. The experimental results are shown in fig. 2, the anti-EGFR × HER2 bispecific antibody and the positive control EGFR antibody P2 bind to EGFR-ECD with EC50 of 0.1952nM and 0.2073nM, respectively, and the affinities of the two are equivalent.

To test the binding capacity of the anti-EGFR x HER2 bispecific antibody to HER2, the recombinant HER2-ECD-Fc protein was diluted to 0.4. mu.g/ml with coating solution, and 50. mu.l/well was added to the plate, overnight at 4 ℃. The plates were washed 3 times with PBST, 200. mu.l/well blocking solution was added, and after 1 hour at 37 ℃ the plates were washed 1 time with PBST for future use. The anti-EGFR XHER 2 bispecific antibody and the control antibody (HER2 antibody T4) were diluted to 100. mu.g/ml with a diluent, and diluted 4-fold to form 12 concentration gradients (maximum concentration of 100000ng/ml and minimum concentration of 0.02ng/ml), and the blocked ELISA plates were added in sequence at 100. mu.l/well and left at 37 ℃ for 1 hour. The plates were washed 3 times with PBST, and HRP-labeled mouse anti-human Fab antibody was added and left at 37 ℃ for 30 minutes. After PBST washing for 3 times, the residual liquid drops are patted dry on absorbent paper, 100 mu l of TMB is added into each hole, and the plate is placed for 5 minutes in a dark place at room temperature (20 +/-5 ℃); add 50. mu.l of 2M H per well₂SO₄Stopping the substrate reaction by the stop solution, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism6, mapping and calculating EC₅₀. The experimental results are shown in fig. 3, and the anti-EGFR is preparedThe HER2 bispecific antibody and the positive control HER2 antibody T4 bound HER2-ECD with EC50 of 0.1758nM and 0.1924nM, respectively, and the anti-EGFR × HER2 bispecific antibody affinity was comparable to HER2 mab.

To test the ability of the anti-EGFR xHER 2 bispecific antibody to bind both HER2 and EGFR, the recombinant HER2-ECD-Fc protein was diluted to 0.4. mu.g/ml with coating solution, 50. mu.l/well was added to the plate, overnight at 4 ℃. The plates were washed 3 times with PBST, 200. mu.l/well blocking solution was added, and after 1 hour at 37 ℃ the plates were washed 1 time with PBST for future use. The anti-EGFR x HER2 bispecific antibody was diluted to 100. mu.g/ml with a diluent, diluted 4-fold to form 12 concentration gradients (maximum concentration of 100000ng/ml and minimum concentration of 0.02ng/ml), and the blocked ELISA plates were added sequentially at 100. mu.l/well and left at 37 ℃ for 1 hour. The plates were washed 3 times with PBST, EGFR-ECD-Fc-biotin was added at 150 ng/well, and left at 37 ℃ for 1 hour. After 3 PBST washes, HRP-labeled Streptavidin was added and left at 37 ℃ for 30 minutes. After PBST washing for 3 times, the residual liquid drops are patted dry on absorbent paper, 100 mu l of TMB is added into each hole, and the plate is placed for 5 minutes in a dark place at room temperature (20 +/-5 ℃); add 50. mu.l of 2M H per well₂SO₄Stopping the substrate reaction by the stop solution, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism6, mapping and calculating EC₅₀. Results of the experiments shown in figure 4, anti-EGFR × HER2 bispecific antibodies were able to bind EGFR and HER2 simultaneously with EC₅₀It was 0.08763 nM. Example 4 preparation of EGFR x cMet bispecific antibody

