Anti-TNFα / anti-IL-17A natural antibody structure-mimicking heterodimer bispecific antibody and method for producing same

Abstract

The present invention provides an anti-TNFα / anti-IL-17A natural antibody structure-like heterodimeric bispecific antibody and a method for producing the same, which can simultaneously bind to two target molecules to treat complex diseases.

Description

[Technical field]

[0001] The present invention relates to an anti-TNFα / anti-IL-17A natural antibody structure-like heterodimeric bispecific antibody and a method for producing the same. Specifically, the present invention provides a highly stable heterodimeric anti-TNFα / anti-IL-17A bispecific antibody that has the properties of natural IgG and has no heavy-light chain mismatch, and a method for producing the same. [Background technology]

[0002] TNFα is a cellular inflammatory factor that induces the secretion of other inflammatory factors by binding to its receptors TNFR1 and TNFR2. TNFα antagonists can inhibit such binding and reduce the activity of the TNFα signal pathway, thereby achieving the purpose of suppressing inflammatory responses. Currently, there are two types of TNFα antagonists on the market: soluble TNFα receptors and TNFα monoclonal antibodies. Soluble TNFα receptors fuse the extracellular domain of TNFR2 with the constant domain of IgG, such as Etanercept from Pfizer, which was marketed in 1998. TNFα monoclonal antibodies are genetically engineered antibodies that specifically recognize TNFα and can neutralize TNFα, and currently, infliximab and golimumab from J&J, certolizumab from UCB, and adalimumab from Abbott (Clinical Immunology, 2008, 126:13-30) are commercially available. Clinical applications show that TNFα antagonists can effectively reduce RA inflammation and alleviate the radiological progression of joints, and the improvement rate of ACR20 index of patients reaches 50% to 70%. However, it has been found that 20-30% of patients show an inadequate response to TNFα antagonists or the efficacy gradually decreases after long-term treatment with TNFα antagonists (Biodrugs, 2009, 23(2):111-124).

[0003] Recent studies have shown that in this portion of RA patients who do not respond well or whose disease gradually lapses after treatment, the levels of Th17 cells and the expression levels of IL-17A cytokine in vivo are elevated. This phenomenon has also been confirmed in animal models, where the levels of Th17 cells and the expression levels of IL-17A cytokine in vivo are also elevated in animals who do not respond well to TNFα antagonists. Many publications have reported that IL-17A can act synergistically with TNFα to induce or aggravate inflammatory responses, and that the increase in Th17 and IL-17A in vivo may be an important mechanism for the ineffective response to TNFα antagonists (Trends Pharmacol Sci.2015 Apr;36(4):189-95.Ann Rheum Dis.2012 Oct;71(10):1741-8.PLoS ONE 2014, 9(5):e95346.). Therefore, simultaneous suppression of TNFα and IL-17A may improve the therapeutic effect and quality of life for RA patients and patients with other autoimmune diseases associated with TNFα and IL-17A, such as psoriasis and psoriatic arthritis. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Clinical Immunology, 2008, 126:13-30 [Non-Patent Document 2] Biodrugs, 2009, 23(2):111-124 [Non-Patent Document 3] Trends Pharmacol Sci.2015 Apr;36(4):189-95 [Non-Patent Document 4] Ann Rheum Dis.2012 Oct;71(10):1741-8.PLoS ONE 2014, 9(5):e95346 Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, it is necessary to investigate novel therapeutic agents that can simultaneously block the TNFα and IL-17A signaling pathways. [Means for solving the problem]

[0006] The present invention provides a novel, highly stable heterodimeric bispecific antibody that has the structural characteristics of natural IgG and is free of heavy chain-light chain mismatches, and is capable of simultaneously blocking TNFα and IL-17A, and a method for producing the same. This bifunctional antibody selectively binds to tumor cells that simultaneously highly express TNFα and IL-17A, thereby exerting an efficient and specific killing effect and tending to have low toxic side effects.

[0007] A first aspect of the present invention relates to a heterodimeric bispecific antibody comprising a first Fc chain and a second Fc chain, a first antigen-binding functional region capable of specifically binding to TNFα, and a second antigen-binding functional region capable of specifically binding to IL-17A.

[0008] Here, the first Fc chain and the second Fc chain are both Fc fragments of immunoglobulin G containing amino acid substitutions, and the first Fc chain and the second Fc chain together form a heterodimer that can bind to an Fc receptor.

[0009] Here, the first Fc chain and the second Fc chain are linked to the first antigen-binding functional region and the second antigen-binding functional region, respectively, via a covalent bond or a linker.

[0010] and one of the first Fc chain and the second Fc chain comprises amino acid substitutions at positions 366 and 399, and the other comprises amino acid substitutions at positions 351, 407 and 409, wherein the amino acid positions are numbered according to the Kabat EU index numbering system.

[0011] In the present specification, the first Fc chain and the second Fc are defined only for the purpose of distinguishing between the two existing Fc chains, and do not imply a different importance or order. Furthermore, the first Fc chain and the second Fc chain may be linked to a first antigen-binding functional region and a second antigen-binding functional region as desired, i.e., the first Fc chain may be linked to the first antigen-binding functional region or to the second antigen-binding functional region, and similarly for the second Fc chain.

[0012] In some embodiments, the amino acid substitutions in the first and second Fc chain are as follows: a) L351G, L351Y, L351V, L351P, L351D, L351E, L351K or L351W; b) T366L, T366P, T366W or T366V; c) D399C, D399N, D399I, D399G, D399R, D399T or D399A; d) Y407L, Y407A, Y407P, Y407F, Y407T or Y407H; and e) K409C, K409P, K409S, K409F, K409V, K409Q or K409R.

[0013] In some embodiments, the amino acid substitutions include the following: a) one of the first Fc chain and the second Fc chain has T366L and D399R substitutions, and the other has L351E, Y407L and K409V substitutions; b) one of the first Fc chain and the second Fc chain has T366L and D399C substitutions, and the other has L351G, Y407L and K409C substitutions; c) one of the first Fc chain and the second Fc chain has T366L and D399C substitutions, and the other has L351Y, Y407A and K409P substitutions; d) one of the first Fc chain and the second Fc chain has T366P and D399N substitutions, and the other has L351V, Y407P and K409S substitutions; e) one of the first Fc chain and the second Fc chain has T366W and D399G substitutions, and the other has L351D, Y407P and K409S substitutions; f) one of the first Fc chain and the second Fc chain has T366P and D399I substitutions, and the other has L351P, Y407F and K409F substitutions; g) one of the first Fc chain and the second Fc chain has T366V and D399T substitutions, and the other has L351K, Y407T and K409Q substitutions; h) One of the first Fc chain and the second Fc chain has the substitutions T366L and D399A, and the other has the substitutions L351W, Y407H and K409R.

[0014] In some embodiments, the amino acid substitutions in one of the first Fc chain and the second Fc chain are T366L and D399R, and the amino acid substitutions in the other are L351E, Y407L, and K409V.

[0015] In some embodiments, the first antigen-binding functional region and the second antigen-binding functional region are selected from a Fab fragment, an scFv fragment, a variable domain fragment Fv, and a heavy chain variable region fragment VHH of a heavy chain antibody.

[0016] In some embodiments, the first antigen-binding functional region and the second antigen-binding functional region are both Fab fragments.

[0017] In some embodiments, one of the first antigen-binding functional region and the second antigen-binding functional region is a Fab fragment, and the other is an scFv.

[0018] In some embodiments, a Fab fragment comprises a first heavy chain variable region and a second heavy chain variable region that are different, and a first light chain variable region and a second light chain variable region that are different.

[0019] In some embodiments, when a first Fc chain and a first antigen-binding functional region linked thereto via a covalent bond, and a second Fc chain and a second antigen-binding functional region linked thereto via a covalent bond are present in a solution containing a reducing agent, and the solution does not contain any polypeptides other than the first Fc chain and the first antigen-binding functional region linked thereto via a covalent bond, and the second Fc chain and the second antigen-binding functional region linked thereto via a covalent bond, the weight percentage of the formed homodimer based on all polypeptide chains is less than 50%.

[0020] In some embodiments, the first antigen-binding functional region comprises the amino acid sequence of SEQ ID NOs:2 and 6.

[0021] In some embodiments, the second antigen-binding functional region comprises the amino acid sequence of SEQ ID NOs:10 and 12.

[0022] In some embodiments, the first antigen-binding functional region further comprises the amino acid sequence of SEQ ID NOs:4 and 8.

[0023] In some embodiments, the second antigen-binding functional region further comprises the amino acid sequence of SEQ ID NOs:4 and 14.

[0024] In some embodiments, the amino acid sequence of the bispecific antibody is a combination of SEQ ID NOs: 2, 4, 6, 8, 10, 12 and 14. For example, SEQ ID Nos: 2, 4, 6 and 8 are combined, SEQ ID Nos: 10, 4, 12 and 14 are combined, and then both of these combined are combined to form a bispecific antibody of the invention.

[0025] A second aspect of the invention relates to an isolated polynucleotide encoding the heterodimeric bispecific antibody according to the first aspect.

[0026] In some embodiments, the nucleotide sequence encoding the amino acids of the first antigen-binding functional region is selected from SEQ ID NOs: 1 and 5.

[0027] In some embodiments, the nucleotide sequence encoding the amino acids of the second antigen-binding functional region is selected from SEQ ID NOs:9 and 11.

[0028] In some embodiments, the nucleotide sequence encoding the amino acids of the first antigen-binding functional region is further selected from SEQ ID NOs: 3 and 7.

[0029] In some embodiments, the nucleotide sequence encoding the amino acids of the second antigen-binding functional region is further selected from SEQ ID NOs: 3 and 13.

[0030] In some embodiments, the sequence of the polynucleotide is a combination of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 13. For example, a combination of SEQ ID Nos: 1, 3, 5, and 7, or a combination of SEQ ID Nos: 9, 3, 11, and 13.

[0031] A third aspect of the invention relates to a recombinant expression vector comprising the isolated polynucleotide according to the second aspect.

[0032] In some embodiments, the expression vector is the plasmid vector X0GC obtained by pCDNA-based modification.

[0033] A fourth aspect of the invention relates to a host cell comprising an isolated polynucleotide according to the second aspect or a recombinant expression vector according to the third aspect.

