Multispecific antibodies and methods of making and uses thereof

Abstract

To provide multispecific antibodies and methods of making and uses thereof.SOLUTION: The multispecific antibody comprises: (a) a Fab fragment capable of specifically binding to a first antigen, where the Fab fragment comprises a light chain, and a CH1 and a variable region of a heavy chain; (b) a first peptide linker fused at N terminus to the heavy chain; and (c) a second peptide linker fused at N-terminus to the light chain, where only one disulfide bond can be formed between the first peptide linker and the second peptide linker, and the first peptide linker and the second peptide linker are independently selected from peptide linkers containing any one of specific amino acid sequences. The antibody can be easily produced by recombinant expression and can simultaneously target and bind to two different antigens, or different epitopes of the same antigen, or multiple epitopes of different antigens.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to Chinese Patent Application No. 201911015236.1, filed on October 24, 2019, the entire text of which is incorporated herein by reference.

[0002] The present invention relates generally to the field of antibodies. More specifically, the present invention relates to multispecific antibodies and methods for their production and use. [Background technology]

[0003] It is known that most natural antibody molecules are bivalent and monospecific. However, over the past half century, advances in antibody engineering have led to the artificial generation of many types of bispecific and multispecific antibody molecules. These antibodies come in a variety of forms, including single-chain Fv antibodies (scFv, Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988)), tetravalent IgG-scFv fusions (Coloma and Morrison, Nat. Biotechnol., 15:15 9-163 (1997)), double-chain antibodies (diabodies) (Holliger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), tandem scFv molecules (see, for example, Bargou et al., Science 321, 974-977 (2008)), tetravalent IgG-like dual variable domain antibodies ("DVD-Ig", Wu et al., Nat. Biotechnol., 25:1290-1297 (2007)), tetravalent Fab tandem immunoglobulins ("FIT-Ig"), (WO 2015 / 103072, Epimab These include bispecific or multispecific antibodies, such as those used in the treatment of various tumors, including those used for "T cell redirection," bispecific or multispecific antibodies, and bivalent rat / mouse heterobispecific IgG (Lindhofer et al., J. Immunol., 155:219-225 (1995)). Bispecific or multispecific antibodies can bind to two or more different epitopes or antigen targets, and also possess novel functions not found in conventional binding molecules (see, for example, the review in Sergey et al., Drug Des Devel Ther., 12:195-208 (2018)). Therefore, bispecific or multispecific antibodies, particularly those used for "T cell redirection," are attracting increasing attention. Such bispecific antibodies can target both surface antigens on tumor cells and activating components of the T-cell surface receptor (TCR) complex (e.g., CD3), resulting in the activation of cytotoxic T lymphocytes (CTLs) that target and attack tumors.A representative form of such a bispecific antibody is a "bispecific T cell conjugate" or "BiTE" antibody, which comprises, for example, two scFv antibodies linked via a glycine-serine (GS) linker, where one scFv provides a binding site for a tumor antigen (e.g., the 17-1A tumor antigen) and the other scFv provides a binding site for the CD3 antigen on T cells (Mack et al., Proc. Natl. Acad. Sci. USA, 92:7021-7025 (1995)). The anti-CD3 x anti-CD19 BiTE antibody blinatumomab has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of a rare form of B-cell acute lymphoblastic leukemia (ALL). Other bispecific antibody formats used in T cell redirection include, but are not limited to, tetravalent tandem double-chain antibodies ("TandAbs," Kipriyanov et al., J. Mol. Biol., 293:41-56 (1999); Arndt et al., Blood, 94:2562-2568 (1999)) and amphipathic retargeting proteins ("DARTs," Johnson et al., J. Mol. Biol., 399:436-449 (2010)).

