WO2021136249A1

WO2021136249A1 - Bispecific antibody induced by fab modification, preparation method therefor and use thereof

Info

Publication number: WO2021136249A1
Application number: PCT/CN2020/140730
Authority: WO
Inventors: 周易
Original assignee: 周易
Priority date: 2019-12-31
Filing date: 2020-12-29
Publication date: 2021-07-08
Also published as: CN113121696A

Abstract

A bispecific antibody induced by Fab modification and a preparation method therefor and use thereof. By comprehensively considering various interactions between interfacial amino acids, such as electrostatic interaction and steric interaction, amino acid modification is introduced at specific positions of an antibody light and heavy chain interaction interface. Furthermore, a linking peptide between the heavy chain VH/CH1 and/or a linking peptide between the light chain VL/CL are optimized, so that the correct pairing ratio of the light and heavy chains is increased to 99% or more, and the influence of the amino acid modification on the expression of the mutant is significantly reduced.

Description

Bispecific antibody induced by Fab transformation and preparation method and application thereof

Technical field

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

Background technique

There are many ways to construct bispecific antibodies. Among them, IgG type bispecific antibodies have similar structure, physicochemical properties and Fc segment functions to common antibodies. Generally, an IgG type bispecific antibody consists of two heavy chains with different amino acid sequences (i.e. heavy chain HC_A against antigen A and heavy chain HC_B against antigen B) and two light chains with different amino acid sequences (i.e. light chain against antigen A). The chain LC_A and the anti-antigen B light chain LC_B) are composed. When 4 polypeptide chains are combined, homodimers and heterodimers will be formed between the two heavy chains, and mismatches will also be formed between the light and heavy chains. Therefore, there will be 8 different combinations, of which only One is the desired target antibody molecule. However, it is extremely inefficient and difficult to separate and purify the target molecule from 8 kinds of molecules.

The progress in the field of antibody engineering has made significant progress in the preparation of IgG-type bispecific antibodies. Most IgG-type bispecific antibodies promote the formation of heterodimers between the two heavy chains of the antibody by modifying the Fc segment (Ridgway, Presta et al. 1996; Carter 2001, US2010286374A1, CN106883297A, US20150307628A1). However, due to the difference between the light and heavy chains Interactions are more complex, making the specific pairing of light and heavy chains more difficult. Specifically, the ideal state is that of the two heavy chains and the two light chains that constitute the bispecific antibody, the light chain LC_A only specifically pairs with the heavy chain HC_A, but not with the heavy chain HC_B. Chain LC_B only specifically paired with heavy chain HC_B, but not with heavy chain HC_A. CN104968677A; WO2016172485A2; WO2014082179A; Nat Biotechnol. 2014Feb; 32(2): 191-8.; Protein Sci. 2017 Oct; 26(10): 2021-2038.; Protein Eng Des Sel. 2017 Sep 1; 30(9): 685 -696.; Disclosed how to correctly pair the light and heavy chains, but the art still needs to find suitable optimizations to further improve the specificity of light chain pairing, reduce the by-products of mismatches, and the yield of bispecific antibodies.

Summary of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art and provide a bispecific antibody induced by Fab transformation and its preparation method and application. In the present invention, by comprehensively considering various interactions between interface amino acids, such as electrostatic interactions, spatial interactions, etc., amino acid modifications are introduced at specific positions on the antibody light-heavy chain interaction interface, and connecting peptides are introduced into the heavy and light chains for optimization. , So that the correct pairing ratio of light and heavy chains is increased to over 99%, and the influence of amino acid modification on the expression of mutants is significantly reduced.

The first aspect of the present invention is to provide a bispecific antibody comprising: a heavy chain A capable of binding to a specific antigen and a light chain a paired with the heavy chain A, and A heavy chain B that can bind to another specific antigen and a light chain b paired with the heavy chain B; both heavy chain A and heavy chain B have the VH domain of the antibody heavy chain variable region and the antibody heavy chain constant region CH1 Domain, CH2 domain, CH3 domain, light chain a and light chain b all have antibody light chain variable region VL domain and light chain constant region CL domain; one of the VH domain and CH1 domain of heavy chain A Insert a connecting peptide between the VH domain and the CH1 domain of the heavy chain B, and/or insert a connecting peptide between the VL domain and the CL domain of the light chain a, and/or the light chain A connecting peptide is inserted between the VL domain and the CL domain of b; compared with the wild-type human antibody, the heavy chain A and light chain a, heavy chain B and light chain b have one of the mutations selected from the following One or more: (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; above The position of the amino acid is determined according to the EU index of KABAT numbering.

It should be understood that, if present, the connecting peptide inserted between the VH domain and CH1 domain of heavy chain A, the connecting peptide inserted between the VH domain and CH1 domain of heavy chain B, and the VL structure of light chain a The connecting peptide inserted between the domain and the CL domain, and the connecting peptide inserted between the VL domain and the CL domain of the light chain b may be all the same, or part of the same, or different from each other.

Preferably, the connecting peptide is 1-4 amino acids in length. That is, if it exists, the length of the connecting peptide inserted between the VH domain and CH1 domain of heavy chain A is 1-4 amino acids; if it exists, the length of the connecting peptide inserted between the VH domain and CH1 domain of heavy chain B is It is 1-4 amino acids; if present, the connecting peptide inserted between the VL domain and CL domain of light chain a is 1-4 amino acids in length; if present, it is between the VL and CL domains of light chain b. The connecting peptide inserted in between is 1-4 amino acids in length.

Preferably, the connecting 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, SAAS, ASSS, SASS, SSAS, SSSA, GA, AG, GGA, GAG, AGG, GAA, AGA, AAG, GGGA, GGAG, GAGG, AGGG, GGAA, AAGG, GAAG, GAGA, AGAG, AGGA, GAAA, AGAA, AAGA, AAAG, or any combination of amino acids.