[0034] In some embodiments, the host cell is selected from human embryonic kidney cells HEK293 or HEK293T, HEK293F, HEK293E obtained by modification based on HEK293 cells, hamster ovary cells CHO or CHO-S, CHO-dhfr-, CHO / DG44, ExpiCHO obtained by modification based on CHO cells, E. coli or E. coli BL21, BL21(DE3), Rosetta, Origami obtained by modification based on E. coli, yeast or Pichia pastoris obtained by modification based on yeast, Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha, insect cells or High5 cells obtained by modification based on insect cells, SF9 cells, plant cells, mammalian mammary gland cells, and somatic cells.

[0035] A fifth aspect of the present invention relates to a composition comprising a heterodimeric bispecific antibody according to the first aspect or an isolated polynucleotide according to the second aspect or a recombinant expression vector according to the third aspect or a host cell according to the fourth aspect and a pharma- ceutically acceptable carrier.

[0036] A sixth aspect of the invention relates to a method for producing a heterodimeric bispecific antibody according to the first aspect comprising the steps of: 1) expressing in a host cell an isolated polynucleotide according to the second aspect or a recombinant expression vector according to the third aspect, respectively; 2) reducing each expressed protein in the host cell; 3) mixing the reduced protein and then oxidizing the mixture.

[0037] In some embodiments, the host cell is selected from human embryonic kidney cells HEK293 or HEK293T, HEK293F, HEK293F obtained by modification based on HEK293 cells, hamster ovary cells CHO or CHO-S, CHO-dhfr-, CHO / DG44, ExpiCHO obtained by modification based on CHO cells, E. coli or E. coli BL21, BL21(DE3), Rosetta, Origami obtained by modification based on E. coli, Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha obtained by modification based on yeast or yeast, insect cells or High5 cells obtained by modification based on insect cells, SF9 cells, plant cells, mammalian mammary gland cells, somatic cells.

[0038] In some embodiments, the reduction step includes 1) performing the reduction reaction in the presence of a reducing agent selected from 2-mercaptoethylamine, dithiothreitol, tris(2-carboxyethyl)phosphine or chemical derivatives thereof, and 2) removing the reducing agent. For example, the reduction reaction is performed in the presence of dithiothreitol at a concentration of 0.1 mM or more at 4° C. for at least 3 hours. The above limitations on reducing agents and reduction reaction conditions are also applicable to other cases of use of reducing agents and reduction reactions herein.

[0039] In some embodiments, the oxidation step is oxidation in air, but further comprises carrying out the oxidation reaction in the presence of an oxidizing agent selected from L-dehydroascorbic acid or a chemical derivative thereof, e.g., carrying out the oxidation reaction in the presence of L-dehydroascorbic acid at a concentration of 0.5 mM or more at 4° C. for at least 5 hours.

[0040] In some embodiments, the method further comprises an isolation and purification step.

[0041] A seventh aspect of the present invention relates to the use of a heterodimeric bispecific antibody according to the first aspect, and / or an isolated polynucleotide according to the second aspect, and / or a recombinant expression vector according to the third aspect, and / or a host cell according to the fourth aspect, and / or a composition according to the fifth aspect in the manufacture of a medicament for preventing and / or treating a disease in a subject.

[0042] An eighth aspect of the present invention relates to a heterodimeric bispecific antibody according to the first aspect, and / or an isolated polynucleotide according to the second aspect, and / or a recombinant expression vector according to the third aspect, and / or a host cell according to the fourth aspect, and / or a composition according to the fifth aspect for use as a medicament for preventing and / or treating a disease in a subject.

[0043] A ninth aspect of the present invention relates to a method for preventing and / or treating a disease comprising the step of administering to a subject in need thereof a heterodimeric bispecific antibody according to the first aspect, and / or an isolated polynucleotide according to the second aspect, and / or a recombinant expression vector according to the third aspect, and / or a host cell according to the fourth aspect, and / or a composition according to the fifth aspect.

[0044] In some embodiments, the subject is a mammal, preferably a human.

[0045] In some embodiments, the disease is selected from rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, gouty arthritis, juvenile rheumatoid arthritis, septic arthritis, psoriasis, type I diabetes, multiple sclerosis, autoimmune encephalomyelitis, Crohn's disease, systemic vasculitis, dermatomyositis, mixed connective tissue disease, lupus erythematosus, idiopathic thrombocytopenic purpura, primary Sjogren's syndrome, glomerulonephritis, gout, organ transplant rejection, asthma, or arteriosclerosis.

[0046] The present invention has designed a novel, highly stable, anti-TNFα / anti-IL-17A native antibody structure-like heterodimer bispecific antibody that has the characteristics of native IgG and has no heavy-light chain mismatch. The bispecific antibody produced by the present invention can simultaneously block the TNFα signal pathway and the IL-17A signal pathway, and when applied to the treatment of complex diseases, it can exert a more effective effect than a single therapeutic agent. In addition, in contrast to the combination therapy of multiple drugs, the bispecific antibody is a single therapeutic molecule, which not only facilitates the use by patients and medical professionals, but also simplifies the complicated new drug development process. The present invention has also discovered that the TNFα neutralizing activity of the anti-TNFα portion of the bispecific antibody is significantly stronger in the presence of IL-17A than in the absence of IL-17A. This bispecific antibody was shown to have stronger TNFα neutralizing activity at the lesion site where IL-17A levels are high, but weaker TNFα neutralizing activity in the circulatory system where IL-17A levels are low, suggesting that this bispecific antibody effectively treats disease while maintaining low toxic side effects. [Brief description of the drawings]

[0047] [Figure 1] 1 shows chromatograms of the elution peaks of anti-TNFα expression products and anti-IL-17A expression products, where A in Fig. 1 is a chromatogram of the elution peak of the anti-TNFα expression product, and B in Fig. 1 is a chromatogram of the elution peak of the anti-IL-17A expression product. [Diagram 2] 1 shows the structure of an anti-TNFα / anti-IL-17A heterodimer antibody molecule. [Diagram 3] 1 shows the structure of a half antibody molecule containing one heavy and one light chain. [Figure 4] 4 shows the results of SEC analysis of half antibody molecules containing one heavy chain and one light chain, where FIG. 4A is an anti-TNFα half antibody molecule with a purity of 98% and FIG. 4B is an anti-IL-17A half antibody molecule with a purity of 90.2%. [Diagram 5]1 shows the results of non-reduced capillary electrophoresis of anti-TNFα / IL-17A heterodimeric antibody molecules, where peak 1 corresponds to one light chain, peak 2 corresponds to one heavy chain+one light chain, peak 3 corresponds to two heavy chains, peak 4 corresponds to two heavy chains+one light chain, and peak 5 corresponds to two heavy chains+two light chains. [Figure 6] The elution peak of the anti-TNFα / anti-IL-17A heterodimer antibody molecule is shown. [Figure 7] The results of non-reduced capillary electrophoresis of the final purified anti-TNFα / anti-IL-17A heterodimer antibody molecule are shown, where peak 1 corresponds to one light chain, peak 2 corresponds to two heavy chains, peak 3 corresponds to two heavy chains + one light chain, and peak 4 corresponds to two heavy chains + two light chains. [Figure 8] SEC analysis of the anti-TNFα / anti-IL-17A heterodimer antibody molecule shows a purity of 99.9%. [Figure 9] 9 shows the in vitro neutralizing activity of the target antigen of anti-TNFα / anti-IL-17A heterodimer antibody BH1657, where FIG. 9A shows the TNFα neutralizing activity of BH1657, FIG. 9B shows the IL-17A neutralizing activity of BH1657, and FIG. 9C shows the neutralizing activity of BH1657 in the simultaneous presence of TNFα and IL-17A. [Figure 10] 10 shows that the anti-TNFα activity of anti-TNFα / anti-IL-17A heterodimer antibody BH1657 is affected by the presence or absence of IL-17A, where FIG. 10A shows the effect of IL-17A on blocking the TNFα / TNFR2 activity of BH1657, and FIG. 10B shows the effect of IL-17A on the TNFα neutralizing activity of BH1657. [Figure 11] Figure 1 shows the in vivo activity of anti-TNFα / anti-IL-17A heterodimer antibody BH1657 to neutralize the target antigen. ##p<0.01 vs. anti-TNFα; **p<0.01 vs. anti-IL-17A. [Figure 12]Efficacy of anti-mouse TNFα / anti-mouse IL-17A heterodimer antibody BH1654 in a type II collagen-induced murine arthritis (mCIA) model. *p<0.5, **p<0.01, ***p<0.001 vs vehicle; #p<0.05, ###p<0.001 vs BH1654 140nmol / kg. [Figure 13] Efficacy of anti-mouse TNFα / anti-mouse IL-17A heterodimer antibody BH1654 in a mouse glucose-6-phosphate isomerase-induced arthritis (mGPI) model. #p<0.5, ##p<0.01, ###p<0.001 vs vehicle; *p<0.05, **p<0.01 vs BH1654 70nmol / kg. [Figure 14] Efficacy of anti-mouse TNFα / anti-mouse IL-17A heterodimer antibody BH1654 in a zymosan-induced SKG mouse psoriatic arthritis model. #p<0.5 vs. vehicle. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Definition: "Covalent bond" refers to the covalent linkage between two Fc chains or between any one of the Fc chains and the antigen-binding functional region linked thereto in a heterodimeric bispecific antibody to form a single molecule, where the Fc chain comprises a first antigen-binding functional region and a second antigen-binding functional region linked by one or more covalent bonds (e.g., disulfide bonds), and the first Fc chain and the second Fc chain are each linked to the antigen-binding functional region via a covalent bond (e.g., imine bond or amide bond).

[0049] An antigen-binding functional region refers to a region that can specifically interact with a target molecule such as an antigen. Its action is highly selective. Usually, a sequence that recognizes one target molecule cannot recognize the sequence of another molecule. Representative antigen-binding functional regions include antibody variable regions, structural variants of antibody variable regions, receptor-binding domains, ligand-binding domains, or enzyme-binding domains.