[0004] Full-length bispecific antibodies containing Fc are known to have longer half-lives and possess Fc effector functions. However, full-length bispecific antibodies require the use of knobs-into-holes (KiH) technology (Ridgway et al., Protein Eng., 9:617-621 (1996)) to enhance the assembly and stability of heterodimerization of the Fc region. However, poor heterodimerization and light chain mismatches can lead to poor stability of the target product and the generation of a series of non-target products, making expression and purification difficult. While the Fc region confers a long half-life to bispecific antibodies, it also hinders heterodimer formation. Furthermore, Fc effector function is not only unnecessary for some drug designs but can also have a detrimental effect on drug function. Therefore, to circumvent Fc effectors, bispecific antibodies such as BiTEs, two-chain antibodies, DARTs, and TandAbs achieve bispecificity by linking different variable domains via a peptide linker in a single-chain format. Compared to full-size monoclonal antibodies, these molecules have the advantage of being smaller, allowing rapid access to tissues and tumors. However, their disadvantages are that they lack the Fc region, their molecular weight is typically less than 60 kDa, their in vivo half-life is very short due to renal clearance, and they are physically unstable (Spiess et al. (2015), cited above). Full-length bispecific antibody Fc forms inactive molecular by-products due to the pairing of uncorrelated heavy chains with dimers, while single-chain bispecific antibodies have poor stability and short half-lives. Therefore, two-chain Fab antibodies, which are intermediate between the two, are expected to simultaneously solve these problems. However, random association of two light chains also results in inactive and undesirable by-products, making constructs in which two or more Fabs are directly fused via a conventional linker less viable.

[0005] Many bispecific or multispecific antibody formats have been identified as possible formats for the research and development of new therapeutic antibodies. However, to date, no format has been able to provide comprehensive properties that would allow its use in the development of new therapeutic antibodies to treat most diseases. Given the increasing availability of bispecific or multispecific antibodies and the varying results associated with currently available formats, potential improved formats are needed to address specific challenges associated with the research and development of antibodies to treat specific diseases. Summary of the Invention

[0006] The present specification provides novel bispecific antibodies that are easily produced by reducing mismatch by-products and increasing the production of target products, and that exhibit higher stability and less aggregation than bispecific antibody fragments known in the art, as well as methods for producing the same. Furthermore, such methods can be applied to existing antibodies without screening for common light or heavy chains. Furthermore, these novel bispecific antibodies have a higher molecular weight than many single-chain bispecific antibody fragments, preventing excessive renal clearance and increasing in vivo half-life.

[0007] According to a first aspect, the present invention provides a method for producing a medicament for a medicament comprising: (a) a Fab fragment capable of specifically binding to a first antigen, the Fab fragment consisting of one light chain and one heavy chain CH1 and variable region; (b) a first peptide linker N-terminally fused to the heavy chain; (c) a second peptide linker fused at the N-terminus to the light chain, The present invention provides an antibody in which only one disulfide bond can be formed between the first peptide linker and the second peptide linker, and the first peptide linker and the second peptide linker are each independently selected from peptide linkers containing any of the sequences shown in Seq ID Nos. 1 to 2 (in which X represents any amino acid except Cys, or is deleted).

[0008] According to a second aspect, the present invention provides a nucleic acid encoding an antibody according to the first aspect.

[0009] According to a third aspect, the present invention provides an expression vector comprising a nucleic acid according to the second aspect.

[0010] According to a fourth aspect, the present invention provides a host cell comprising a nucleic acid according to the second aspect or an expression vector according to the third aspect.

[0011] In some embodiments, the host cell is a mammalian cell, which can include, but is not limited to, CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, and PER.C6 cells.

[0012] According to a fifth aspect, the present invention provides a method for producing a cellular membrane comprising: (a) culturing a host cell according to the fourth aspect; and (b) recovering said antibody from said host cell or from the supernatant of a culture of said host cell.

[0013] According to a sixth aspect, the present invention provides a pharmaceutical composition comprising an antibody according to the first aspect, a nucleic acid according to the second aspect, an expression vector according to the third aspect, or a host cell according to the fourth aspect, and a pharmaceutically acceptable carrier.

[0014] According to a seventh aspect, the present invention provides the use of an antibody according to the first aspect, a nucleic acid according to the second aspect, an expression vector according to the third aspect, or a host cell according to the fourth aspect in the manufacture of a medicament for treating, ameliorating or preventing a tumor, an autoimmune disease or an infectious disease.

[0015] According to an eighth aspect, the present invention provides a method for treating, ameliorating or preventing a tumour, an autoimmune disease or an infectious disease in an individual comprising administering to the individual an antibody according to the first aspect, a nucleic acid according to the second aspect, an expression vector according to the third aspect or a host cell according to the fourth aspect.