4.1 light and heavy chain pairing experiment

To construct EGFR × cMet bispecific antibodies, the anti-EGFR antibody combinations P2, P3, and anti-cMet antibody combinations O2, O3 of example 2 were randomly selected for screening. To verify whether the light and heavy chains of the EGFR × cMet bispecific antibody were perfectly matched correctly, this example co-transfects the EGFR antibody and the light chain of the cMet antibody and the heavy chain of the EGFR antibody as shown in table 9, and observes whether the light chain of the cMet antibody would interfere with the correct matching of the EGFR antibody. The EGFR antibody and the cMet antibody light chain were additionally co-transfected with the cMet antibody heavy chain and it was observed whether the EGFR antibody light chain would interfere with the correct pairing of the cMet antibody. Expression vectors were transfected with PEI into 293E cells in suspension culture at a: b ═ 1:1:1. After culturing for 5-6 days, collecting transient expression culture supernatant, and purifying Protein by Protein A affinity chromatography. The ratio of the two light chains was analyzed by reductive capillary gel electrophoresis. The results are shown in table 9, the light chain of cMet antibodies O2, O3 was fully paired correctly with the heavy chain of O2, O3 and was not interfered by the expression of the light chain of EGFR antibodies P2, P3. The EGFR antibodies P2 and P3 light chain can be correctly paired with the EGFR antibodies P2 and P3 heavy chain, wherein the EGFR antibody P2 is minimally affected, and when the cMet antibody light chain and the P2 antibody light chain are transfected in equal proportion, the pairing of the P2 antibody is completely undisturbed.

TABLE 9 EGFR × cMet bispecific antibody light and heavy chain pairing experiments

Construction, expression and purification of EGFR x cMet bispecific antibody molecules

EGFR antibody P2 and cMet antibody O2 were selected to construct an EGFR × cMet bispecific antibody. Introduction of S364R + E357S + Y349C + I253N point mutation in the heavy chain CH3 domain of P2 and F405E + K409F + K370D + S354C point mutation in the heavy chain CH3 domain of O2 promoted preferential heterodimer formation of the heavy chains of P2 and O2 as shown in table 10. Expression vectors were transfected into suspension cultured 293E cells with PEI in the ratio heavy chain a: light chain a: heavy chain B: and (3) culturing the light chain b ═ 2:2:1:1 for 5-6 days, collecting transient expression culture supernatant, and purifying the Protein by Protein A affinity chromatography. LC-MS analysis showed that no light heavy chain mismatches and heavy chain homodimers were detected and the bispecific antibody was about 100% pure.

TABLE 10 construction method of EGFR × cMet bispecific antibody molecule

4.3 detection of EGFR x cMet bispecific antibody binding antigen Activity by ELISA method

The recombinant EGFR-ECD-Fc protein was diluted to 3. mu.g/ml with the coating solution, and 50. mu.l/well was added to the microplate at 4 ℃ overnight. PBST wash plate 3 times, add 200 ul/well blocking solution, standing at 37 ℃ for 1 hour, and then washing the plate for 1 time by PBST for later use. The anti-EGFR x cMet bispecific antibody and the control antibody (EGFR antibody P2) were diluted to 100. mu.g/ml with a diluent, diluted 4-fold to form 12 concentration gradients (maximum concentration 100000ng/ml, minimum concentration 0.02ng/ml), and the blocked ELISA plates were added sequentially, 100. mu.l/well, and left at 37 ℃ for 1 hour. The plates were washed 3 times with PBST, and HRP-labeled mouse anti-human Fab antibody was added and left at 37 ℃ for 30 minutes. After PBST washing for 3 times, the residual liquid drops are patted dry on absorbent paper, 100 mu l of TMB is added into each hole, and the plate is placed for 5 minutes in a dark place at room temperature (20 +/-5 ℃); adding stop solution into each hole to stop the reaction of the substrate, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism6, mapping and calculating EC₅₀. The results are shown in FIG. 5, EC binding to EGFR-ECD by anti-EGFR x cMet bispecific antibody and positive control EGFR antibody P2₅₀0.2339nM and 0.2073nM, respectively, with comparable affinities.