[0050] Interchain binding of one or more disulfide bonds means that the first Fc chain and the second Fc chain are linked by one or more disulfide bonds to form a heterodimeric fragment. In the present invention, the one or more disulfide bonds can be formed when the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and the antigen-binding functional region linked thereto, are synthesized in the same cell, or can be formed by an in vitro reduction-oxidation process after the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and the antigen-binding functional region linked thereto are separately synthesized in different cells.

[0051] The first Fc chain and the second Fc chain refer to a binding fragment formed by a covalent bond including a disulfide bond, and each chain includes at least a part of the heavy chain constant region of an immunoglobulin. The first Fc chain and the second Fc chain also include those having different amino acid sequences and differing at at least one amino acid position. In the first Fc chain and the second Fc chain of the present invention, since there is a strong repulsive force between the same chains while there is an attractive force between different chains, the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and the antigen-binding functional region linked thereto tend to form a heterodimer when co-expressed in a cell. When the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and the antigen-binding functional region linked thereto, are expressed separately in two host cells, the first Fc chain or the first Fc chain and the antigen-binding functional region linked thereto do not tend to form homodimers, and the second Fc chain or the second Fc chain and the antigen-binding functional region linked thereto do not tend to form homodimers. In the present invention, when the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and the antigen-binding functional region linked thereto are expressed separately in two host cells in the presence of a reducing agent, the proportion of homodimers is less than 50%, i.e., the proportion of monomers (one Fc chain or one Fc chain and the antigen-binding functional region linked thereto) is more than 50%.

[0052] Immunoglobulins have a symmetrical structure with four polypeptide chains, including two identical heavy chains that are longer and have a larger relative molecular weight, contain 450-550 amino acid residues, and have a relative molecular weight between 55000 Da and 70000 Da, and two identical light chains (L chains) that are shorter and have a smaller relative molecular weight, contain about 210 amino acid residues, and have a relative molecular weight of about 24000 Da. The sequence of about 110 amino acids near the N-terminus varies greatly in the heavy and light chains of different immunoglobulins and is called the variable region (V region). In contrast, the remaining amino acid sequence near the C-terminus is relatively stable and is called the constant region (C region). In the heavy chain, the variable region occupies about 1 / 4 of the length of the heavy chain, while the constant region occupies about 3 / 4 of the length of the heavy chain. Regarding the five known Igs, namely IgG(γ), IgA(α), IgD(δ), IgM(μ) and IgE(ε), the H chains of the first three Igs have three constant regions, namely CH1, CH2 and CH3. The H chains of the latter two isotypes (IgM and IgE) have one VH region and four constant regions, namely CH1 to CH4. The constant region is both the framework of the immunoglobulin molecule and one of the regions that activates the immune response. The examples of the present invention relate to IgG, but it is clear to those skilled in the art that the class of the antibody of the present invention can be converted by known methods. For example, the initial IgM antibody of the present invention can be converted into the IgG antibody of the present invention. It should be noted that the IgG subclass can be converted into another subclass by class conversion technology, for example, IgG1 can be converted into IgG2. Thus, the effector functions of the antibodies of the invention can be converted by isotype switching to IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibodies for various therapeutic applications, hi one example, the antibody of the invention is an IgG1 antibody, e.g., IgG1, κ.

[0053] In the present invention, the portion of the constant region includes at least the region where the first Fc chain and the second Fc chain interact with each other. For IgG, the region is a portion of amino acids located in the CH3 region, including at least GLN347, TYR349, THR350, LEU351, SER354, ARG355, ASP356, GLU357, LYS360, SER364, THR366, LEU368, LYS370, ASN390, LYS392, THR394, PR0395, VAL397, ASP399, SER400, PHE405, TYR407, LYS409, and LYS439.

[0054] "The first Fc chain and the second Fc chain are each linked to one antigen-binding functional region via a covalent bond or a linker" refers to the first Fc chain and the second Fc chain being each linked to an antigen-binding fragment of an antibody, or a single-chain antibody capable of recognizing an antigen, or a structural variant of another antibody fragment capable of recognizing an antigen, or a receptor capable of recognizing a ligand, or a ligand capable of recognizing a receptor, via a covalent bond or a linker. Here, the covalent bond is a type of chemical bond in which two or more atoms share their outer electrons, ideally reaching an electron-saturated state, thereby forming a relatively stable chemical structure called a covalent bond. In other words, a covalent bond is an interaction formed between atoms by sharing an electron pair. Atoms of the same element or different elements may be linked together via a covalent bond. The covalent bond between the first Fc chain and the second Fc chain of the present invention includes, but is not limited to, an amide bond formed by a dehydration reaction between the amino group of one amino acid molecule and the carboxyl group of another amino acid molecule, or an amide bond or imine bond formed between an aldehyde group of ethylene glycol, polyethylene glycol, or other compounds or polymers thereof and an amino group of one amino acid molecule. Here, the linker is an amino acid sequence or compound or polymer of compounds that can link two polypeptide chains by a covalent bond, where the amino acid sequence includes, but is not limited to, a small peptide segment such as GGGSGGGGSGGGGGS. The first Fc chain or the second Fc chain can be linked to a single chain antibody capable of recognizing an antigen or a structural variant of another antibody fragment capable of recognizing an antigen via an amide bond.

[0055] The first Fc chain and the second Fc chain tend to form heterodimers rather than homodimers, since in the first Fc chain and the second Fc chain, there is a repulsive force between the same polypeptide chains, while there is an attractive force between different polypeptide chains, when co-expressed in a cell, the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and the antigen-binding functional region linked thereto tend to form a heterodimer. When the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and the antigen-binding functional region linked thereto are separately expressed in two host cells, the first Fc chain or the first Fc chain and the antigen-binding functional region linked thereto do not tend to form homodimers, and the second Fc chain or the second Fc chain and the antigen-binding functional region linked thereto do not tend to form homodimers.

[0056] The Kabat EU index numbering system refers to the method used by Kabat to assign a number to each amino acid in an antibody sequence, and such a method of assigning a number to each residue has become a standard method in the art. The Kabat scheme can be extended beyond his work to other antibodies. Based on conserved amino acids, the target antibody is aligned with one of the consensus sequences identified by Kabat.

[0057] Fc domain refers to the fragment crystallizable region (Fc) corresponding to the CH2 and CH3 domains of Ig, which is the portion of Ig that interacts with effector molecules or cells.

[0058] IgG, short for Immunoglobulin G (IgG), is the major antibody component in serum. Human IgG is classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, based on antigenic differences in the r chain of the IgG molecule.

[0059] A half antibody molecule is a structure formed by one heavy chain and one light chain of an antibody, which may or may not be linked via a covalent bond, and is a monovalent antibody structure that recognizes an antigen.

[0060] Fab fragment is an antigen binding fragment (Fab) and is a molecular recognition sequence corresponding to the two arms of an antibody molecule, which consists of an intact light chain and the VH and CHI domains of the heavy chain. scFv is a molecular recognition sequence and is a structural variant of an antibody fragment obtained by genetic engineering modification of the light chain variable region and the heavy chain variable region of an antibody. The extracellular region of a membrane receptor is a molecular recognition sequence. A membrane receptor usually includes an extracellular region located outside the cell that can recognize and bind to the corresponding antigen or ligand, a transmembrane region that anchors the receptor to the surface of the cell, and an intracellular region located inside the cell that has kinase activity or can transmit a signal. The ligand of a cell membrane receptor refers to a protein, small peptide, or compound that is recognized and bound by the extracellular region of the membrane receptor. Cytokines are low molecular weight soluble proteins produced by various cells induced by immunogens, mitogens, or other stimuli, and have various functions such as innate immunity, adaptive immunity, hematopoiesis, cell growth and regulation of adult pluripotent stem cells (APSCs), and repair of damaged tissues. Cytokines are classified as interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors, etc. Protein expression tags refer to segments of amino acid sequence, either small peptides or long amino acids, that are added to the N- or C-terminus of a target protein. Addition of tags can be advantageous for correct folding of proteins, isolation and purification of proteins, and reduction of intracellular degradation of proteins. Commonly used tags include, but are not limited to, HA, SUMO, His, GST, GFP, and Flag.

[0061] There is no limitation on the antibodies applicable to the heterodimeric bispecific antibodies of the present invention. Preferably, any antibody that can be used for the treatment and / or prevention of diseases known in the prior art may be used in the present invention.

[0062] The heterodimeric bispecific antibodies of the present invention may have one or more substitutions, deletions, additions, and / or insertions. For example, some amino acids can be substituted with other amino acids in the protein structure without significant loss of binding ability to other polypeptides (e.g., antigens) or cells. Since the biological functional activity of a protein is determined by the binding ability and characteristics of the protein, some amino acid sequence substitutions can be made to the protein sequence without significant loss of their biological effectiveness or activity.

[0063] Often, a polypeptide variant will contain one or more conservative substitutions, which refers to the replacement of an amino acid with another amino acid having similar characteristics, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydrophilicity of the polypeptide to remain substantially unchanged.

[0064] Amino acid substitutions are typically based on the relative similarity of the amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account various such characteristics are known to those of skill in the art and include arginine and lysine, glutamic acid and aspartic acid, serine and threonine, glutamine and asparagine, as well as valine, leucine, and isoleucine.

[0065] The term "identity" as used in the present invention has the meaning commonly known in the art, and the rules and standards for measuring identity between different sequences are also known to those skilled in the art, and refers to the percentage of identical residues between a polynucleotide or polypeptide sequence variant and a non-variant sequence after aligning the sequences and introducing gaps (if necessary to achieve the maximum homology percentage). In the present invention, if the definition of identity is satisfied, the resulting variant sequence is also required to have the biological activity possessed by the parent sequence. Methods and means for screening the above variant sequences with activity are known to those skilled in the art. Those skilled in the art can easily obtain such variant sequences from the teachings disclosed herein. In specific embodiments, polynucleotide and polypeptide variants have at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or at least about 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% polynucleotide or polypeptide identity to the polynucleotides or polypeptides described herein. Due to redundancy in the genetic code, variants exist that encode the same amino acid sequences as these sequences.