Preferably, the heavy chain A and heavy chain B, light chain a and light chain b in the bispecific antibody of the present invention contain mutations selected from the following group: (1) The L145 mutation of heavy chain A has positive Charged amino acids, V133 of light chain a is mutated to a negatively charged amino acid, and L145 of heavy chain B is mutated to a negatively charged amino acid, and V133 of light chain b is mutated to a positively charged amino acid; (2) L128 of heavy chain A is mutated to A positively charged amino acid, V133 of light chain a is mutated to a negatively charged amino acid, and L128 of heavy chain B is mutated to a negatively charged amino acid, and V133 of light chain b is mutated to a positively charged amino acid; (3) L145 of heavy chain A Mutation is a positively charged amino acid, V133 of light chain a is mutated to a negatively charged amino acid, and L128 of heavy chain B is mutated to a negatively charged amino acid, and V133 of light chain b is mutated to a positively charged amino acid; (4) Heavy chain A The L128 mutation of light chain a is a positively charged amino acid, the V133 of light chain a is mutated to a negatively charged amino acid, the L145 of heavy chain B is mutated to a negatively charged amino acid, and the V133 of light chain b is mutated to a positively charged amino acid; (5) Heavy Q39 and L145 of chain A are mutated to positively charged amino acids, Q38 and V133 of light chain a are mutated to negatively charged amino acids, and Q39 and L145 of heavy chain B are mutated to negatively charged amino acids, and Q38, V133 of light chain b are mutated to negatively charged amino acids. Are positively charged amino acids; (6) Q39 and L128 of heavy chain A are mutated to positively charged amino acids, Q38 and V133 of light chain a are mutated to negatively charged amino acids, and Q39 and L128 of heavy chain B are mutated to negatively charged Amino acids, Q38 and V133 of light chain b are mutated to positively charged amino acids; (7) Q39 and L145 of heavy chain A are mutated to positively charged amino acids, and Q38 and V133 of light chain a are mutated to negatively charged amino acids, and the heavy chain Q39 and L128 of B are mutated to negatively charged amino acids, and Q38 and V133 of light chain b are mutated to positively charged amino acids; (8) Q39 and L128 of heavy chain A are mutated to positively charged amino acids, and Q38, V133 of light chain a The mutation is a negatively charged amino acid, and the Q39 and L145 of the heavy chain B are mutated to negatively charged amino acids, and the Q38 and V133 of the light chain b are mutated to positively charged amino acids.

Further preferably, the positively charged amino acid refers to K (lysine) or R (arginine), and the negatively charged amino acid refers to D (aspartic acid) or E (glutamic acid). Chain A and heavy chain B, light chain a and light chain b contain mutations selected from the following group: 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 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).

Among them, Q39R means Gln39 is replaced with arginine (R), Q39K means Gln39 is replaced with lysine (K), Q39E means Gln39 is replaced with glutamic acid (E), Q39D means Gln39 is replaced Aspartic acid (D), Q38R means Gln38 is replaced with arginine (R), Q38K means Gln38 is replaced with lysine (K), Q38E means Gln38 is replaced with glutamic acid (E) , Q38D means Gln38 is replaced with aspartic acid (D), L145R means Leucine 145 is replaced with Arginine (R), L145K means Leucine 145 is replaced with Lysine (K), L145E means Leucine 145 is replaced with Glutamate (E), L145D means Leucine 145 is replaced with Aspartic acid (D), L128R means Leucine 128 is replaced with Arginine (R) , L128K refers to the replacement of leucine 128 with lysine (K), L128E refers to the replacement of leucine 128 with glutamic acid (E), and L128D refers to the replacement of leucine 128 with aspartic acid ( D), V133R means valine 133 is replaced with arginine (R), V133K means valine 133 is replaced with lysine (K), V133E means valine 133 is replaced with glutamic acid (E), V133D means that valine 133 is replaced with aspartic acid (D).

Preferably, the CH3 domains of heavy chain A and heavy chain B are respectively named as CH3_A domain and CH3_B domain. Compared with the wild-type human antibody heavy chain constant region CH3 domain, the CH3_A and CH3_B domains contain The mutations that facilitate the formation of bispecific antibodies are not limited to, for example, WO9627011, CN101198698B, CN102459346B, CN105051069A, US2016177364A1, US2010286374A1, CN106883297A, US20150307628A1, CN104968677A, Nat Biotechnol. 2014 Feb; 32(2):191-8. In the embodiment of the present invention, one of the amino acid mutations at the following positions is involved to facilitate the formation of 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 structure 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 structure 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 is replaced with arginine (R), Y349C refers to tyrosine Tyr349 is replaced with cysteine (C), S354C refers to serine Ser354 is replaced with cysteine (C) , E356K means glutamic acid Glu356 is replaced with lysine (K), E357K means glutamic acid Glu357 is replaced with lysine (K), E357S means glutamic acid Glu357 is replaced with serine (S), K360E means lysine Lys360 is replaced with lysine (K), S364R means serine Ser364 is replaced with arginine (R), S364K means serine Ser364 is replaced with lysine (K), L368D means Leucine Leu368 is replaced with aspartic acid (D), K370D means lysine Lys370 is replaced with aspartic acid (D), K392D means lysine Lys392 is replaced with aspartic acid (D) , D399K means that Asp399 is replaced with Lysine (K), K439E means Lys439 is replaced with Glutamic acid (E), F405E means Phe405 is replaced with Glutamic acid (E), K409F refers to the substitution of lysine Lys409 with phenylalanine (F).

The second aspect of the present invention is to provide a composition comprising: (1) the heterodimer according to the first aspect of the present invention, and (2) a pharmaceutically acceptable carrier and/or diluent And/or excipients.

The third aspect of the present invention is to provide a polynucleotide comprising: a nucleotide molecule A encoding the heavy chain A of the bispecific antibody of the first aspect of the present invention, encoding the present invention The nucleotide molecule a of the light chain a of the bispecific antibody of the first aspect, which encodes the nucleotide molecule B of the heavy chain B of the bispecific antibody of the first aspect of the present invention, encodes the present invention The nucleotide molecule b of the light chain b of the bispecific antibody of the first aspect.

The fourth aspect of the present invention is to provide a vector combination comprising: a recombinant vector selected from the group consisting of a recombinant vector containing a nucleotide molecule A, a recombinant vector containing a nucleotide molecule a, and the nucleotide molecule at the same time Recombination vector of A and nucleotide molecule a, a recombination vector containing said nucleotide molecule B, a recombination vector containing nucleotide molecule b, recombination of said nucleotide molecule B and nucleotide molecule b at the same time Two or more of the vectors, and the combination of vectors contains a nucleotide molecule A, a nucleotide molecule a, a nucleotide molecule B, and a nucleotide molecule b at the same time.