To test the binding capacity of the anti-EGFR x cMet bispecific antibody to cMet, the recombinant cMet-ECD-Fc protein was diluted to 0.4. mu.g/ml with coating solution, 50. mu.l/well was added to the plate, overnight at 4 ℃. The plates were washed 3 times with PBST, 200. mu.l/well blocking solution was added, and after 1 hour at 37 ℃ the plates were washed 1 time with PBST for future use. The anti-EGFR x cMet bispecific antibody and the control antibody (cMet antibody O2) were diluted to 100. mu.g/ml with a diluent, diluted 4-fold to form 12 concentration gradients (maximum concentration 100000ng/ml, minimum concentration 0.02ng/ml), and the blocked ELISA plates were added sequentially, 100. mu.l/well, and left at 37 ℃ for 1 hour. The plates were washed 3 times with PBST, and HRP-labeled mouse anti-human Fab antibody was added and left at 37 ℃ for 30 minutes. After PBST washing for 3 times, the residual liquid drops are patted dry on absorbent paper, 100 mu l of TMB is added into each hole, and the plate is placed for 5 minutes in a dark place at room temperature (20 +/-5 ℃); add 50. mu.l of 2M H per well₂SO₄Stopping the substrate reaction by the stop solution, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism6, mapping and calculating EC₅₀. The results of the experiment are shown in FIG. 6, EC binding to cMet-ECD for anti-EGFR x cMet bispecific antibody and positive control cMet antibody O2₅₀The anti-EGFR x cMet bispecific antibody affinities were comparable to cMet mab at 0.8635nM and 0.5524nM, respectively.

To detect anti-EGFR x cMetAbility of bispecific antibody to bind to both cMet and EGFR simultaneously, recombinant cMet-ECD-Fc protein was diluted to 0.4 μ g/ml with coating solution, 50 μ l/well added to plate, overnight at 4 ℃. The plates were washed 3 times with PBST, 200. mu.l/well blocking solution was added, and after 1 hour at 37 ℃ the plates were washed 1 time with PBST for future use. The anti-EGFR x cMet bispecific antibody was diluted to 100. mu.g/ml with a diluent, diluted 4-fold to form 12 concentration gradients (maximum concentration of 100000ng/ml and minimum concentration of 0.02ng/ml), and the blocked ELISA plates were added sequentially at 100. mu.l/well and left at 37 ℃ for 1 hour. The plates were washed 3 times with PBST, EGFR-ECD-Fc-biotin was added at 150 ng/well, and left at 37 ℃ for 1 hour. After 3 PBST washes, HRP-labeled Streptavidin was added and left at 37 ℃ for 30 minutes. After PBST washing for 3 times, the residual liquid drops are patted dry on absorbent paper, 100 mu l of TMB is added into each hole, and the plate is placed for 5 minutes in a dark place at room temperature (20 +/-5 ℃); add 50. mu.l of 2M H per well₂SO₄Stopping the substrate reaction by the stop solution, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism6, mapping and calculating EC₅₀. The results are shown in FIG. 7, the anti-EGFR x cMet bispecific antibody can bind to EGFR and cMet simultaneously, and EC₅₀It was 0.3744 nM.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Sequence listing

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Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

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Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

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Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

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Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

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Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

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Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

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Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

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Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

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Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

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Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

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Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

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Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe

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Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

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Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

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Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

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Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

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Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

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Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

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Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

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Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

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Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

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Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

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Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

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Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

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Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

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Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

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Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

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Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

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Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

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Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

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65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 5

<211> 445

<212> PRT

<213> Artificial

<400> 5

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Asp Met Ser Trp Gly Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Phe Met Ser Ser Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Asp Arg Phe Gly Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 6

<211> 214

<212> PRT

<213> Artificial

<400> 6

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Thr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Arg

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 7

<211> 448

<212> PRT

<213> Artificial

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Met Ile Asp Pro Ser Asn Ser Asp Thr Arg Phe Asn Pro Asn Phe

50 55 60

Lys Asp Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Tyr Arg Ser Tyr Val Thr Pro Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 11

<211> 220

<212> PRT

<213> Artificial

<400> 11

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr

20 25 30

Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ala Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Arg Phe Asn Arg Gly Glu Cys

210 215 220

<210> 9

<211> 449

<212> PRT

<213> Artificial

<400> 9

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Ile Asp Thr Ser Lys Thr Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