[0066] In another embodiment of the present invention, a polynucleotide composition is provided that can hybridize to the polynucleotide sequence provided by the present invention, or a fragment thereof, or a complementary sequence thereof, under moderate to high stringency conditions. Hybridization techniques are known in the field of molecular biology. For illustrative purposes, suitable moderately stringent conditions for testing hybridization of the polynucleotide of the present invention with other polynucleotides include pre-washing with a solution of 5xSSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridizing overnight under 5xSSC conditions at 50°C to 60°C, and washing twice for 20 minutes with 2x, 0.5x, and 0.2xSSC containing 0.1% SDS at 65°C. Those skilled in the art will appreciate that the stringency of hybridization can be easily manipulated, for example, by changing the salt content of the hybridization solution and / or the hybridization temperature. For example, in another embodiment, suitable highly stringent hybridization conditions include those described above, but differ by increasing the hybridization temperature, for example, to 60-65°C or 65-70°C.

[0067] The host cell of the present invention may be any cell that expresses a foreign gene, including, but not limited to, E. coli, yeast, insect cells, plant cells, and mammalian cells.

[0068] Vectors of the invention include vectors capable of replicating in any type of cell or organism, including, for example, plasmids, phages, cosmids, and minichromosomes. In some embodiments, a vector comprising a polynucleotide of the invention is a vector suitable for propagation or replication of a polynucleotide, or a vector suitable for expression of a polypeptide of the invention. Such vectors are known in the art and commercially available.

[0069] "Vector" includes shuttle vectors and expression vectors. Typically, a plasmid construct further comprises an origin of replication (e.g., CoEl origin of replication) for plasmid replication and selection in bacteria, and a selection marker (e.g., ampicillin or tetracycline resistance). "Expression vector" refers to a vector that contains the control sequences or regulatory elements necessary to express the antibodies of the invention, including antibody fragments, in bacteria or eukaryotic cells.

[0070] The vector of the present invention may be any vector for expressing a foreign gene, including but not limited to a plasmid vector, where the plasmid vector includes at least a replication origin, a promoter, a target gene, multiple cloning sites, and a selection marker gene, and preferably the vector of the present invention includes but is not limited to a plasmid vector obtained by modification based on pCDNA, such as X0GC vector.

[0071] Subjects of the present invention include birds, reptiles, mammals, etc. Preferably, mammals include rodents, primates, and more preferably, primates include humans.

[0072] The scope of the disease according to the present invention includes, but is not limited to, inflammation and autoimmune diseases. Preferably, the disease is selected from rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, gouty arthritis, juvenile rheumatoid arthritis, suppurative arthritis, psoriasis, type I diabetes, multiple sclerosis, autoimmune encephalomyelitis, Crohn's disease, systemic vasculitis, dermatomyositis, mixed connective tissue disease, lupus erythematosus, idiopathic thrombocytopenic purpura, primary Sjogren's syndrome, glomerulonephritis, gout, organ transplant rejection, asthma, or arteriosclerosis. More preferably, the disease is selected from rheumatoid arthritis, osteoarthritis, psoriatic arthritis, gouty arthritis, juvenile rheumatoid arthritis, and suppurative arthritis.

[0073] The pharma- ceutically acceptable carrier refers to a common pharmaceutical carrier in the pharmaceutical field, such as a diluent, excipient, water, etc., a filler such as starch, sucrose, lactose, microcrystalline cellulose, etc., a binder such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone, a wetting agent such as glycerin, a disintegrant such as sodium carboxymethyl starch, hydroxypropyl cellulose, crosslinked carboxymethyl cellulose, agar, calcium carbonate, and sodium bicarbonate, an absorption promoter such as a quaternary ammonium compound, a surfactant such as hexadecanol, sodium dodecyl sulfate, an adsorption carrier such as kaolinite, bentonite, etc., a lubricant such as talc, calcium stearate, and magnesium stearate, finely powdered silica gel, and polyethylene glycol, etc. In addition, other adjuvants such as flavoring agents and sweeteners may be added to the composition.

[0074] The present invention will be further described below with reference to the following non-limiting examples. Those skilled in the art will recognize that various modifications can be made to the present invention without departing from the spirit of the present invention. Such modifications are also included within the scope of the present invention.

[0075] The following experimental methods are all conventional methods unless otherwise stated. The experimental materials used are readily available from commercial companies unless otherwise stated. The antibodies used in the following examples of the present invention are all commercially available standard antibodies.

[0076] Example 1 Vector construction of anti-TNFα / anti-IL-17A heterodimer antibody molecule X0GC expression vectors were constructed containing the heavy and light chains of anti-human TNFα antibody (Adalimumab), where the antibody variable region sequences are derived from https: / / www.drugbank.ca / drugs / DB00051. The nucleotide sequence of the light chain variable region is shown in SEQ ID NO:1, the amino acid sequence is shown in SEQ ID NO:2, the nucleotide sequence of the light chain constant region is shown in SEQ ID NO:3, the amino acid sequence is shown in SEQ ID NO:4, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO:5, the amino acid sequence is shown in SEQ ID NO:6, the nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO:7, and the amino acid sequence is shown in SEQ ID NO:8. The light chain variable region and the light chain constant region, and the heavy chain variable region and the heavy chain constant region were amplified by PCR, respectively. Phusion high fidelity DNA polymerase (F-530L) from NEB was used in all PCR reactions in this study. PCR primers were conventionally designed according to the principles of complementary base pairing and the requirements of the enzyme cleavage site. All reactions were carried out in 8.9 μL of H 2 0, 4 μL of 5× Phusion High Fidelity DNA Polymerase Buffer, 4 μL of 1 mM dNTP, 1 μL of upstream primer, 1 μL of downstream primer, 0.1 μL of Phusion High Fidelity DNA Polymerase and 1 μL of template. The PCR products of the variable and constant regions were subjected to 1.5% agarose gel electrophoresis, and the corresponding fragments were recovered using a DNA recovery kit (Promega, A9282, the same below). Using the recovered variable and constant region fragments as templates, further PCR reactions were performed using the upstream primer of the variable region and the downstream primer of the constant region, and then the corresponding fragments were recovered to obtain the full-length fragments of the light chain or heavy chain. The X0GC vector and the full-length fragment were enzymatically cleaved with EcoRI (NEB, catalog number R3101L) and HindIII (NEB, catalog number R3104L). The enzyme digestion reaction system consisted of 2 μL of 10× buffer 3, 0.5 μL each of EcoRI and HindIII, 3 μL of full-length fragment recovered from the gel, and 14.5 μL of H 2The enzyme digestion reaction system was reacted at 37°C for 3 hours. The enzyme digestion product was ligated with T4 DNA ligase (NEB, catalog number M0202V) (hereinafter the same). The reaction system consisted of 2 μL of 10× ligase buffer, 0.5 μL of ligase, 3 μL of full-length fragment recovered from the gel, 3 μL of X0GC vector recovered from the gel, and 11.5 μL of H 2 The temperature was 0°C. Ligation was carried out at room temperature for 12 hours. The ligated products were transformed into E. coli DH5α competent cells (Tiangen, CB104, the same below). The antibody heavy and light chain X0GC expression vectors were obtained to express the antibody heavy and light chains in eukaryotic cells, respectively.

[0077] X0GC expression vectors containing the heavy and light chains of anti-human IL-17A antibody (BJHM) were constructed, respectively, where the antibody variable region sequences were derived from patent application WO2018050028. The nucleotide sequence of the light chain variable region is shown in SEQ ID NO:9, the amino acid sequence is shown in SEQ ID NO:10, the nucleotide sequence of the light chain constant region is shown in SEQ ID NO:3, the amino acid sequence is shown in SEQ ID NO:4, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO:11, the amino acid sequence is shown in SEQ ID NO:12, the nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO:13, and the amino acid sequence is shown in SEQ ID NO:14. The X0GC expression vectors of the heavy and light chains of the antibody were obtained to express the heavy and light chains of the antibody in eukaryotic cells, respectively.

[0078] Example 2 Expression of anti-TNFα / anti-IL-17A heterodimeric antibody molecules Expression vectors containing the heavy and light chains of anti-human or mouse TNFα antibodies were transfected into ExpiCHO cells (ExpiCHO TMThe expression vectors containing the heavy and light chains of anti-human or mouse IL-17A antibodies were also transfected into ExpiCHO cells. The day before transfection, the cells were cultured at 3.5 × 10 6 On the day of transfection, fresh ExpiCHO expression medium (ExpiCHO TM Expression Medium (catalog no. A29100-01, Invitrogen) at 6 × 10 cells 6 The plasmid was taken according to the transfection volume and the final concentration of the plasmid was 0.5 μg / mL, and the OptiPRO TM SFM medium (OptiPRO TM Plasmids were diluted to 4% of the transfection volume in SFM (Invitrogen, Catalog No. 12309-019) and mixed by inversion. TM Transfection reagent (ExpiFectamine TM CHO Transfection Kit, Catalog No. A29129, Invitrogen) and OptiPRO TM The transfection reagent was diluted with SFM medium to 4% of the transfection volume and mixed by inversion. The diluted transfection reagent was added to the diluted plasmid, mixed gently, left at room temperature for 1 to 5 min, and gradually added dropwise to the cells. Then, the cells were incubated in a cell incubator (CO 2 The mixture was incubated at 37°C for 20 hours in a shaker at 120 rpm. TM Enhancer (ExpiFectamine TM CHO Transfection Kit, Catalog No. A29129, Invitrogen) and 0.24x the transfection volume of ExpiCHO TM Feed (ExpiCHO TMFeed (cat. no. A29101-02, Invitrogen) was slowly added dropwise to the cells. They were incubated at 32°C in a shaker at 120 rpm. The supernatant of the 10-day transfected cell culture was collected by centrifugation.

[0079] The expression level was measured by ELISA. Before purification by chromatography column, the precipitate was removed by filtration through a 0.2 μm filter membrane. This step was performed at 4°C.