Among them, the expression vector used in each of the above-mentioned recombinant vectors is a conventional expression vector in the art, which means that it contains appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and / Or expression vector of sequence and other appropriate sequence. The expression vector may be a virus or a plasmid, such as an appropriate phage or phagemid. For more technical details, please refer to, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. For many known technologies and solutions for nucleic acid manipulation, please refer to Current Protocols in Molecular Biology, second edition, edited by 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 pTT5.

The fifth aspect of the present invention is to provide a recombinant host cell containing the vector combination.

The original host cell of the recombinant host cell of the present invention can be various conventional host cells in the art, as long as it can make the above-mentioned recombinant vector stably replicate by itself, and the nucleotides carried can be effectively expressed. . The original host cell may be a prokaryotic expression cell or a eukaryotic expression cell. The host cell preferably includes: COS, CHO (Chinese Hamster Ovary), NS0, sf9, sf21, DH5α, BL21 (DE3 ) Or TG1, more preferably E. coli TG1, BL21 (DE3) cells (expressing single-chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full-length IgG antibodies). The aforementioned expression vector is transformed into a host cell to obtain the preferred recombinant host cell of the present invention. The transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an electrotransformation method.

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

The sixth aspect of the present invention is to provide the bispecific antibody according to the first aspect of the present invention, the composition according to the second aspect of the present invention, the polynucleotide according to the third aspect of the present invention, the present invention Use of the vector combination according to the fourth aspect or the recombinant host cell according to the fifth aspect of the present invention for preparing bispecific antibodies, bispecific fusion proteins and antibody-fusion protein chimeras.

The seventh aspect of the present invention provides a method for preparing the bispecific antibody according to the first aspect of the present invention, characterized in that the recombinant host cell according to the fifth aspect of the present invention is used to express the bispecific antibody. Sex antibody.

In the present invention, the recombinant host cell contains both the nucleotide molecule A encoding the heavy chain A of the bispecific antibody according to the first aspect of the present invention, and the nucleotide molecule A encoding the bispecific antibody according to the first aspect of the present invention. The nucleotide molecule a of the light chain a of the antibody, 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 bispecific according to the first aspect of the present invention The nucleotide molecule b of the light chain b of the antibody is expressed by the recombinant host cell and recovered to obtain a bispecific antibody.

Wherein, the bispecific antibody can be purified from the recombinant host cell by standard experimental means. For example, when the heterodimeric protein contains an antibody Fc fragment, protein A can 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 appropriate combinations of the above methods.

In the present invention, the molar ratio of nucleotide molecule A, nucleotide molecule a, nucleotide molecule B and nucleotide molecule b in the recombinant host cell is (1-3):(1-3):( 1-3):(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.

In an embodiment of the present invention, wherein the light chain is selected from κ chain or λ 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 antibody Fab fragments, preferably from human antibody Fab fragments. In general, the CH1 and CL domains of human antibody Fab fragments are derived from wild-type human antibody Fab fragments. The human antibody Fab fragment of the present invention also includes individual amino acid changes to the wild-type human antibody Fab sequence, for example, including certain amino acid mutations at the glycosylation site, or other nonsense mutations. In addition to the mutations mentioned in the present invention, it may also contain other mutations that do not affect the function of the antibody Fab segment.

In the present invention, the CH3 is derived from an antibody Fc fragment, preferably 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. Wild-type human antibody Fc refers to the amino acid sequence that exists in the human population. Of course, there are some subtle differences in Fc fragments among individuals. The human antibody Fc fragment of the present invention also includes individual amino acid changes to the wild-type human antibody Fc sequence, for example, including certain amino acid mutations at the glycosylation site, or other nonsense mutations. In addition to the mutations mentioned in the present invention, the CH3 and CH2 domains may also contain other mutations that do not affect the function of the antibody, especially the Fc segment.

In the present invention, the numbering of the amino acid position is determined according to the position of the Kabat EU numbering index. Those skilled in the art know that even if the amino acid sequence is changed due to the insertion or deletion of amino acids or other mutations in the above-mentioned region, the position number of each amino acid corresponding to the standard sequence determined according to the Kabat EU numbering index remains unchanged. The EU index is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD. (1991).

The beneficial effects of the present invention:

In the present invention, by comprehensively considering various interactions between interface amino acids, such as electrostatic interactions, steric interactions, etc., amino acid modifications are introduced at specific positions on the antibody light-heavy chain interaction interface, and the heavy chain VH/CH1 connecting peptide and/ Or the connecting peptide between light chain VL/CL has been optimized, so that the correct pairing ratio of light and heavy chains is increased to more than 99%, and the influence of amino acid modification on the expression of mutants is significantly reduced, thereby increasing the yield of antibodies and reducing production costs. .

Description of the drawings

Figure 1 shows the results of electrophoresis analysis of light and heavy chain pairing. 4-12% SDS-PAGE protein gel electrophoresis. The lanes from left to right are: protein molecular weight standard, A ₂ b, A ₆ b, B ₁₂ a, B ₁₆ a.

Figure 2 shows the ELISA detection of the binding activity of the EGFR×HER2 bispecific antibody to the antigen EGFR-ECD-Fc.

Figure 3 shows the binding activity of the EGFR×HER2 bispecific antibody to the antigen HER2-ECD-Fc detected by ELISA.

Figure 4 shows the ELISA detection of EGFR×HER2 bispecific antibody binding activity to the antigens HER2-ECD-Fc and EGFR-ECD-Fc at the same time.

Figure 5 shows the binding activity of the EGFR×cMet bispecific antibody to the antigen EGFR-ECD-Fc detected by ELISA.

Figure 6 shows the binding activity of EGFR×cMet bispecific antibody to antigen cMet-ECD-Fc detected by ELISA.

Figure 7 shows the ELISA detection of EGFR×cMet bispecific antibody binding activity to the antigens cMet-ECD-Fc and EGFR-ECD-Fc at the same time.

Detailed ways

The following examples and experimental examples are to further illustrate the present invention, and should not be construed as limiting the present invention. The examples do not include detailed descriptions of traditional 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. Such methods are useful in the art Persons with ordinary skills in this field are well known and described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 ^nd edition, Cold spring Harbor Laboratory Press.

The experimental materials and sources used in the following examples and the preparation method of the experimental reagents are specifically described as follows.

1. Experimental materials:

293E cells: from NRC Biotechnology Research Institute.

2. Experimental reagents:

PBS: purchased from Shenggong Biological Engineering (Shanghai) Co., Ltd., catalog number B548117.