85 90 95

Cys Val Arg Asp Arg Val Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 10

<211> 214

<212> PRT

<213> Artificial

<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln His Phe Asp His Leu Pro Leu

85 90 95

Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Arg

195 200 205

Phe Asn Arg Gly Glu Cys

210

Claims

1. A bispecific antibody, wherein said bispecific antibody comprises: a heavy chain A capable of being combined with a specific antigen and a light chain a matched with the heavy chain A, and a heavy chain B capable of being combined with another specific antigen and a light chain B matched with the heavy chain B;

heavy chain A and heavy chain B both have an antibody heavy chain variable region VH domain, an antibody heavy chain constant region CH1 domain, a CH2 domain, a CH3 domain, and light chain a and light chain B both have an antibody light chain variable region VL domain and a light chain constant region CL domain;

a connecting peptide is inserted between the VH domain of heavy chain A and the CH1 domain, and/or a connecting peptide is inserted between the VH domain of heavy chain B and the CH1 domain, and/or a connecting peptide is inserted between the VL domain of light chain a and the CL domain, and/or a connecting peptide is inserted between the VL domain of light chain B and the CL domain;

the heavy chain a and light chain a, heavy chain B and light chain B have one or more of the following mutations compared to a wild-type human antibody:

(a) q39 of the VH domain is mutated and Q38 of the VL domain is mutated;

(b) l145 and/or L128 of the CH1 domain is mutated, and V133 of the CL domain is mutated;

the positions of the above-mentioned amino acids are determined according to the EU index of KABAT numbering.

2. The bispecific antibody of claim 1, wherein the linker peptide is 1-4 amino acids in length, preferably: g, GG, GS, SG, SS, GGG, GGS, GSG, SGG, GSS, SGS, SSG, SSS, GGGG, GGGS, GGSG, GSGG, SGGG, GGSS, SSGG, GSSG, GSGS, SGSG, SGGS, GSSS, SGSS, SSGS, SSSG, A, AA, AS, SA, SS, AAA, AAS, ASA, SAA, ASS, SAS, SSA, SSS, AAAA, AAAS, AASA, ASAA, SAAA, AASS, SSAA, ASSA, ASAS, SASA, ASSS, SASS, SSAS, SSSA, GA, AG, GGA, GAG, GAA, AGA, AAG, GGGA, GGAG, GAGG, AGGG, AAAA, AAGG, GAAG, AGGA, AGAA, AAGA, AAAG, or any combination of other amino acids.

3. The bispecific antibody of claim 1, wherein the heavy chain a and heavy chain B, light chain a and light chain B comprise mutations selected from the group consisting of:

(1) the L145 of the heavy chain A is mutated into an amino acid with positive charge, the V133 of the light chain a is mutated into an amino acid with negative charge, the L145 of the heavy chain B is mutated into an amino acid with negative charge, and the V133 of the light chain B is mutated into an amino acid with positive charge;

(2) the L128 mutation of the heavy chain A is amino acid with positive charge, the V133 mutation of the light chain a is amino acid with negative charge, the L128 mutation of the heavy chain B is amino acid with negative charge, and the V133 mutation of the light chain B is amino acid with positive charge;

(3) the L145 of the heavy chain A is mutated into an amino acid with positive charge, the V133 of the light chain a is mutated into an amino acid with negative charge, the L128 of the heavy chain B is mutated into an amino acid with negative charge, and the V133 of the light chain B is mutated into an amino acid with positive charge;

(4) the L128 mutation of the heavy chain A is an amino acid with positive charge, the V133 mutation of the light chain a is an amino acid with negative charge, the L145 mutation of the heavy chain B is an amino acid with negative charge, and the V133 mutation of the light chain B is an amino acid with positive charge;

(5) the Q39 and L145 of the heavy chain A are mutated into amino acids with positive charges, the Q38 and V133 of the light chain a are mutated into amino acids with negative charges, the Q39 and L145 of the heavy chain B are mutated into amino acids with negative charges, and the Q38 and V133 of the light chain B are mutated into amino acids with positive charges;