[0016] According to a ninth aspect, the present invention provides an antibody according to the first aspect, a nucleic acid according to the second aspect, an expression vector according to the third aspect, or a host cell according to the fourth aspect in use for treating, ameliorating or preventing a tumour, an autoimmune disease or an infectious disease in an individual. [Brief explanation of the drawings]

[0017] [Figures 1A-1C] 1A shows the structures of bispecific or multispecific antibodies constructed in the present invention. FIG. 1A shows the structure of an anti-TNFα x anti-IL-17A bispecific antibody (E1), FIG. 1B shows the structure of an anti-CD137 x PD-1 ECD protein bispecific antibody (E2), and FIG. 1C shows the structure of an anti-CD3 x anti-CD19 bispecific antibody (E3). [Figure 1D-1E] Figure 1D shows a schematic diagram of the structure of the anti-PD-L1 x anti-CD137 bispecific antibody (E4), and Figure 1E shows a schematic diagram of the structure of the anti-CD3 x anti-CD137 x PD-1 ECD protein trispecific antibody (E5). [Figure 2] The results of purification and SDS-PAGE electrophoresis of anti-TNFα × anti-IL-17A bispecific antibody (E1) are shown. Figure 2A shows the Capto L affinity chromatogram of antibody E1, and Figure 2B shows the results of SDS-PAGE electrophoresis of antibody E1. Lane M is a DNA marker, lane 1 shows the results of SDS-PAGE electrophoresis under non-reducing conditions, and lane 3 shows the results of SDS-PAGE electrophoresis under reducing conditions. [Figure 3] 1 shows the results of detecting the purity of antibody E1 by HPLC. [Figure 4]The binding-dissociation curves of binding affinity measured by Fortebio are shown in Figure 4A, which shows the binding-dissociation curve when antibody E1 binds to the antigen TNF-α, Figure 4B, which shows the binding-dissociation curve when adalimumab binds to the antigen TNF-α, Figure 4C, which shows the binding-dissociation curve when antibody E1 binds to the antigen IL-17A, and Figure 4D, which shows the binding-dissociation curve when secukinumab binds to the antigen IL-17A. [Figure 5] Figure 5A shows the results of flow cytometry of antibody E2 binding to cell surface antigens, Figure 5A shows the results of flow cytometry of antibody E2 binding to MC38-PDL-1 cells, and Figure 5B shows the results of flow cytometry of antibody E2 binding to B8-CD137 cells. [Figure 6] 1 shows the results of measuring the affinity constants between each specific antibody constructed in the present invention and its corresponding antigen.

[0018] Detailed Description of the Invention The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise specified, the terms of the present invention are to be understood according to common practice by those of ordinary skill in the art.

[0019] <Definition> As used herein, the term "about" refers to ±10% of the stated number, for example, about 1% refers to a range of 0.9% to 1.1%.

[0020] The term "antibody" as used herein refers to any form of antibody or fragment thereof capable of exhibiting the desired biological activity. Therefore, it is used in the broadest sense to cover monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity. Therefore, as will be understood by those skilled in the art, the term "antibody" as used herein can also refer to a fusion protein of any form of the same or different antibodies or their fragments capable of exhibiting the desired biological activity, thereby achieving the function of a multispecific antibody.

[0021] As used herein, the term "antigen" refers to a molecule or portion of a molecule that can bind to a selective binding agent, such as an antibody, and can also be used in an animal to produce antibodies capable of binding to an epitope of the antigen. An antigen can have one or more binding epitopes. Antigens described herein can include, but are not limited to, most proteins, bacteria, viruses, bacterial exotoxins, polysaccharides (such as the capsular polysaccharide of Streptococcus pneumoniae), lipoids, and the like. The term "specific binding" as used herein is a term familiar to those skilled in the art, and methods for measuring specific binding, such as between an antibody and an antigen, are also familiar to those skilled in the art. For example, in some embodiments, "specific binding" means that an antibody binds to its intended target but does not significantly bind to other targets. The antibody binds to its intended target epitope with significantly increased affinity and / or longer duration than its binding to other epitopes.

[0022] As used herein, the term "antigen-binding fragment" includes fragments or derivatives of antibodies that substantially retain their binding activity. Thus, the term "antigen-binding fragment" refers to a portion of a full-length antibody, typically its antigen-binding or variable region. Examples of antigen-binding fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies, single-chain antibody molecules such as sc-Fv, and multispecific antibodies formed from antibody fragments. It is also understood that antigen-binding fragments may contain conservative amino acid substitutions that do not substantially alter their binding activity.

[0023] As used herein, the term "Fab fragment" includes one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with other heavy chain molecules.