Citric acid: purchased from Sinopharm Chemical Reagent Co., Ltd.

Prime star HS DNA polymerase: purchased from Takara, product number R010A.

Endotoxin-free plasmid large-scale extraction kit: purchased from TIANGEN company, item number DP117.

3. Experimental equipment:

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.

Centrifuge: purchased from Eppendorf.

G1600AX capillary electrophoresis instrument: purchased from Agilent.

MicroCal PEAQ-DSC micro calorimeter scanning calorimeter: purchased from Malvern Company.

Octet molecular interaction system: purchased from ForteBio.

Xevo G2-XS Tof Time-of-Flight Mass Spectrometer: purchased from Waters.

Example 1. CH1/CL point mutation design

When two different antibody molecules are co-expressed in one cell, the four polypeptide chains heavy chain A, light chain a, heavy chain B and light chain b will be randomly paired to produce bispecific antibodies and mismatched antibodies. In order to reduce the occurrence of mismatches, we hope that of the two heavy chains and the two light chains that constitute the bispecific antibody, the light chain a will only specifically pair with the heavy chain A, but will not pair with the heavy chain B. Chain b only specifically pairs with heavy chain B, but not with heavy chain A. Therefore, the antibody light and heavy chains need to be engineered. Table 1 lists the amino acids that interact on the CH1/CL interface of the antibody. By mutating these amino acids, it is possible to promote the correct pairing of the light and heavy chains of the bispecific antibody. The antibody light chain is selected from kappa chain and lambda chain, and the amino acids located on the interaction interface in the light chain constant regions Cκ and Cλ are highly conserved. Therefore, although all mutations on the antibody light chain in the present invention are completed on the kappa chain, the same applies to the lambda chain.

Table 1. Amino acids interacting on the CH1/CL interface

CH1CH1	CLCL
F170,P171,A172,V173F170, P171, A172, V173	S162S162
F170,S183,V185F170, S183, V185	S176S176
L128,L145,S183L128, L145, S183	V133V133
L128,A129,P130,A141L128, A129, P130, A141	F118F118
A141,V185,T187A141,V185,T187	L135L135
V173,Q175V173,Q175	Q160Q160

S136S136	K206K206
S136,T139,A141S136, T139, A141	F116F116

1.1 Construction of recombinant antibodies with CH1/Cκ mutations

The heavy chain HC (SEQ ID NO: 1) and light chain LC (SEQ ID NO: 2) of the anti-HER2 antibody Trastuzumab were subcloned into the mammalian cell expression vector pTT5 to obtain a recombinant expression vector for mammalian cells to express 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 cells to express Cetuximab. Subcloned the heavy chain (SEQ ID NO: 5) and light chain (SEQ ID NO: 6) of the anti-IL17 antibody anti-IL17Ab into the mammalian cell expression vector pTT5 to obtain a recombinant expression vector for mammalian cell expression of anti-IL17Ab . According to Table 1 of Example 1, there exists between Val (or V) at position 133 of the EU numbering of the antibody light chain and Leu (or L) at position 128 of the EU numbering of the antibody heavy chain and Leu at position 145. interaction. The heavy chain L128 or L145 of Trastuzumab, Cetuximab and anti-IL17Ab and the V133 of the light chain were mutated by overlapping PCR method, and finally the mutant vector for expression in mammalian cells corresponding to the mutants shown in Table 2 was obtained.

1.2 Transient expression of Trastuzumab, Cetuximab, anti-IL17Ab wild-type and mutants, and detect the effect of different mutation combinations on antibody expression

The expression vector corresponding to the mutation combination of step 1 was transfected into suspension cultured 293E cells with PEI, and the co-transformation ratio of the recombinant expression vector of the heavy chain and the light chain was 1:1. After culturing for 5-6 days, the transient expression culture supernatant was collected, and the antibody expression level was detected by Fortebio using the Fc capture method. The results are shown in Table 2. 1) The wild-type and mutants of all antibodies can be expressed normally, indicating that the introduction of oppositely charged amino acids in the heavy chain (L128 or L145) and light chain V133 does not affect the pairing of the antibody light and heavy chains. 2) The expression of the mutant of Trastuzumab is significantly lower than that of the wild type, which shows similar solutions to Figures 1A and 1B in WO2016172485A2. Specifically, WO2016172485A2 reported that when the heavy chain L128 or L145 of the anti-VEGF antibody Ranibizumab is mutated to negatively charged amino acids such as D or E, and the light chain V133 is mutated to positively charged amino acids such as R or K, the mutant protein is expressed The amount is significantly reduced. 3) However, this example found that after Cetuximab and anti-IL17Ab heavy chain (L128 or L145) and light chain V133 were introduced with oppositely charged amino acids, the expression level of the mutant did not decrease. Therefore, the introduction of oppositely charged amino acids in the antibody heavy chain (L128 or L145) and light chain V133 does not necessarily affect the expression of the antibody. The results shown in Figure 1A and 1B in WO2016172485A2 are not universal.

Table 2 The effect of CH1/CL mutations on antibody expression levels

Example 2 Light and heavy chain pairing experiment

2.1 VH/VL and CH1/CL mutation combinations

The introduction of mutations in the amino acids of the antibody VH/VL interface can promote the correct pairing of the light and heavy chains of the bispecific antibody. CN104968677A and WO2016172485A2 disclose the introduction of oppositely charged amino acids in Q39 of the heavy chain of an antibody and Q38 of the light chain, which is conducive to the correct pairing of the light and heavy chains of the bispecific antibody. On the basis of Example 1, in this example, the anti-EGFR antibody Panitumumab, the anti-HER2 antibody Trastuzumab, and the anti-cMet antibody Onartuzumab were selected as templates, and the mutation combinations shown in Table 3 were further designed. Trastuzumab heavy chain HC (SEQ ID NO: 1) and light chain LC (SEQ ID NO: 2), Onartuzumab heavy chain HC (SEQ ID NO: 7) and light chain LC (SEQ ID NO: 8), Panitumumab The heavy chain HC (SEQ ID NO: 9) and the light chain LC (SEQ ID NO: 10) of the above were subcloned into the mammalian cell expression vector pTT5 to obtain a recombinant expression vector for mammalian cell expression. The heavy chain HC and light chain LC encoding genes of Trastuzumab, Panitumumab and Onartuzumab were combined and mutated by overlapping PCR method, and finally recombinant expression vectors for expressing the mutants in mammalian cells were obtained.