[0080] Example 3. Purification of anti-TNFα / anti-IL-17A heterodimer antibody molecule expression product Purification was performed at room temperature using an AKTA explorer 100 protein purification system (GE Healthcare) and an affinity chromatography column Mabselect SuRe (16 mm ID, 27 mL, GE Healthcare). First, the chromatography column was equilibrated with mobile phase A (20 mM sodium phosphate buffer, pH 7.4) and the baseline was stabilized, and then the supernatant of the cells treated as described above was loaded at a flow rate of 5 mL / min, and after loading, mobile phase A was used for equilibration. The samples were the anti-TNFα expression product and the anti-IL-17A expression product expressed in Example 2, respectively. Then, the column was washed with 3 column volumes of mobile phase B1 (mobile phase A containing 1 M sodium chloride), and then washed with 1 column volume of equilibrated solution A (20 mM sodium phosphate buffer, pH 7.4), and finally, elution was performed with 5 column volumes of mobile phase B2 (100 mM glycine, 10 mM sodium chloride pH 3.3), and the elution peak, i.e., the peak of the target protein, was collected. In the above elution steps, the flow rate was 5 mL / min. The chromatogram of the elution peak of anti-TNFα is shown in FIG. 1A, and the chromatogram of the elution peak of anti-IL-17A is shown in FIG. 1B. The elution peaks shown (gray areas shown in the figure) were collected, and the pH was adjusted to 7.0 by adding 1 M Tris solution dropwise.

[0081] Example 4. Purification of anti-TNFα / anti-IL-17A heterodimeric antibody molecules The structure of the anti-TNFα / anti-IL-17A heterodimer antibody molecule is shown in FIG.

[0082] Heterodimers were obtained by in vitro reconstitution of anti-TNFα and anti-IL-17A expression products purified by Mabselect SuRe (16 mm ID, 27 mL, GE Healthcare) column in Example 3 above. First, the protein solution purified and collected as described above was concentrated by ultrafiltration through an ultrafiltration concentration tube (nominal cut-off molecular weight of 10 kDa), and the solution was replaced with phosphate buffer saline (PBS) (pH = 7.4). The PBS was added to the obtained anti-TNFα and anti-IL-17A expression products, respectively, to adjust the concentration to 1 mg / mL, and 1 M DTT was added at 1 / 200 times the final volume so that the final concentration of DTT was 5 mM. Reduction was performed at 4°C (3 to 8 hours), and disulfide bonds were cleaved by the reduction process. The disulfide bonds in the hinge regions of the homodimeric antibody molecules contained in small amounts in the anti-TNFα and anti-IL-17A expression products were also cleaved to form half antibody molecules containing one heavy chain and one light chain, the structures of which are shown in Figure 3. The reduced samples were analyzed by SEC-HPLC in which the mobile phase buffer contained 1 mM DTT reducing agent, and the results are shown in Figures 4A and 4B, where the weight percentages of the anti-TNFα and anti-IL-17A homodimeric molecules were both less than 10%, and correspondingly, the weight percentages of the half antibody molecules were both greater than 0%.

[0083] The reduced anti-TNFα and anti-IL-17A half antibody molecules were then mixed in an equimolar ratio and reconstituted at 4°C for 24 hours. In the process of reconstitution, a heterodimeric bispecific antibody containing both anti-TNFα and anti-IL-17A half antibody molecules was formed from the anti-TNFα and anti-IL-17A half antibody molecules via non-covalent interactions between CH2 / CH3. The protein solution was then concentrated by ultrafiltration through an ultrafiltration concentration tube (nominal cut-off molecular weight of 10 kDa). The solution was replaced with a phosphate solution (PBS, pH=7.4) to stop the reduction. An oxidation reaction was performed with air or an oxidizing agent to reorganize the disulfide bonds of the heterodimeric bispecific antibody. The oxidation reaction was performed under the conditions of adding 100 mM L-dehydroascorbic acid as an oxidizing agent, making the final protein concentration 1 mg / mL, and making the final oxidizing agent concentration 1 mM, and the oxidation reaction was performed at 4°C for 24 hours. The samples obtained by the above oxidation reaction were analyzed by capillary electrophoresis, and the results are shown in FIG.

[0084] The heterodimer molecules obtained by reduction-oxidation of the above anti-TNFα and anti-IL-17A half antibody molecules were concentrated by ultrafiltration through an ultrafiltration concentration tube (nominal cut-off molecular weight of 10 kDa). The solution was replaced with 10 mM sodium phosphate buffer (pH = 5.8). Purification was performed at 4 ° C. using an AKTA explorer 100 type protein purification system (GE Healthcare) and an ion chromatography column Source 15S (10 mm ID, 2 mL, GE Healthcare). First, the chromatography column was equilibrated with mobile phase A (10 mM sodium phosphate buffer, pH 7.0). After stabilizing the baseline, the protein solution treated as described above was loaded at a flow rate of 1 mL / min. After loading, mobile phase A was used for equilibration. The column was then washed with a gradient of A (10 mM sodium phosphate, pH 5.8) to B (10 mM sodium phosphate, pH 5.8) (0% B to 100% B, 80 min, flow rate 0.5 mL / min) for 20 column volumes. The main elution peak was collected (shown in Figure 6), and the collected protein solution was concentrated by ultrafiltration through an ultrafiltration concentration tube (nominal cut-off molecular weight of 10 kDa). The solution was replaced with a phosphate solution (PBS, pH = 7.4), sterilized by filtration, and stored at 4 °C. This anti-human TNFα / anti-human IL-17A heterodimer antibody was named BH1657. The purified product was analyzed by capillary electrophoresis method, and the results are shown in Figure 7. Purity analysis by SEC-HPLC showed the results in Figure 8, and the purity was 99.9%.

[0085] Example 5. Preparation of anti-mouse TNFα / anti-mouse IL-17A heterodimer antibody molecule According to the method described in Examples 1 to 4, the present invention simultaneously constructed an X0GC expression vector containing the heavy and light chains of an anti-mouse TNFα antibody (whose heavy and light chain variable region sequences are obtained by cloning from V1q hybridoma cells secreting rat anti-mouse TNFα antibody (Echtenacher et al., J. Immunol. 145: 3762-3766 (1990)) and an X0GC expression vector containing the heavy and light chains of an anti-mouse IL-17A antibody (whose heavy and light chain variable region sequences are derived from patent US7846443, 1D10). The nucleotide sequence of the light chain constant region of the anti-mouse TNFα antibody is shown in SEQ ID NO: 3, the amino acid sequence is shown in SEQ ID NO: 4, the nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8. The nucleotide sequence of the light chain variable region of the anti-mouse IL-17A antibody is shown in SEQ ID NO: 1. The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO:15, the amino acid sequence is shown in SEQ ID NO:16, the nucleotide sequence of the light chain constant region is shown in SEQ ID NO:3, the amino acid sequence is shown in SEQ ID NO:4, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO:17, the amino acid sequence is shown in SEQ ID NO:18, the nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO:13, and the amino acid sequence is shown in SEQ ID NO:14. Then, X0GC expression vectors containing the heavy and light chains of these antibodies were obtained and used to express the heavy and light chains of the antibodies in eukaryotic cells, respectively. Then, an anti-mouse TNFα / anti-mouse IL-17A heterodimer antibody was obtained by expression / purification and named BH1654, which has a purity similar to that of BH1657 (data not shown).

[0086] Example 6. Stability of anti-TNFα / anti-IL-17A heterodimeric antibody molecules 1 mg / mL of the well-sealed anti-TNFα / anti-IL-17A heterodimer antibody (BH1657) sample obtained in Example 4 was placed in a 40°C incubator (Boxun Biochemical Incubator BSP-400), and 10 μg of the sample was taken at the corresponding time points (baseline (0th week), 1st week, 4th week, 6th week, and 8th week) and isolated by high performance size exclusion chromatography (SEC-HPLC). The above SEC-HPLC conditions were as follows: (1) Size exclusion chromatography column: Waters xbridge BHE200 3.5 um, 7.8 mm x 30 cm; (2) Mobile phase: 0.1 M PB pH 6.7 + 0.1 M Na 2 SO 4 , pH 6.7; (3) flow rate: 0.6 mL / min; (4) UV detection wavelength: 280 nm; (5) collection time: 40 min. The instrument used was an Agilent 1200 Infinity Chromatography, and the chromatogram was recorded by Agilent ChemStation to calculate the remaining monomer ratio. As shown in Table 1, under the experimental conditions of 40°C, the change in the ratio of the main peak was small, so that the anti-TNFα / anti-IL-17A heterodimer antibody is considered to have good thermal stability.

[0087] [Table 1]

[0088] Example 7. Target binding activity of anti-TNFα / anti-IL-17A heterodimer antibodies The binding kinetic constants of anti-TNFα / anti-IL-17A heterodimer antibody (BH1657) with its antigens TNFα and IL-17A were measured using a Biacore X100 instrument. This instrument detects the binding and dissociation between molecules bound and coated on a biochip and test molecules by optical surface plasmon resonance technology. The main reagent used is a Protein A chip (GE Healthcare, 29-1275-57). The experimental process is outlined as follows: BH1657 sample and standard (anti-TNFα monoclonal antibody and anti-IL-17A monoclonal antibody), human TNFα and human IL-17A were respectively diluted in 1×HBS-EP+ solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4). In the capture and binding phase, BH1657 samples or standards with a concentration of 1 μg / mL were injected at a rate of 10 μL / min for 60 seconds. In the binding phase, different concentrations of human TNFα and solvent control (1×HBS-EP+ solution) were injected at a rate of 30 μL / min for 120 seconds, respectively, or different concentrations of human IL-17A and solvent control were injected at a rate of 30 μL / min for 60 seconds, respectively. In the dissociation phase, 1×HBS-EP+ solution was used for injection at a rate of 10 μL / min for 1800 seconds. The regeneration condition was 10 mM glycine salt solution, pH 1.5. The binding and dissociation kinetic constants were combined and analyzed and calculated by Biacore X100 control software.

[0089] The binding kinetic constants, dissociation kinetic constants and equilibrium dissociation constants for BH1657, human TNFα and human IL-17A are shown in Tables 2 and 3, respectively. The results demonstrated that the anti-TNFα / anti-IL-17A heterodimer retained antigen-binding activity consistent with the parent monoclonal antibody.

[0090] [Table 2]

[0091] [Table 3]

[0092] The binding ability of anti-TNFα / anti-IL-17A heterodimer antibody BH1657 to different types of single antigens was measured by enzyme-linked immunosorbent assay (ELISA).