(6) the Q39 and L128 of the heavy chain A are mutated into amino acids with positive charges, the Q38 and V133 of the light chain a are mutated into amino acids with negative charges, the Q39 and L128 of the heavy chain B are mutated into amino acids with negative charges, and the Q38 and V133 of the light chain B are mutated into amino acids with positive charges;

(7) the Q39 and L145 of the heavy chain A are mutated into amino acids with positive charges, the Q38 and V133 of the light chain a are mutated into amino acids with negative charges, the Q39 and L128 of the heavy chain B are mutated into amino acids with negative charges, and the Q38 and V133 of the light chain B are mutated into amino acids with positive charges;

(8) the Q39 and L128 of the heavy chain A are mutated into amino acids with positive charges, the Q38 and V133 of the light chain a are mutated into amino acids with negative charges, the Q39 and L145 of the heavy chain B are mutated into amino acids with negative charges, and the Q38 and V133 of the light chain B are mutated into amino acids with positive charges.

4. The bispecific antibody of claim 3, wherein said positively charged amino acid is K or R, said negatively charged amino acid is D or E, and said heavy chain A and heavy chain B, light chain a and light chain B contain mutations selected from the group consisting of:

1) heavy chain A: L145K or L145R, light chain a: V133D or V133E, and heavy chain B: L145D or L145E, light chain b:

V133K or V133R;

2) heavy chain A: L128K or L128R, light chain a: V133D or V133E, and heavy chain B: L128D or L128E, light chain b: V133K or V133R;

3) heavy chain A: L145K or L145R, light chain a: V133D or V133E, and heavy chain B: L128D or L128E, light chain b: V133K or V133R;

4) heavy chain A: L128K or L128R, light chain a: V133D or V133E, and heavy chain B: L145D or L145E, light chain b: V133K or V133R;

5) heavy chain A: (Q39K or Q39R) + (L145K or L145R), light chain a: (Q38D or Q38E) + (V133D or V133E), and heavy chain B: (Q39D or Q39E) + (L145D or L145E), light chain b: (Q38K or Q38R) + (V133K or V133R);

6) heavy chain A: (Q39K or Q39R) + (L128K or L128R), light chain a: (Q38D or Q38E) + (V133D or V133E), and heavy chain B: (Q39D or Q39E) + (L128D or L128E), light chain b: (Q38K or Q38R) + (V133K or V133R);

7) heavy chain A: (Q39K or Q39R) + (L145K or L145R), light chain a: (Q38D or Q38E) + (V133D or V133E), and heavy chain B: (Q39D or Q39E) + (L128D or L128E), light chain b: (Q38K or Q38R) + (V133K or V133R);

8) heavy chain A: (Q39K or Q39R) + (L128K or L128R), light chain a: (Q38D or Q38E) + (V133D or V133E), and heavy chain B: (Q39D or Q39E) + (L145D or L145E), light chain b: (Q38K or Q38R) + (V133K or V133R).

5. The bispecific antibody of any one of claims 1 to 4, wherein the CH3 domains of heavy chain A and heavy chain B, designated as CH3_ A domain and CH3_ B domain, respectively, have the following mutations compared to the wild-type human antibody heavy chain constant region CH3 domain:

(a1) CH3_ a domain: F405E + K409F + K370D, CH3_ B domain: S364R + E357S;

(a2) CH3_ a domain: F405E + K409F + K370D + S354C, CH3_ B domain: S364R + E357S + Y349C;

(a3) CH3_ a domain: F405E + K409F + K370D + Y349C, CH3_ B domain: S364R + E357S + S354C (b1) CH3_ a domain: F405E + K409F + K392D, CH3_ B domain: D399K;

(b2) CH3_ a domain: F405E + K409F + K392D + S354C, CH3_ B domain: D399K + Y349C;

(b3) CH3_ a domain: F405E + K409F + K392D + Y349C, CH3_ B domain: D399K + S354C;

(c1) CH3_ a domain: F405E + K409F + K439D, CH3_ B domain: E356K + E357K;