table 3

2.2 The introduction of connecting peptides between antibodies VH/CH1 and or between VL/CL is beneficial to increase the expression level of mutants

Example 1 shows that the introduction of mutations in the antibody heavy chain (L128 or L145) and light chain V133 into oppositely charged amino acids does not necessarily affect the expression of the antibody. The present invention further creatively discovered that when the antibody heavy chain (L128 or L145) and light chain V133 mutations are introduced into oppositely charged amino acids resulting in a significant decrease in expression, they can be introduced between antibody VH/CH1 and or between VL/CL The connecting peptide is beneficial to increase the expression level of the mutant. Specifically, the linker peptide linker1 is inserted before the Ala at position 118 of the EU numbering of the antibody heavy chain, and the linker peptide linker2 is inserted before the Arg at position 108 of the EU numbering of the antibody light chain. Linker1 and linker2 are linker peptides with a length of 0-4 amino acids. , A connecting peptide sequence of 2 amino acids and 4 amino acids, preferably GG and GGGS. Among them, linker1 is the most obvious linker peptide such as "GG" with 2 amino acids. Accordingly, the mutants shown in Table 4 were constructed. The expression vector corresponding to the mutation combination was transfected into suspension cultured 293E cells with PEI, and the co-transformation ratio of the recombinant expression vector of the heavy chain and the light chain was 1:1. After culturing for 5-6 days, collect the transient expression culture supernatant, use the Fc capture method to detect the expression of the antibody by Fortebio, purify the protein by Protein A affinity chromatography, and use the MicroCal PEAQ-DSC Measure the Tm value. The results are shown in Table 4. 1) There is no significant difference between the expression levels of combination P0 and combination P1, indicating that the connecting peptide does not affect the expression of the antibody; 2) the connecting peptide is inserted between the antibodies VH/CH1 and/or between VL/CL, Especially inserting the connecting peptide GG between VH/CH1 can significantly increase the expression of the mutant; 3) Point mutations do not affect the thermal stability of Fab, inserting the connecting peptide only affects the coupling of the melting peak of Fab and CH3, but not Will affect the thermal stability of Fab.

Table 4 The effect of the combination of VH/VL and CH1/CL mutations on expression level and thermal stability

Note: *The melting peak coupling of Fab and CH3

2.3 Orthogonal experiment

In Table 3 of Example 2, the mutants with the connecting peptide "GG" introduced between the antibody VH/CH1 can all be expressed normally, indicating that the light and heavy chains can be paired correctly. Therefore, the light and heavy chains of combination P2, combination P6, combination P12, and combination P16 were further selected for orthogonal experiment. Specifically, the heavy chain of the combination P2 and the combination P6 is positively charged by mutation, and the light chain is negatively charged, and the heavy chain of the opposite combination P12 and P16 is negatively charged by the mutation, and the light chain is positively charged. Therefore, theoretically, the combination P2, the light chain of the combination P6 and the heavy chain of the combination P12, and the heavy chain of the combination P16 cannot be paired due to the repulsion of the same charge. The light chain of the combination 12, the light chain of the combination P16 and the light chain of the combination P2 and the heavy chain of the combination P6 cannot be paired. There are also repulsions of the same charge that cannot be paired.

As shown in Table 5, the heavy chain A of P2 and P6 is named heavy chain A ₂ and A ₆ , the light chain is named light chain a, and the heavy chain B of P12 and P16 is named heavy chain B ₁₂ and B ₁₆ , The light chain is named light chain b. In order to further investigate the impact of the newly introduced mutations on the correct pairing of the light and heavy chains, we transiently expressed A ₂ b and A ₆ b, and B ₁₂ a and B ₁₆ a. By comparing the expressions of Ab and Ba under the same transient conditions, Investigate the correct pairing of light and heavy chains. The expression vector was transfected into suspension cultured 293E cells with PEI, and the co-transformation ratio of heavy chain and light chain recombinant expression vectors was 1:1. After culturing for 5-6 days, the transient expression culture supernatant was collected, and the protein was purified by Protein A affinity chromatography. SDS-PAGE electrophoresis detection results are shown in Figure 1. Judging from the molecular weight, A ₂ b, A ₆ b, B ₁₂ a, and B ₁₆ a are about 100 kD, all of which are heavy chain dimers. It shows that heavy chain A cannot be paired with light chain b, and heavy chain B cannot be paired with light chain a. Therefore, the light and heavy chains of the combination P2 and P6 and the light and heavy chains of the combination P12 and P16 are completely orthogonal.

table 5

Example 3. Preparation of EGFR×HER2 bispecific antibody

3.1 Light and heavy chain pairing experiment

In order to construct the EGFR×HER2 bispecific antibody, the anti-EGFR antibody combination P2, P3, P5 and the anti-HER2 antibody combination T4 in Table 4 of Example 2 were randomly selected for light and heavy chain pairing experiments. In order to verify whether the light and heavy chains of the EGFR×HER2 bispecific antibody are completely paired correctly, the light chain of the EGFR antibody and the HER2 antibody and the heavy chain of the EGFR antibody were co-transfected as shown in Table 7 to observe whether the light chain of the HER2 antibody Will interfere with the correct pairing of EGFR antibodies. In addition, the EGFR antibody and the light chain of the HER2 antibody were co-transfected with the heavy chain of the HER2 antibody to observe whether the light chain of the EGFR antibody would interfere with the correct pairing of the HER2 antibody. The expression vector was transfected into suspension cultured 293E cells with PEI, and the transfection ratio was A:a:b=1:1:1. After culturing for 5 to 6 days, the transient expression culture supernatant was collected, and the protein was purified by Protein A affinity chromatography. The ratio of the two light chains was analyzed by reducing capillary gel electrophoresis. The results are shown in Table 7. The light chain of HER2 antibody T4 can completely pair with the heavy chain of T4 without interference from the light chain of EGFR antibodies P2, P3, and P5. EGFR antibody P2, P3, and P5 light chains can be correctly paired with EGFR antibody P2, P3, and P5 heavy chains. Among them, EGFR antibody P2 is the least affected. When HER2 antibody light chain and P2 antibody light chain are transfected in equal proportions, P2 antibody The pairing is completely undisturbed.