[0024] As used herein, the term "Fab' fragment" contains one light chain and a portion or fragment of one heavy chain, said portion or fragment containing the VH domain and the CH1 domain, as well as the region between the CH1 and CH2 domains, which allows interchain disulfide bond formation between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0025] As used herein, the term "F(ab')2 fragment" refers to a fragment containing two light chains and two heavy chains, each of which contains a portion of the constant region between the CH1 and CH2 domains such that an interchain disulfide bond is formed between the two heavy chains. Thus, an F(ab')2 fragment is composed of two Fab' fragments linked together via disulfide bonds between the two heavy chains.

[0026] As used herein, the term "Fv fragment" comprises the variable regions from the heavy and light chains, but lacks the constant regions.

[0027] As used herein, the term "single-chain Fv" or "scFv" refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in the form of a single polypeptide chain. Typically, the Fv polypeptide also contains a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0028] As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites, which comprises a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, each domain is forced to pair with the complementary domain of the other chain, generating two antigen-binding sites.

[0029] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions are amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (LCDR-1), 50-56 (LCDR-2), and 89-97 (LCDR-3) of the light chain variable region, and residues 31-35 (HCDR-1), 50-65 (HCDR-2), and 95-102 (HCDR-3) of the heavy chain variable region; see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md.), and / or amino acid residues from the "hypervariable ring" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) of the light chain variable domain, and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy chain variable domain, Chothia and Lesk, (1987) J. Mool. Biol. 196:901-917). "Frame Region" or "FR" residues refer to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0030] The term "peptide linker" as used herein refers to a relatively flexible peptide molecule used to link two polypeptides. The peptide linker used in the present invention contains only one cysteine, allowing the formation of a stable disulfide bond between the two peptide linkers.

[0031] As used herein, the term "binding moiety" refers to a moiety that can specifically bind to another substance, and can include, but is not limited to, antibodies or antigen-binding fragments thereof, ligands and receptors, etc. The binding moiety contained in an antibody of the invention enables the antibody to be targeted to the target to which the binding moiety specifically binds.

[0032] The term "tumor-associated antigen" as used herein refers to any molecule (e.g., protein, peptide, lipid, carbohydrate, etc.) that is expressed solely, predominantly, or excessively by tumor cells, making the antigen associated with the tumor. A tumor-associated antigen may be an antigen expressed only by one type of tumor, making the tumor antigen associated with or unique to only one type of tumor. Optionally, a tumor antigen may be associated with or unique to multiple types of tumors. For example, a tumor-associated antigen may be expressed by both breast cancer cells and colon cancer cells, but not by normal, non-tumor, or non-cancer cells. Exemplary tumor-associated antigens are tumor cell surface antigens, and such antigens are more likely to be recognized by therapeutic and diagnostic antibodies.

[0033] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., each antibody in the population is identical except for minor variations that may occur naturally. Monoclonal antibodies are highly specific to a single antigen epitope. The monoclonal antibodies disclosed herein are not limited by their source or method of production (e.g., hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes intact immunoglobulins and fragments thereof within the definition of "antibody."

[0034] An "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporated into the recombinant polynucleotide. DETAILED DESCRIPTION OF THE INVENTION

[0035] Conventional bispecific antibody fragments, in which the VL and VH of an antibody are linked in tandem via a (G4S)n linking peptide, suffer from poor stability, low gene expression levels, a tendency for expression products to aggregate, and a short in vivo half-life. The role of the hinge region in antibody heavy chains is known to provide a flexible structure that ensures sufficient antigen binding for both antibody arms and to provide two or more pairs of disulfide bonds for the generation of stable homodimer structures. However, when constructing bispecific antibodies, it is desirable to maximize heterodimer formation and reduce homodimer formation. Previous methods have promoted heterodimer formation by introducing a knobs-into-holes structure through amino acid mutation in CH3 or by introducing oppositely charged amino acids through mutation, but these methods did not solve the problem of light chain mismatch. In addition, some antibodies do not require Fc effector function and require additional mutations to remove the Fc effector, or directly remove the Fc to adopt the F(ab)2 or Fab form, making it difficult to introduce amino acid mutations that promote heterodimerization into the Fab segment. The present invention creatively utilizes the stabilizing effect of disulfide bonds in the antibody hinge region by fusing a mutated antibody hinge region to the C-terminus of the heavy and light chains of an Fab antibody, respectively, so that (1) a pair of disulfide bonds is formed between the heavy and light chains only through the mutated antibody hinge region, leaving the pair of disulfide bonds formed between the native CH1 and CL unchanged, and (2) the non-covalent interaction between the heavy chain variable region and the light chain variable region provides additional stabilizing factors. Overall, the absence of the second stabilizing factor (the two pairs of disulfide bonds naturally occurring in the hinge region) and the third stabilizing factor (the non-covalent dimerization action of the Fc segment of the native heavy chain, particularly the CH3 portion) significantly reduces the stability of the homodimer between the heavy chains, while the addition of a pair of disulfide bonds between the light chain and the heavy chain increases stability. As a result, the stability of the resulting light-heavy chain heterodimer is significantly higher than that of the homodimer, with the heterodimer accounting for a very high proportion of the expression product, while the homodimer accounts for a very low proportion of the expression product, and furthermore, no stable product is produced.Trace amounts of homodimers of the heavy or light chain themselves can be effectively removed using affinity purification media for CH1 or CL. Therefore, according to the present invention, the target bispecific antibody can be easily obtained with high purity and stability.