[0093] The specific implementation process is as follows: Recombinant human TNFα, monkey TNFα, human IL-17A, and monkey IL-17A (all purchased from Beijing Sino Biological Co., Ltd.) were coated and reconstituted on a 96-well high-adsorption ELISA plate using a carbonate buffer solution of pH=9.6 at a coating concentration of 1 μg / mL at 100 μL / well, and the coating was performed overnight at 4°C. Washed five times with PBST. Blocked with PBST containing 1% BSA at 300 μL / well and incubated at 25°C for 1 hour. Washed five times with PBST. BH1657 samples and controls serially diluted in PBST containing 1% BSA were added at 100 μL / well and incubated at 25°C for 1 hour. Washed five times with PBST. Then, 100 μL / well of anti-human IgG antibody labeled with horseradish peroxidase (Chemicon, Cat. No. AP309P) diluted 1:10000 in PBST containing 1% BSA was added and incubated for 1 hour at 25°C. The plate was washed 5 times with PBST. 100 μL / well of the color-developing substrate TMB was added and developed for 10 minutes at room temperature. 1M H 2 SO 4 Color development was stopped by adding 100 μL / well of 100 μL of 100 mM NaCl. The absorbance at 450 nm was read in a microplate reader.

[0094] The binding ability of BH1657 to different types of TNFα and different types of IL-17A is shown in Table 4. The anti-TNFα / anti-IL-17A heterodimer had good binding ability to human TNFα, monkey TNFα, human IL-17A, and monkey IL-17A, and the activity was similar to that of the parent monoclonal antibody.

[0095] [Table 4]

[0096] Example 8. In vitro neutralizing activity of anti-TNFα / anti-IL-17A heterodimer antibodies Human foreskin fibroblasts HFF-1, capable of secreting a series of cytokines and chemokines under stimulation with TNFα or IL-17A, were used to measure the neutralizing activity of the anti-TNFα / anti-IL-17A heterodimer antibody BH1657 against its targets.

[0097] TNFα neutralizing activity: BH1657 samples and control samples were serially diluted in DMEM (GIBCO, Cat. No. 11995-073) complete medium containing 15% FBS (Hyclone, Cat. No. SH30084.03) and added to cell culture plates at 50 μL / well. Human TNFα was also added to the cell culture plates at a concentration of 2 ng / mL (final concentration 0.5 ng / mL) diluted in the same complete medium at 50 μL / well. Culture was incubated at 37°C with 5% CO 2 Incubated in an incubator for 1 hour. HFF-1 cells (ATCC, Catalog No. SCRC-1041) were then resuspended in complete medium and seeded into 96-well cell culture plates at 5000 cells / well, 100 μL per well. Culture was incubated at 37°C with 5% CO 2 The cells were incubated in an incubator for 24 hours. After the end of incubation, the cell culture plates were centrifuged at 250g for 5 minutes, the culture supernatants were taken out, and the levels of human IL-6 were measured according to the instructions of the human IL-6 ELISA kit (R&D systems, Cat. No. S6050). The results are shown in Figure 9A, and BH1657 significantly blocked TNFα-induced IL-6 secretion and exhibited good TNFα neutralizing activity, which was slightly weaker than its bivalent anti-TNFα parent monoclonal antibody.

[0098] IL-17A neutralizing activity: BH1657 samples and control samples were serially diluted in DMEM (GIBCO, Cat. No. 11995-073) complete medium containing 15% FBS (Hyclone, Cat. No. SH30084.03) and added to cell culture plates at 50 μL / well. Human IL-17A diluted in the same complete medium at a concentration of 20 ng / mL (final concentration 0.5 ng / mL) was added to cell culture plates at 50 μL / well. Cultures were incubated at 37°C in 5% CO 2 Incubated in an incubator for 1 hour. HFF-1 cells (ATCC, Catalog No. SCRC-1041) were then resuspended in complete medium and seeded into 96-well cell culture plates at 5000 cells / well, 100 μL per well. Culture was incubated at 37°C with 5% CO 2 The cells were incubated in an incubator for 24 hours. After the end of incubation, the cell culture plates were centrifuged at 250g for 5 minutes, the culture supernatants were taken out, and the levels of human IL-6 were measured according to the instructions of the human IL-6 ELISA kit (R&D systems, Cat. No. S6050). The results are shown in Figure 9B, and BH1657 significantly blocked IL-17A-induced IL-6 secretion and showed good IL-17A neutralizing activity, which was slightly weaker than its bivalent anti-IL-17A parent monoclonal antibody.

[0099] TNFα and IL-17A have synergistic effects on HFF-1. To measure the neutralizing activity in the presence of TNFα and IL-17A simultaneously: BH1657 samples and control samples were serially diluted in DMEM (GIBCO, Cat. No. 11995-073) complete medium containing 15% FBS (Hyclone, Cat. No. SH30084.03) and added to the cell culture plate at 50 μL / well. Human TNFα at a concentration of 2 ng / mL (final concentration of 0.5 ng / mL) and human IL-17A at a concentration of 10 ng / mL (final concentration of 2.5 ng / mL) diluted in the same complete medium were added to the cell culture plate at 50 μL / well. Culture was performed at 37°C and 5% CO 2The cells were then resuspended in complete medium and seeded into 96-well cell culture plates at 5000 cells / well, 100 μL per well. The cells were incubated in a 5% CO incubator at 37°C. 2 The cells were incubated in an incubator for 24 hours. After the incubation, the cell culture plate was centrifuged at 250g for 5 minutes, the culture supernatant was taken out, and the level of human IL-6 was measured according to the instructions of the human IL-6 ELISA kit (R&D systems, catalog number S6050). The results are shown in Figure 9C, and BH1657 significantly blocked TNFα and IL-17A-induced IL-6 secretion in HFF-1, and at high concentrations it could completely block the level of IL-6, which was stronger than its bivalent parent monoclonal antibody.

[0100] Example 9. Effect of the presence or absence of IL-17A on the anti-TNFα activity of anti-TNFα / anti-IL-17A heterodimer antibody The effect of IL-17A on the TNFα / TNFR2 blocking activity of anti-TNFα / IL-17A heterodimer antibody BH1657 was measured by ELISA. The specific implementation process is as follows: Recombinant human TNFα (purchased from Beijing Sino Biological Co., Ltd.) was coated and reconstituted on a 96-well high-adsorption ELISA plate using carbonate buffer solution at pH=9.6 at a coating concentration of 1μg / mL at 100μL / well, and the coating was performed overnight at 4℃. Washed five times with PBST. Blocked with PBST containing 1% BSA at 300μL / well and incubated at 25℃ for 1 hour. Washed five times with PBST. 25 μL of BH1657 samples and controls serially diluted in PBST containing 1% BSA were added, and 25 μL of human IL-17A (purchased from Beijing Sino Biological Co., Ltd.) (16 μg / mL, final concentration 4 μg / mL) and 50 μL of biotin-labeled TNFR2-Fc (purchased from Beijing Sino Biological Co., Ltd.) (0.3 μg / mL, final concentration 0.15 μg / mL) were added and incubated for 1 hour at 25°C. The plates were washed five times with PBST. 100 μL / well of horseradish peroxidase-labeled streptavidin (BD, Cat. No. 554066) diluted 1:1000 in PBST containing 1% BSA was then added and incubated for 1 hour at 25°C. The plates were washed five times with PBST. 100 μL / well of the color-developing substrate TMB was added and allowed to develop at room temperature for 10 minutes. 1M H 2 SO 4 Color development was stopped by adding 100 μL / well of 100 μL of 100 mM NaCl. Absorbance at 450 nm was read on a microplate reader. The results are shown in FIG. 10A, and in the absence of IL-17A, the TNFα / TNFR2 blocking activity was lower than that of the parental monoclonal antibody. In the presence of IL-17A, the activity was enhanced and comparable to that of the parental monoclonal antibody. The TNFα / TNFR2 blocking activity of the anti-TNFα parental monoclonal antibody was not affected by IL-17A.

[0101] L-929 cells (purchased from the Basic Medical Cell Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were used to measure the effect of IL-17A on the TNFα neutralizing activity of anti-TNFα / anti-IL-17A heterodimer antibody BH1657. The specific implementation process is as follows: The complete medium for L-929 cells was MEM (GIBCO, catalog number 10370-021) medium containing 10% FBS (Hyclone, catalog number SH30084.03). L-929 cells were resuspended in complete medium and seeded into 96-well cell culture plates at 15000 cells / well, 100 μL per well. Culture was incubated at 37°C with 5% CO 2 The cells were incubated overnight in an incubator. The culture supernatant was discarded, and the BH1657 samples and control samples were serially diluted in complete medium containing 2 ng / mL human TNFα and 1 μg / mL actinomycin D, and added to the cell culture plate at 100 μL / well. The cells were incubated at 37°C with 5% CO 2 The cells were then incubated in an incubator for 24 hours. At the end of the incubation, 20 μL of MTS (CellTiter96 Aqueous One Solution, Promega, Cat. No.: G358B) was added to each well of the cell culture plate to detect cell vitality. The results are shown in FIG. 10B, and show that in the presence of IL-17A, the TNFα neutralizing activity of the anti-TNFα moiety in the anti-TNFα / anti-IL-17A heterodimer bispecific antibody is significantly stronger than that in the absence of IL-17A. This difference in activity of the anti-TNFα / anti-IL-17A bispecific antibody suggests that the bispecific antibody effectively treats the disease while maintaining low toxic side effects, since it has stronger TNFα neutralizing activity at the lesion site where the level of IL-17A is high, but shows weaker TNFα neutralizing activity in the circulatory system where the level of IL-17A is low.

[0102] Example 10. In vivo neutralizing activity of anti-TNFα / anti-IL-17A heterodimer antibodies Female BALB / c mice aged 6-8 weeks purchased from Beijing HFK Bioscience were used as experimental materials. After adapting to the environment for 1 week, the mice were randomly divided into groups (10 mice per group). Each group was intravenously administered a single dose of anti-TNFα / IL-17A heterodimer antibody BH1657, anti-TNFα monoclonal antibody, anti-IL-17A monoclonal antibody, and a combination of anti-TNFα monoclonal antibody and anti-IL-17A monoclonal antibody at three dose levels (0.1 mg / kg, 1 mg / kg, and 10 mg / kg for monoclonal antibodies, and 0.2 mg / kg, 2 mg / kg, and 20 mg / kg for bispecific antibodies). One hour after administration, 0.2 μg of TNFα and 20 μg of IL-17A were subcutaneously injected per mouse. After 2 hours, blood was collected from the orbital vein without anticoagulation, and the blood sample was left at room temperature for 30 minutes to 1 hour. After clotting, the blood sample was centrifuged at 3000 rpm for 10 minutes to obtain serum samples. The concentration of mouse CXCL1 in serum was measured according to the instructions of the Mouse CXCL1 ELISA Kit (RayBiotech, Catalog No. ELM-KC).