Table 7. EGFR×HER2 bispecific antibody light and heavy chain pairing experiment

3.2. EGFR×HER2 bispecific antibody molecule construction, expression and purification

EGFR antibody P2 and HER2 antibody T4 were selected to construct EGFR×HER2 bispecific antibody. The introduction of the S364R+E357S+Y349C+I253N point mutation in the CH3 domain of the heavy chain of P2 and the F405E+K409F+K370D+S354C point mutation in the CH3 domain of the heavy chain of T4 promoted the preferential formation of heterologous two in the heavy chains of P2 and T4. Aggregate, as shown in Table 8. The expression vector was transfected into suspension cultured 293E cells with PEI, the transfection ratio was heavy chain A: light chain a: heavy chain B: light chain b=3:3:1:1. After 5-6 days of culture, the transient expression was collected Culture the supernatant and purify the protein by Protein A affinity chromatography. LC-MS analysis results showed that no mismatches of light and heavy chains and homodimers of heavy chains were detected, and the purity of the bispecific antibody was about 100%.

Table 8. EGFR×HER2 bispecific antibody molecular construction method

3.3. ELISA method to detect EGFR×HER2 bispecific antibody binding antigen activity

Dilute the recombinant EGFR-ECD-Fc protein with the coating solution to 3μg/ml, add 50μl/well to the microtiter plate at 4°C overnight. Wash the plate 3 times with PBST, add 200μl/well blocking solution, and place it at 37°C for 1 hour, then wash the plate once with PBST for use. Dilute the anti-EGFR×HER2 bispecific antibody and control antibody (EGFR antibody P2) to 100μg/ml with diluent, 4 times the dilution to form 12 concentration gradients (the highest concentration is 100000ng/ml, the lowest concentration is 0.02ng/ml) , Add the blocked ELISA plate successively, 100μl/well, and place at 37°C for 1 hour. Wash the plate 3 times with PBST, add HRP-labeled mouse anti-human Fab antibody, and place at 37°C for 30 minutes. After washing the plate 3 times with PBST, pat dry the remaining droplets on absorbent paper as much as possible, add 100μl of TMB to each well, and place it in the dark at room temperature (20±5°C) for 5 minutes; add stop solution to each well to stop the substrate reaction, instrument read OD at 450nm, GraphPad Prism6 data analysis, plotting and calculation of EC _50. The experimental results are shown in Figure 2. The EC50 of the anti-EGFR×HER2 bispecific antibody and the positive control EGFR antibody P2 binding to EGFR-ECD are 0.1952nM and 0.2073nM, respectively, and the affinity of the two is equivalent.

In order to detect the binding ability of the anti-EGFR×HER2 bispecific antibody to HER2, the recombinant HER2-ECD-Fc protein was diluted to 0.4 μg/ml with coating solution, 50 μl/well was added to the enzyme-labeled plate, and overnight at 4°C. Wash the plate 3 times with PBST, add 200μl/well blocking solution, and place it at 37°C for 1 hour, then wash the plate once with PBST for use. Dilute the anti-EGFR×HER2 bispecific antibody and control antibody (HER2 antibody T4) to 100μg/ml with diluent, 4 times the dilution to form 12 concentration gradients (the highest concentration is 100000ng/ml, the lowest concentration is 0.02ng/ml) , Add the blocked ELISA plate successively, 100μl/well, and place at 37°C for 1 hour. Wash the plate 3 times with PBST, add HRP-labeled mouse anti-human Fab antibody, and place at 37°C for 30 minutes. After washing the plate 3 times with PBST, pat dry the remaining droplets on absorbent paper as much as possible, add 100μl of TMB to each well, and leave it in the dark at room temperature (20±5℃) for 5 minutes; add 50μl of 2M H ₂ SO ₄ stop solution to each well to stop substrate reaction, microplate read OD at 450nm, GraphPad Prism6 data analysis, plotting and calculation of EC _50. The experimental results are shown in Figure 3. The EC50 of the anti-EGFR×HER2 bispecific antibody and the positive control HER2 antibody T4 binding to HER2-ECD were 0.1758nM and 0.1924nM, respectively, and the anti-EGFR×HER2 bispecific antibody affinity and HER2 monoclonal antibody quite.

In order to test the ability of the anti-EGFR×HER2 bispecific antibody to simultaneously bind to HER2 and EGFR, the recombinant HER2-ECD-Fc protein was diluted to 0.4μg/ml with coating solution, 50μl/well was added to the enzyme-labeled plate, and overnight at 4°C. Wash the plate 3 times with PBST, add 200μl/well blocking solution, and place it at 37°C for 1 hour, then wash the plate once with PBST for use. Dilute the anti-EGFR×HER2 bispecific antibody to 100μg/ml with diluent, 4 times the dilution to form 12 concentration gradients (the highest concentration is 100000ng/ml, the lowest concentration is 0.02ng/ml), and the blocked enzyme label is added in sequence The plate, 100μl/well, was placed at 37°C for 1 hour. Wash the plate 3 times with PBST, add EGFR-ECD-Fc-biotin at 150ng/well, and place at 37°C for 1 hour. After washing the plate 3 times with PBST, HRP-labeled Streptavidin was added and placed at 37°C for 30 minutes. After washing the plate 3 times with PBST, pat dry the remaining droplets on absorbent paper as much as possible, add 100μl of TMB to each well, and leave it in the dark at room temperature (20±5℃) for 5 minutes; add 50μl of 2M H ₂ SO ₄ stop solution to each well to stop substrate reaction, microplate read OD at 450nm, GraphPad Prism6 data analysis, plotting and calculation of EC _50. The experimental results are shown in Figure 4, the anti-EGFR×HER2 bispecific antibody can simultaneously bind to EGFR and HER2, and the EC ₅₀ is 0.08763 nM.