[0036] Thus, the present invention provides improved bispecific or multispecific antibodies that can be readily produced by recombinant expression and that are capable of simultaneously targeting two different antigens, or different epitopes on the same antigen, or multiple epitopes on more different antigens.

[0037] According to a first aspect, the present invention provides a method for producing a medicament for a medicament comprising: (a) a Fab fragment capable of specifically binding to a first antigen, the Fab fragment consisting of one light chain and one heavy chain CH1 and variable region; (b) a first peptide linker N-terminally fused to the heavy chain; (c) a second peptide linker fused at the N-terminus to the light chain, The present invention provides an antibody in which only one disulfide bond can be formed between the first peptide linker and the second peptide linker, and the first peptide linker and the second peptide linker are each independently selected from the group consisting of peptide linkers containing any of the sequences shown in Seq ID NOs. 1 to 2 (wherein Seq ID NOs. 1 to 2 are XPPCPAPE and EPAPCPPX, respectively, where X represents any amino acid except Cys, or is deleted).

[0038] In some embodiments, the first peptide linker and / or the second peptide linker may be a hinge region of a native antibody that can be subjected to a deletion mutation that retains only one cysteine.

[0039] In a preferred embodiment, the first peptide linker and / or the second peptide linker are C239 It may also be a deletion mutant IgG1 hinge region.

[0040] In some embodiments, the first peptide linker and the second peptide linker are the same.

[0041] In some embodiments, the first peptide linker and the second peptide linker are different.

[0042] The amino acid sequence of an antibody is numbered to identify identical positions, and currently there are several different numbering methods for antibodies. The Kabat method (Kabat et al., 1991) was developed based on the location of regions of high sequence variation between sequences of the same domain type. The variable domains of the heavy chain (VH) and light chain (Vλ and Vκ) of an antibody are numbered differently. The Chothia method (Al-Lazikani, 1997) is the same as the Kabat method, except that the position of annotations around the first VH complementarity-determining region (CDR) is modified to correspond to the structural ring. The antibodies of the present invention are numbered according to the Kabat method.

[0043] In some embodiments, the antibody according to the first aspect can be fused to other binding moieties via the C-terminus of the first peptide linker and / or the second peptide linker, making the antibody bivalent or trivalent.

[0044] For example, the antibody described in the first aspect can be fused to a first binding moiety via the C-terminus of the first peptide linker or the second peptide linker to form a bispecific antibody. The first binding moiety can be selected from an antibody or an antigen-binding fragment thereof, a ligand, and a receptor. In some embodiments, the antibody described in the first aspect can be fused to a first binding moiety and a second binding moiety via the C-terminus of the first peptide linker and the C-terminus of the second peptide linker, respectively, to form a bispecific (when the first binding moiety and the second binding moiety are identical) or trispecific (when the first binding moiety and the second binding moiety are different) antibody. The first binding moiety and the second binding moiety can each independently be selected from an antibody or an antigen-binding fragment thereof, a ligand, and a receptor.

[0045] In some embodiments, the antibody of the first aspect further comprises a third binding moiety that binds to the N-terminus of the light chain or the heavy chain of the Fab fragment. Preferably, the third binding moiety binds to the N-terminus of the light chain of the Fab fragment.

[0046] In some embodiments, the first binding moiety is a heavy chain variable region (VH) capable of specifically binding to an antibody of a second antigen, and the second binding moiety is a light chain variable region (VL) capable of specifically binding to an antibody of a second antigen.