[0103] The results are shown in Figure 11, which shows that BH1657 significantly blocked the synergistic stimulation of CXCL1 secretion in mice by TNFα and IL-17A, and at high concentrations could completely block CXCL1 levels, more potently than its bivalent parent monoclonal antibody.

[0104] Example 11. Target binding activity of anti-mouse TNFα / anti-mouse IL-17A heterodimer antibodies The binding ability of anti-mouse TNFα / anti-mouse IL-17A heterodimer antibody (i.e., BH1654) with different kinds of single antigens was measured by enzyme-linked immunosorbent assay (ELISA). The specific implementation process is as follows: Recombinant mouse TNFα, rat TNFα, mouse IL-17A, and rat IL-17A (all purchased from Beijing Sino Biological Co., Ltd.) were coated and reconstituted on a 96-well high-adsorption ELISA plate using carbonate buffer solution with a pH of 9.6 at a coating concentration of 1 μg / mL at 100 μL / well, and the coating was performed overnight at 4 ° C. Washed five times with PBST. Blocked with PBST containing 1% BSA at 300 μL / well and incubated at 25 ° C. for 1 hour. Washed five times with PBST. BH1657 samples and standards (anti-mouse TNFα monoclonal antibody and anti-mouse IL-17A monoclonal antibody) serially diluted in PBST containing 1% BSA were added at 100 μL / well and incubated at 25°C for 1 hour. The plate was washed five times with PBST. Horseradish peroxidase-labeled anti-human IgG antibody (Chemicon, Cat. No. AP309P) diluted 1:10000 in PBST containing 1% BSA was then added at 100 μL / well and incubated at 25°C for 1 hour. The plate was washed five times with PBST. The color-developing substrate TMB was added at 100 μL / well and allowed to develop for 10 minutes at room temperature. 1M H 2 SO 4 Color development was stopped by adding 100 μL / well of 100 μL of 100 mM NaCl. The absorbance at 450 nm was read in a microplate reader.

[0105] The binding ability of BH1654 to different types of TNFα and different types of IL-17A is shown in Table 5. The anti-mouse TNFα / anti-mouse IL-17A heterodimer has good binding ability to mouse TNFα and mouse IL-17A, which is similar to the activity of the parent monoclonal antibody. It does not bind to rat TNFα and has weak binding to rat IL-17A.

[0106] [Table 5]

[0107] Example 12. Pharmacodynamic study of anti-mouse TNFα / anti-mouse IL-17A heterodimer antibodies in a type II collagen-induced murine arthritis (mCIA) model The mouse CIA model can well mimic human rheumatic diseases and predict the therapeutic effects of drugs on such human diseases, and has been widely applied in studying the pathogenesis of rheumatoid arthritis and screening of therapeutic drugs. The experimental animals used were 8-week-old male DBA1 / J mice (purchased from Shanghai SLAC Laboratory Animal Co., Ltd.). After adapting to the environment for one week, five mice were randomly selected as normal control mice, and the remaining mice were used to establish the mouse CIA model. The CIA model was obtained by performing primary and booster immunizations on the mice. For the primary immunization, 70 μg of type II bovine collagen (Chondrex, catalog number 20022) was mixed with Freund's complete adjuvant (Sigma-Aldrich, catalog number F5881) to form an emulsion, which was then injected intradermally into the tail base of the mice. Three weeks later, booster immunizations were performed. For booster immunization, 70 μg of type II bovine collagen was mixed with Freund's incomplete adjuvant (Sigma-Aldrich, Cat. No. F5506) to form an emulsion, which was then injected intradermally into the back of the mice. After booster immunization, the mice were observed for clinical symptoms of arthritis, such as erythroderma in the limbs and feet, and then divided into groups of 8 mice each.

[0108] Each group of CIA model mice was administered the drug. The drug solvent (PBS), anti-mouse TNFα monoclonal antibody (70nmol / kg), anti-mouse IL-17A monoclonal antibody (70nmol / kg), anti-mouse TNFα monoclonal antibody and anti-mouse IL-17A monoclonal antibody combination (70nmol / kg+70nmol / kg), and anti-mouse TNFα / mouse IL-17A heterodimer antibody BH1654 (70nmol / kg, 140nmol / kg) were administered intraperitoneally once every two days for a total of seven times. After administration, the body weight was weighed once every two days, and the lesion status of the front and back limbs was observed, and the arthritis index was evaluated. 0 = no erythema; 1 = erythema and mild swelling in the ankle or tarsal joint; 2 = erythema and slight swelling from the ankle to the ankle; 3 = erythema and moderate swelling from the ankle to the metatarsal joint; 4 = erythema and severe swelling of the paw including the ankle, interphalangeal joint, or stiffness in the limb. Mice were given an inflammation score for each limb, with a maximum score of 16 for each mouse.

[0109] The experimental results are shown in Figure 12. The mice in the normal control group did not develop an inflammatory response, while the CIA model mice in the vehicle group developed obvious inflammatory responses and the arthritis index rapidly increased, while the arthritis index of the CIA model mice in the anti-mouse TNFα / anti-mouse IL-17A heterodimer antibody BH1654-administered group was significantly suppressed, showing better inflammation-alleviating effects than anti-mouse TNFα monoclonal antibody and anti-mouse IL-17A monoclonal antibody.

[0110] Example 13. Pharmacodynamic study of anti-mouse TNFα / anti-mouse IL-17A heterodimer antibodies in a glucose-6-phosphate isomerase-induced mouse arthritis (mGPI) model Glucose-6-phosphate isomerase (GPI) or its 325-339 peptide fragment (GPI 325-339) to establish a mouse GPI model. This model is an animal model that has been developed in recent years and can closely mimic human rheumatic diseases, and is considered to be suitable for studying the pathogenesis of rheumatoid arthritis and screening of therapeutic drugs. Eight-week-old male DBA1 / J mice (purchased from Shanghai SLAC Laboratory Animal Co., Ltd.) were used as experimental animals. After adapting to the environment for one week, five mice were randomly selected as normal control mice, and the remaining mice were used to establish the mouse CIA model. The GPI model was obtained by first immunizing mice. The immunization consisted of 100 μg of GPI 325-339 (Synthesized by the chemical group of Beijing Hanmi Pharm Co., Ltd., with the amino acid sequence IWYINCFGCETHAML (SEQ ID No:19)) was mixed with Freund's complete adjuvant (Sigma-Aldrich, Cat. No. F5881) to form an emulsion, which was then injected intradermally into the tail base of mice. On the 1st (same day) and 3rd day after immunization, mice were administered pertussis toxin (Merck, Cat. No. 516560) via intraperitoneal injection. On the 5th day after immunization, the mice were randomly divided into groups with 8 mice per group.

[0111] Each group of GPI model mice was administered prophylactically. The drug solvent (PBS), anti-mouse TNFα monoclonal antibody (35nmol / kg), anti-mouse IL-17A monoclonal antibody (35nmol / kg), combination of anti-mouse TNFα monoclonal antibody and anti-mouse IL-17A monoclonal antibody (35nmol / kg+35nmol / kg), and anti-mouse TNFα / mouse IL-17A heterodimer antibody BH1654 (35nmol / kg, 70nmol / kg) were administered intraperitoneally once every two days for a total of seven times. After administration, the body weight was weighed once a day, and the lesion status of the front and back limbs was observed, and the arthritis index was evaluated. 0 = no erythema; 1 = erythema and mild swelling in the ankle or tarsal joint; 2 = erythema and slight swelling from the ankle to the ankle; 3 = erythema and moderate swelling from the ankle to the metatarsal joint; 4 = erythema and severe swelling of the paw including the ankle, interphalangeal joint, or stiffness in the limb. Mice were given an inflammation score for each limb, with a maximum score of 16 for each mouse.

[0112] The experimental results are shown in Figure 13. The normal control mice did not develop inflammatory reactions, while the vehicle GPI model mice developed obvious inflammatory reactions. The anti-mouse TNFα / anti-mouse IL-17A heterodimer antibody BH1654 showed good anti-inflammatory effects at 35 nmol / kg and 70 nmol / kg, and was dose-dependent. The inflammation-relieving effect of BH1654 was stronger than that of anti-mouse TNFα monoclonal antibody and anti-mouse IL-17A monoclonal antibody.

[0113] Example 14. Pharmacodynamic study of anti-mouse TNFα / anti-mouse IL-17A heterodimer antibodies in a zymosan-induced SKG mouse psoriatic arthritis (PsA) model A mouse psoriatic arthritis model was established by immunizing SKG mice with zymosan (dextran). This model is an animal model that mimics human spondyloarthritis (a type of immune disease, including ankylosing spondylitis, psoriatic arthritis, etc.) that has been developed in recent years, and in this example, it was used to evaluate a therapeutic drug for psoriatic arthritis. The experimental animals used were 9-10 week old female SKG / Jcl mice (purchased from Japan CLEA Co., Ltd.), and after adapting to the environment for one week, four mice were randomly selected as normal control mice, and the remaining mice were used to establish a mouse PsA model. The PsA model was obtained by performing primary immunization on the mice. For immunization, 3 mg zymosan (purchased from Sigma-Alrich, catalog number Z4250) dissolved in PBS buffer solution was administered by intraperitoneal injection. On the 12th day after immunization, the mice were randomly divided into groups of eight mice each.