Example 4 Preparation of EGFR×cMet bispecific antibody

4.1 Light and heavy chain pairing experiment

In order to construct the EGFR×cMet bispecific antibody, the anti-EGFR antibody combination P2 and P3 in Example 2 and the anti-cMet antibody combination O2 and O3 were randomly selected for screening. In order to verify whether the light and heavy chains of the EGFR×cMet bispecific antibody are completely paired correctly, the light chain of the EGFR antibody and the cMet antibody and the heavy chain of the EGFR antibody were co-transfected as shown in Table 9 to observe whether the light chain of the cMet antibody Will interfere with the correct pairing of EGFR antibodies. In addition, co-transfect the light chain of EGFR antibody and cMet antibody with the heavy chain of cMet antibody to observe whether the light chain of EGFR antibody interferes with the correct pairing of cMet antibody. The expression vector was transfected into suspension cultured 293E cells with PEI, and the transfection ratio was A:a:b=1:1:1. After culturing for 5 to 6 days, the transient expression culture supernatant was collected, and the protein was purified by Protein A affinity chromatography. The ratio of the two light chains was analyzed by reducing capillary gel electrophoresis. The results are shown in Table 9. The light chains of cMet antibodies O2 and O3 can be completely paired with the heavy chains of O2 and O3, and are not interfered by the expression of EGFR antibodies P2 and P3 light chains. EGFR antibody P2 and P3 light chain can be correctly paired with EGFR antibody P2 and P3 heavy chain. Among them, EGFR antibody P2 is the least affected. When cMet antibody light chain and P2 antibody light chain are transfected in equal proportions, the pairing of P2 antibody is completely different. Be disturbed.

Table 9. EGFR×cMet bispecific antibody light and heavy chain pairing experiment

4.2. EGFR×cMet bispecific antibody molecule construction, expression and purification

Select EGFR antibody P2 and cMet antibody O2 to construct EGFR×cMet bispecific antibody. The introduction of the S364R+E357S+Y349C+I253N point mutation in the CH3 domain of the heavy chain of P2 and the F405E+K409F+K370D+S354C point mutation in the CH3 domain of the heavy chain of O2 promoted the preferential formation of heterologous two in the heavy chains of P2 and O2 Aggregate, as shown in Table 10. The expression vector was transfected into suspension cultured 293E cells with PEI, the transfection ratio was heavy chain A: light chain a: heavy chain B: light chain b=2:2:1:1, after 5-6 days of culture, the transient expression was collected Culture the supernatant and purify the protein by Protein A affinity chromatography. LC-MS analysis results showed that no mismatches of light and heavy chains and homodimers of heavy chains were detected, and the purity of the bispecific antibody was about 100%.

Table 10. EGFR×cMet bispecific antibody molecule construction method

4.3. ELISA method to detect EGFR×cMet bispecific antibody binding antigen activity

Dilute the recombinant EGFR-ECD-Fc protein with the coating solution to 3μg/ml, add 50μl/well to the microtiter plate at 4°C overnight. Wash the plate 3 times with PBST, add 200μl/well blocking solution, and place it at 37°C for 1 hour, then wash the plate once with PBST for use. Dilute the anti-EGFR×cMet bispecific antibody and the control antibody (EGFR antibody P2) to 100μg/ml with the diluent, 4 times the dilution to form 12 concentration gradients (the highest concentration is 100000ng/ml, the lowest concentration is 0.02ng/ml) , Add the blocked ELISA plate successively, 100μl/well, and place at 37°C for 1 hour. Wash the plate 3 times with PBST, add HRP-labeled mouse anti-human Fab antibody, and place at 37°C for 30 minutes. After washing the plate 3 times with PBST, pat dry the remaining droplets on absorbent paper as much as possible, add 100μl of TMB to each well, and place it in the dark at room temperature (20±5°C) for 5 minutes; add stop solution to each well to stop the substrate reaction, enzyme label instrument read OD at 450nm, GraphPad Prism6 data analysis, plotting and calculation of EC _50. _{The experimental results are shown in Figure 5. The EC 50 of the} anti-EGFR×cMet bispecific antibody and the positive control EGFR antibody P2 binding to EGFR-ECD are 0.2339 nM and 0.2073 nM, respectively, and the affinities of the two are equivalent.

In order to detect the binding ability of the anti-EGFR×cMet bispecific antibody to cMet, the recombinant cMet-ECD-Fc protein was diluted to 0.4 μg/ml with coating solution, 50 μl/well was added to the enzyme-labeled plate, and overnight at 4°C. Wash the plate 3 times with PBST, add 200μl/well blocking solution, and place it at 37°C for 1 hour, then wash the plate once with PBST for use. Dilute the anti-EGFR×cMet bispecific antibody and control antibody (cMet antibody O2) to 100μg/ml with diluent, 4 times the dilution to form 12 concentration gradients (the highest concentration is 100000ng/ml, the lowest concentration is 0.02ng/ml) , Add the blocked ELISA plate successively, 100μl/well, and place at 37°C for 1 hour. Wash the plate 3 times with PBST, add HRP-labeled mouse anti-human Fab antibody, and place at 37°C for 30 minutes. After washing the plate 3 times with PBST, pat dry the remaining droplets on absorbent paper as much as possible, add 100μl of TMB to each well, and leave it in the dark at room temperature (20±5℃) for 5 minutes; add 50μl of 2M H ₂ SO ₄ stop solution to each well to stop substrate reaction, microplate read OD at 450nm, GraphPad Prism6 data analysis, plotting and calculation of EC _50. _{The experimental results are shown in Figure 6. The EC 50 of the} anti-EGFR×cMet bispecific antibody and the positive control cMet antibody O2 bound to cMet-ECD are 0.8635 nM and 0.5524 nM, respectively, and the affinity of the anti-EGFR×cMet bispecific antibody is the same as that of the cMet single Anti-equivalent.

In order to test the ability of the anti-EGFR×cMet bispecific antibody to simultaneously bind cMet and EGFR, the recombinant cMet-ECD-Fc protein was diluted to 0.4μg/ml with coating solution, 50μl/well was added to the enzyme-labeled plate, and overnight at 4°C. Wash the plate 3 times with PBST, add 200μl/well blocking solution, and place it at 37°C for 1 hour, then wash the plate once with PBST for use. Dilute the anti-EGFR×cMet bispecific antibody to 100μg/ml with diluent, and dilute by 4 times to form 12 concentration gradients (the highest concentration is 100000ng/ml, the lowest concentration is 0.02ng/ml), and the blocked enzyme label is added in sequence The plate, 100μl/well, was placed at 37°C for 1 hour. Wash the plate 3 times with PBST, add EGFR-ECD-Fc-biotin at 150ng/well, and place at 37°C for 1 hour. After washing the plate 3 times with PBST, HRP-labeled Streptavidin was added and placed at 37°C for 30 minutes. After washing the plate 3 times with PBST, pat dry the remaining droplets on absorbent paper as much as possible, add 100μl of TMB to each well, and leave it in the dark at room temperature (20±5℃) for 5 minutes; add 50μl of 2M H ₂ SO ₄ stop solution to each well to stop substrate reaction, microplate read OD at 450nm, GraphPad Prism6 data analysis, plotting and calculation of EC _50. The results shown in Figure 7, the bispecific antibody can simultaneously bind EGFR × cMet of cMet and EGFR, and EC ₅₀ is 0.3744nM.