[0047] In some embodiments, the first binding moiety, the second binding moiety, and / or the third binding moiety can be independently selected from bivalent, trivalent, or higher antibody fragments, such that the final antibody is trivalent, tetravalent, or higher. One skilled in the art can select appropriate antibody fragments to be fused to the first peptide linker and / or second peptide linker, as needed.

[0048] In some embodiments, the antigen-binding fragment is selected from a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a diabody, and a single-chain antibody molecule such as an sc-Fv.

[0049] The Fab fragment, the first binding moiety, the second binding moiety, and the third binding moiety capable of specifically binding to a first antigen can each independently be derived from a monoclonal antibody.

[0050] In some embodiments, the monoclonal antibody used in the present invention is selected from the group consisting of adalimumab, secukinumab, rituximab, trastuzumab, gemtuzumab ozogamicin, alemtuzumab, bevacizumab, cetuximab, panitumumab, ofatumumab, ipilimumab, brentuximab-vedotin, and cetiimab. Vedotin, denosumab, pertuzumab, obinutuzumab, ramucirumab, 3F8, abagovomab, adecatumumab, afutuzumab, alacizumab (pegol), amatuximab, apolizumab, bavituximab, bectumomab, belimumab, bevacizumab, cantuzumab-mertansine mertansine, cantuzumab (ravtansine), capromab (pendetide), catumaxomab, sitatuzumab (bodatox), cixutumumab, clivatuzumab (tetraxetan), conatumumab, dacetuzumab, dalotuzumab, detumomab, drozitumab, ecromeximab, edrecolomab, elotuzumab,Enavatuzumab, ensituximab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, FBTA05, flanvotumab, galiximab, gemtuzumab, ganitumab, girentuximab, glembatumumab (vedotin), ibritumomab-tiuxetan tiuxetan, icrucumab, igovomab, indatuximab ravtansine, intetumumab, inotuzumab ozogamicin ozogamicin, ipilimumab (MDX-101), iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab (mertansine), lucatumumab, lumiliximab, mapatumumab, matuzumab, milatuzumab, mitumomab, mogamuri mogamulizumab, moxetumomab (pasudotox), nacolomab (tafenatox), naptumomab (estafenatox), narnatumab, necitumumab, nimotuzumab, nivolumab, NR-LU-10, olaratumab, oportuzumab (monatox),Oregovomab, panitumumab, pertuzumab, pritumumab, racotumomab, radretumab, robatumumab, omalizumab, sibrotuzumab, siltuximab, taplitumomab (paptox), tenatumomab, teprotumumab The antibody may be one or more selected from the group consisting of teprotumumab, ticilimumab, tremelimumab, tigatuzumab, tucotuzumab (celmoleukin), ublituximab, urelumab, veltuzumab, volociximab, votumumab, and zalutumumab.

[0051] Antigens that can be bound by the antibodies of the present invention may be cell-associated proteins, such as cell surface proteins on the membrane of cells (T cells, endothelial cells, or tumor cells), or may be soluble proteins. Antigens may also be any medically relevant proteins that are upregulated during disease or infection, such as receptors and / or their corresponding ligands. Specific examples of cell surface proteins include, but are not limited to, adhesion molecules such as integrins, E-selectin, P-selectin, or L-selectin, CD2, CD3, CD4, CD5, CD7, CD8, CD11a, CD11b, CD18, CD19, CD20, CD23, CD25, CD33, CD38, CD40, CD45, CD69, CD134, ICOS, CD137, CD27, carcinoembryonic antigen (CEA), TCR, class MHC1 and class MHC2 antigens, VEGF, and receptors for these proteins. Soluble proteins include interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-16, or IL-17), viral antigens (e.g., respiratory syncytial virus or cytomegalovirus antigens), immunoglobulins (e.g., IgE), interferons (e.g., interferon α, interferon β, or interferon γ), tumor necrosis factor-α (TNFα), tumor necrosis factor-β, colony-stimulating factors (e.g., G-CSF or GM-CSF), and platelet-derived growth factors (e.g., PDGF-α and PDGF-β), and their receptors, where appropriate. Other antigens include bacterial cell surface antigens, bacterial toxins, viruses (e.g., influenza virus, EBV, HepA, B, and C), bioterrorism agents, radionuclides and heavy metals, and snake and spider venoms and toxins.