[0114] Each group of PsA model mice was administered prophylactically. The drug solvent (PBS), anti-mouse TNFα monoclonal antibody (35 nmol / kg), anti-mouse IL-17A monoclonal antibody (35 nmol / kg), and anti-mouse TNFα / mouse IL-17A heterodimer antibody BH1654 (70 nmol / kg) were administered by intraperitoneal injection twice a week for a total of eight times. After administration, the body weight was weighed twice a week, and the lesion status of the front and back limbs was observed and the arthritis index was evaluated. 0 points: no abnormal phenomena such as swelling of the joints or toes. 0.1 points: there was a phenomenon of erythrocytosis in the toes, and each toe was recorded as 0.1 points. 0.5 points: there was slight swelling in the ankle joint, and the lateral side of the ankle joint developed first. 1 point: there was obvious swelling in the ankle joint. 1.5: A score of degree, where obvious swelling occurs in the ankle joints, with a tendency to spread to the toes, and the degree of swelling is significantly greater than 1 and less than 2. 2: Moderate swelling occurs in the ankle joints, and the swelling continues to spread to the toes, but there is no swelling in the toe joints. 2.5: A score of degree, where the degree of swelling is significantly greater than 2, but there is no swelling in the toe joints. 3: There is severe swelling from the ankle joints to the toe joints. The most severe swelling of the joints is scored as 3 and is generally accompanied by severe swelling of the toes. This scoring system scored joint swelling and toe swelling separately, superimposing the joint swelling with a swelling degree of 0.5 points and scoring 0.1 points per toe. Score was recorded as joint score + toe score.

[0115] The experimental results are shown in Figure 14. The normal control group of mice did not develop an inflammatory response, while the vehicle group of PsA model mice developed obvious inflammatory responses and showed symptoms of psoriasis and psoriatic arthritis. The anti-mouse TNFα / mouse IL-17A heterodimer antibody BH1654 showed good anti-inflammatory effects at 70 nmol / kg, but the anti-mouse TNFα monoclonal antibody and anti-mouse IL-17A monoclonal antibody had almost no anti-inflammatory effect in this model.

[0116] Example 15. Apoptosis-inducing effect of anti-TNFα / anti-IL-17A heterodimer antibody on transmembrane TNFα cells After binding to the transmembrane TNFα, the TNFα antagonist can induce apoptosis of immune cells expressing the transmembrane TNFα by outside-to-inside signaling via the transmembrane TNFα, and such apoptosis is related to the reduction of host defense by the TNFα antagonist. TM The apoptosis-inducing effect of Enbrel TM The risk of infection after taking Humira is higher than that of Enbrel. TM This embodiment relates to the detection of the apoptosis-inducing effect of anti-TNFα / anti-IL-17A heterodimer antibody BH1657. The specific implementation process is as follows: Referring to the literature (Arthritis & Rheumatism, 2008, 58(5):1248-1257.), a cell line expressing transmembrane TNFα was constructed in Jurkat cells (purchased from Basic Medical Cell Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), which was named Jurkat / tmTNFα, and incubated in RPMI 1640 medium (purchased from GIBCO, catalog number 22400-089) containing 10% FBS (purchased from Hyclone, catalog number SH30084.03). On the day of the experiment, Jurkat / tmTNFα cells in logarithmic growth phase were collected and resuspended in RPMI 1640 medium containing 2% FBS, and 1 mL of each solution was inoculated into wells of a 12-well cell culture plate at 400,000 cells / well. The test samples were serially diluted in RPMI 1640 medium containing 2% FBS, and then 1 mL of each solution was added into wells of a 12-well cell culture plate. After 24 hours of co-incubation, the cells were collected and stained with an apoptosis detection kit (purchased from Sigma-Alrich, catalog number APOAF-50TST), and the staining method was described in the kit's instructions. The cell apoptosis status was then analyzed by flow cytometer.

[0117] The experimental results are shown in Table 6. From the data, Humira TM It was shown that Humira has a stronger effect of inducing apoptosis in Jurkat / tmTNFα. TMThis is because the anti-TNFα of BH1657 is bivalent and may be able to crosslink different transmembrane TNFα to induce a stronger outside-in apoptotic signal. In contrast, the anti-TNFα of BH1657, an anti-TNFα / anti-IL-17A heterodimer antibody, is monovalent and has a weak apoptosis-inducing effect, whereas the anti-TNFα of BH1657 is monovalent and has a weak apoptosis-inducing effect. TM This suggests that anti-TNFα / anti-IL-17A heterodimer antibodies are effective against host resistance. TM It is speculated that it may have a lower inhibitory effect than HIV-1 and thus may have a lower risk of infection.

[0118] [Table 6]

Claims

1. A heterodimeric bispecific antibody comprising a first Fc chain and a second Fc chain, a first antigen-binding functional region capable of specifically binding to TNFα, and a second antigen-binding functional region capable of specifically binding to IL-17A, The heterodimeric bispecific antibody specifically binds to TNFα and IL-17A simultaneously; the first Fc chain and the second Fc chain are both Fc fragments of immunoglobulin G containing amino acid substitutions, and the first Fc chain and the second Fc chain together form a heterodimer capable of binding to an Fc receptor; the first Fc chain and the second Fc chain are linked to the first antigen-binding functional region and the second antigen-binding functional region, respectively, via a covalent bond or a linker; and the amino acid substitutions in one of the first Fc chain and the second Fc chain are T366L and D399R, and the amino acid substitutions in the other are L351E, Y407L, and K409V, wherein the amino acid positions are numbered according to the Kabat EU index numbering system; The first antigen-binding functional region comprises the amino acid sequence of SEQ ID NO: 2 and 6; The second antigen-binding functional region comprises the amino acid sequence of SEQ ID NO: 10 and 12; Heterodimeric bispecific antibodies.

2. The heterodimeric bispecific antibody of claim 1, wherein the first antigen-binding functional region and the second antigen-binding functional region are selected from a Fab fragment, an scFv fragment, and a variable domain fragment Fv.

3. The heterodimeric bispecific antibody according to claim 1 or 2, wherein the first antigen-binding functional region and the second antigen-binding functional region are both Fab fragments.

4. The heterodimeric bispecific antibody according to claim 1 or 2, wherein one of the first antigen-binding functional region and the second antigen-binding functional region is a Fab fragment, and the other is an scFv.

5. The first Fc chain and the first antigen-binding functional region linked thereto via a covalent bond, and the second Fc chain and the second antigen-binding functional region linked thereto via a covalent bond are present in a solution containing a reducing agent, and when the solution does not contain any polypeptide other than the first Fc chain and the first antigen-binding functional region linked thereto via a covalent bond and the second Fc chain and the second antigen-binding functional region linked thereto via a covalent bond, the weight percentage based on all polypeptide chains of the formed homodimer is less than 50%. The heterodimeric bispecific antibody according to any one of claims 1 to 4.

6. The heterodimeric bispecific antibody of claim 1, wherein the first antigen-binding functional region further comprises the amino acid sequence of SEQ ID NO:

4.

7. The heterodimeric bispecific antibody of claim 1, wherein the second antigen-binding functional region further comprises the amino acid sequence of SEQ ID NO:

4.

8. An isolated polynucleotide encoding the heterodimeric bispecific antibody of any one of claims 1 to 7.

9. The isolated polynucleotide of claim 8, wherein the nucleotide sequence encoding the amino acid of the first antigen-binding functional region has SEQ ID NO: 1 and 5.

10. The isolated polynucleotide of claim 8, wherein the nucleotide sequence encoding the amino acid of the second antigen binding functional region has SEQ ID NO: 9 and 11.

11. The isolated polynucleotide of claim 9, wherein the nucleotide sequence encoding the amino acid of the first antigen-binding functional region is further selected from SEQ ID NO:

3.

12. The isolated polynucleotide of claim 10, wherein the nucleotide sequence encoding the amino acid of the second antigen-binding functional region is further selected from SEQ ID NO:

3.

13. A recombinant expression vector comprising the isolated polynucleotide of any one of claims 8 to 12.

14. 14. A host cell comprising an isolated polynucleotide according to any one of claims 8 to 12 or a recombinant expression vector according to claim 13.

15. 15. The host cell according to claim 14, which is selected from human embryonic kidney cells HEK293, hamster ovary cells CHO, Escherichia coli, yeast, insect cells, plant cells, mammalian mammary cells, and mammalian somatic cells.

16. A composition comprising a heterodimeric bispecific antibody according to any one of claims 1 to 7, or an isolated polynucleotide according to any one of claims 8 to 12, or a recombinant expression vector according to claim 13, or a host cell according to claim 14 or 15, and a pharma- ceutically acceptable carrier.

17. 1) expressing the isolated polynucleotide of any one of claims 8 to 12 or the recombinant expression vector of claim 13 in a host cell; 2) reducing each expressed protein in the host cell; 3) mixing the reduced proteins and then oxidizing the mixture. A method for producing a heterodimeric bispecific antibody described in any one of claims 1 to 7, comprising the steps of:

18. 18. The method of claim 17, wherein the host cell is selected from human embryonic kidney cells HEK293, hamster ovary cells CHO, Escherichia coli, yeast, insect cells, plant cells, mammalian mammary cells, and mammalian somatic cells.

19. 19. The method of claim 17 or 18, wherein the reducing step comprises the steps of: 1) performing a reduction reaction in the presence of a reducing agent selected from 2-mercaptoethylamine, dithiothreitol, tris(2-carboxyethyl)phosphine, or chemical derivatives thereof; and 2) removing the reducing agent.

20. The method according to any one of claims 17 to 19, wherein the oxidizing step is an oxidation in air, but further comprises a step of carrying out the oxidation reaction in the presence of an oxidizing agent selected from L-dehydroascorbic acid or a chemical derivative thereof.

21. 21. The method of any one of claims 17 to 20, further comprising the step of isolating and purifying the protein.

22. Use of a heterodimeric bispecific antibody according to any one of claims 1 to 7, and / or an isolated polynucleotide according to any one of claims 8 to 12, and / or a recombinant expression vector according to claim 13, and / or a host cell according to claim 14 or 15, and / or a composition according to claim 16 in the manufacture of a medicament for preventing and / or treating a disease in a subject, comprising The disease is selected from rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, gouty arthritis, juvenile rheumatoid arthritis, suppurative arthritis, psoriasis, type I diabetes, multiple sclerosis, autoimmune encephalomyelitis, Crohn's disease, systemic vasculitis, dermatomyositis, mixed connective tissue disease, lupus erythematosus, idiopathic thrombocytopenic purpura, primary Sjogren's syndrome, glomerulonephritis, gout, rejection after organ transplantation, asthma, or arteriosclerosis.

23. 23. The use of claim 22, wherein the subject is a mammal.

24. 23. The use according to claim 22, wherein the subject is a human.

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