The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should all fall within the scope of the present invention.

Claims

A bispecific antibody, characterized in that the bispecific antibody comprises: a heavy chain A that can bind to a specific antigen and a light chain a that is paired with the heavy chain A, and a light chain a that can be paired with another specific antigen. The heavy chain B bound to the sex antigen and the light chain b paired with the heavy chain B;

Both 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 both have antibody light chain variable Region VL domain and light chain constant region CL domain;

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

Compared with wild-type human antibodies, the heavy chain A and light chain a, heavy chain B and light chain b have one or more mutations selected from the following:

(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.
The bispecific antibody according to claim 1, wherein the connecting 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, SAAS, ASSS, SASS, SSAS, SSSA, GA, AG, GGA, GAG , AGG, GAA, AGA, AAG, GGGA, GGAG, GAGG, AGGG, GGAA, AAGG, GAAG, GAGA, AGAG, AGGA, GAAA, AGAA, AAGA, AAAG, or any combination of amino acids.
The bispecific antibody according to claim 1, wherein the heavy chain A and heavy chain B, and light chain a and light chain b contain mutations selected from the following group:

(1) L145 of heavy chain A is mutated to a positively charged amino acid, V133 of light chain a is mutated to a negatively charged amino acid, and L145 of heavy chain B is mutated to a negatively charged amino acid, and V133 of light chain b is mutated to a positively charged amino acid. Amino acid

(2) L128 of heavy chain A is mutated to a positively charged amino acid, V133 of light chain a is mutated to a negatively charged amino acid, and L128 of heavy chain B is mutated to a negatively charged amino acid, and V133 of light chain b is mutated to a positively charged amino acid. Amino acid

(3) L145 of heavy chain A is mutated to a positively charged amino acid, V133 of light chain a is mutated to a negatively charged amino acid, and L128 of heavy chain B is mutated to a negatively charged amino acid, and V133 of light chain b is mutated to a positively charged amino acid. Amino acid

(4) L128 of heavy chain A is mutated to a positively charged amino acid, V133 of light chain a is mutated to a negatively charged amino acid, and L145 of heavy chain B is mutated to a negatively charged amino acid, and V133 of light chain b is mutated to a positively charged amino acid. Amino acid

(5) Q39 and L145 of heavy chain A are mutated to positively charged amino acids, Q38 and V133 of light chain a are mutated to negatively charged amino acids, and Q39 and L145 of heavy chain B are mutated to negatively charged amino acids, Q38 and V133 are mutated into positively charged amino acids;

(6) Q39 and L128 of heavy chain A are mutated to positively charged amino acids, Q38 and V133 of light chain a are mutated to negatively charged amino acids, and Q39 and L128 of heavy chain B are mutated to negatively charged amino acids, and of light chain b Q38 and V133 are mutated into positively charged amino acids;

(7) Q39 and L145 of heavy chain A are mutated to positively charged amino acids, Q38 and V133 of light chain a are mutated to negatively charged amino acids, and Q39 and L128 of heavy chain B are mutated to negatively charged amino acids, and Q39 and L128 of heavy chain B are mutations to negatively charged amino acids. Q38 and V133 are mutated into positively charged amino acids;

(8) Q39 and L128 of heavy chain A are mutated to positively charged amino acids, Q38 and V133 of light chain a are mutated to negatively charged amino acids, and Q39 and L145 of heavy chain B are mutated to negatively charged amino acids, and of light chain b Q38 and V133 are mutated to positively charged amino acids.
The bispecific antibody of claim 3, wherein the positively charged amino acid refers to K or R, the negatively charged amino acid refers to D or E, and the heavy chain A, heavy chain B, and light chain a and light chain b contain mutations selected from the following group:

1) Heavy chain A: L145K or L145R, light chain a: V133D or V133E, 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, 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, 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, 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).
The bispecific antibody according to any one of claims 1 to 4, wherein the CH3 domains of heavy chain A and heavy chain B are respectively named as CH3_A domain and CH3_B domain, which are compatible with wild-type human antibodies. Compared with the CH3 domain of the constant region of the body weight chain, the CH3_A and CH3_B domains have the following mutations:

(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.
A composition, characterized in that it contains: (1) the bispecific antibody according to any one of claims 1-5, and (2) a pharmaceutically acceptable carrier and/or diluent and/or excipient.
A polynucleotide, vector combination, or recombinant host cell, wherein the polynucleotide comprises: a nucleotide encoding the heavy chain A of the bispecific antibody of any one of claims 1-5 Molecule A, the nucleotide molecule a encoding the light chain a of the bispecific antibody of any one of claims 1-5, encoding the heavy chain of the bispecific antibody of any one of claims 1-5 The nucleotide molecule B of chain B, which encodes the nucleotide molecule b of the light chain b of the bispecific antibody according to any one of claims 1 to 5;

The vector combination is selected from a recombinant vector containing a nucleotide molecule A, a recombinant vector containing a nucleotide molecule a, a recombinant vector containing both the nucleotide molecule A and a nucleotide molecule a, and the nucleoside Two or more of the recombinant vector containing the acid molecule B, the recombinant vector containing the nucleotide molecule b, and the recombinant vector containing the nucleotide molecule B and the nucleotide molecule b at the same time, and the combination of vectors simultaneously contains the nucleus Nucleotide molecule A, nucleotide molecule a, nucleotide molecule B and 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, more preferably a CHO cell or a 293E cell.
The bispecific antibody according to any one of claims 1-5, the composition according to claim 6, the polynucleotide, vector combination or recombinant host cell according to claim 7 are used in the preparation of bispecific antibodies, double Use in specific fusion proteins and antibody-fusion protein chimeras.
A method for preparing the bispecific antibody according to any one of claims 1 to 5, characterized in that the recombinant host cell according to claim 7 is used to express the bispecific antibody.
The method according to claim 9, wherein the molar ratio of nucleotide molecule A, nucleotide molecule a, nucleotide molecule B and nucleotide molecule b in the recombinant host cell is (1-3) :(1-3):(1-3):(1-3), for example 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.