(c2) CH3_ a domain: F405E + K409F + K439D + S354C, CH3_ B domain: E356K + E357K + Y349C;

(c3) CH3_ a domain: F405E + K409F + K439D + Y349C, CH3_ B domain: E356K + E357K + S354C;

(d1) CH3_ a domain: F405E + K409F + L368D, CH3_ B domain: S364R;

(d2) CH3_ a domain: F405E + K409F + L368D + S354C, CH3_ B domain: S364R + Y349C;

(d3) CH3_ a domain: F405E + K409F + L368D + Y349C, CH3_ B domain: S364R + S354C;

(e1) CH3_ a domain: F405E + K409F + L368D, CH3_ B domain: S364K;

(e2) CH3_ a domain: F405E + K409F + L368D + S354C, CH3_ B domain: S364K + Y349C;

(e3) CH3_ a domain: F405E + K409F + L368D + Y349C, CH3_ B domain: S364K + S354C;

(f1) CH3_ a domain: F405E + K409F + K360E, CH3_ B domain: Q347R;

(f2) CH3_ a domain: F405E + K409F + K360E + S354C, CH3_ B domain: Q347R + Y349C;

(f3) CH3_ a domain: F405E + K409F + K360E + Y349C, CH3_ B domain: Q347R + S354C;

(g1) CH3_ a domain: F405E + K409F + K370D + K360E, CH3_ B domain: S364R + E357S + Q347R;

(g2) CH3_ a domain: F405E + K409F + K370D + K360E + S354C, CH3_ B domain: S364R + E357S + Q347R + Y349C;

(g3) CH3_ a domain: F405E + K409F + K370D + K360E + Y349C, CH3_ B domain: S364R + E357S + Q347R + S354C.

6. A composition characterized in that it comprises: (1) the bispecific antibody of any one of claims 1-5, and (2) a pharmaceutically acceptable carrier and/or diluent and/or excipient.

7. A polynucleotide, vector combination, or recombinant host cell, wherein the polynucleotide comprises: a nucleotide molecule a encoding the heavy chain a of the bispecific antibody according to any one of claims 1 to 5, a nucleotide molecule a encoding the light chain a of the bispecific antibody according to any one of claims 1 to 5, a nucleotide molecule B encoding the heavy chain B of the bispecific antibody according to any one of claims 1 to 5, a nucleotide molecule B encoding the light chain B of the bispecific antibody according to any one of claims 1 to 5;

the vector combination is selected from two or more of a recombinant vector containing a nucleotide molecule A, a recombinant vector containing a nucleotide molecule a, a recombinant vector containing the nucleotide molecule A and the nucleotide molecule a, a recombinant vector containing the nucleotide molecule B, a recombinant vector containing a nucleotide molecule B and a recombinant vector containing the nucleotide molecule B and the nucleotide molecule B, and the vector combination contains the nucleotide molecule A, the nucleotide molecule a, the nucleotide molecule B and the nucleotide molecule B;

the recombinant host cell contains the vector combination; wherein, the original host cell of the recombinant host cell is preferably a eukaryotic cell, and is further preferably a CHO cell or 293E cell.

8. Use of the bispecific antibody of any one of claims 1 to 5, the composition of claim 6, the polynucleotide, vector combination or recombinant host cell of claim 7 for the preparation of bispecific antibodies, bispecific fusion proteins and antibody-fusion protein chimeras.

9. A method for producing the bispecific antibody of any one of claims 1 to 5, wherein the bispecific antibody is expressed using the recombinant host cell of claim 7.

10. The method of claim 9, wherein the nucleotide molecule a, the nucleotide molecule B and the nucleotide molecule B are present in the recombinant host cell in a molar ratio of (1-3) to (1-3), such as 1:1:1:1, 1:1:1.5:1.5, 1:1:2:2, 1:1:2.5:2.5, 1:1:3:3, 3:3:1:1, 2.5:2.5:1:1, 2:2:1:1, or 1.5:1.5:1: 1.