[0052] Other antigens that may be bound by the antibodies of the invention include serum carrier proteins, polypeptides that enable the recruitment of cell-mediated effector functions, and nuclide chelating proteins.

[0053] In some embodiments, antigens that may be bound by the antibodies described herein include CD20, HER2, EGFR, CD33, CD52, VEGF, CTLA-4, CD30, RANKL, HER2, VEGF-R2, Her3, A33 antigen, CD5, CD19, CD22, CD23 (IgE receptor), CA242 antigen, 5T4, VEGFR-1, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, NPC-1C, vimentin, insulin-like growth factor-1 receptor (IGF-1R), alpha-fetoprotein, carcinoembryonic antigen (CEA), integrin alpha v and tumor-associated antigens including one or more of: β3, integrin α5β1, fibroblast activation protein, FAP-α, TAG-72, MUC1, MUC16, prostate-specific membrane antigen (PMSA), EGP40 pan-cancer antigen, glycoprotein EpCAM, programmed death-1, liver regeneration phosphatase 3 (PRL-3), Lewis-Y antigen, GD2, glypican-3 (GPC3), and mesothelin.

[0054] The first binding moiety, the second binding moiety and the third binding moiety may each independently be selected from a ligand and a receptor.

[0055] "Receptor" refers to a biological macromolecule that can bind to hormones, neurotransmitters, drugs, or intracellular signaling molecules and cause changes in cellular function. The receptor itself contains at least two active sites, one of which recognizes and binds to the ligand, and the other is a functional active site that generates a response reaction. This site can only generate a response reaction after binding to the ligand to form a binary complex and becoming allosteric, thereby initiating a series of biochemical reactions and ultimately producing a biological effect in the target cell. The receptor can specifically bind to its ligand. Usually, the extracellular region of the receptor is the binding moiety in this invention.

[0056] The term "ligand" refers to any molecule that can bind to its receptor. Most ligands are hydrophilic biopolymers, such as cytokines, protein polypeptide hormones, water-soluble hormones, prostaglandins, and hydrophilic neurotransmitters, and cannot penetrate the target cell membrane to enter the cell. Therefore, the receptors for these ligand signaling molecules are localized on the target cell membrane.

[0057] PD-1 (programmed death receptor-1), an important immunosuppressive molecule, is a membrane protein of 268 amino acid residues belonging to the immunoglobulin superfamily. Immunomodulation targeting PD-1 is important for anti-tumor, anti-infection, anti-autoimmune disease, and organ transplant survival. Its ligand, PD-L1, can also be targeted, and corresponding antibodies can play a similar role. PD-1 or PD-L1 can function as a binding moiety, e.g., a first binding moiety and / or a second binding moiety, in the present invention. Preferably, the extracellular domain of PD-1, i.e., the PD-1 ECD, functions as a binding moiety in the present invention.

[0058] The antibody of the present invention can have a CH2-CH3 domain at the C-terminus of its Fab. The CH2-CH3 domain may be linked to a peptide linker or to the C-terminus of the first binding moiety and / or the second binding moiety. The CH2-CH3 domain may optionally be mutated with KiH to introduce a cysteine ​​residue and / or one or more salt bridge mutations to promote heterodimerization, and such additions improve the stability of the heterodimer. As used herein, salt bridges include hydrogen bonds and electrostatic interactions; for example, a salt bridge may occur between a glutamic acid and a lysine residue.

[0059] The heavy and light chains of a natural antibody each contain a variable region (i.e., V region) and a constant region (i.e., C region). The constant region of the heavy chain is called CH, and the constant region of the light chain is called CL. The CL lengths of different types of Ig (κ or λ) are nearly identical, but the CH lengths of different types of Ig vary; for example, IgG, IgA, and IgD contain CH1, CH2, and CH3, while IgM and IgE contain CH1, CH2, CH3, and CH4.

[0060] According to a second aspect, the present invention provides a nucleic acid encoding an antibody according to the first aspect.

[0061] In a preferred embodiment, the nucleic acid may be a codon-optimized nucleic acid suitable for expression in a host cell. For example, depending on the degree of codon degeneracy, the codons still encode the same protein. Methods for optimizing codons based on the host cell used are known to those skilled in the art.

[0062] According to a third aspect, the present invention provides an expression vector comprising a nucleic acid according to the second aspect.

[0063] Any suitable expression vector can be used. For example, prokaryotic cloning vectors include E. coli-derived plasmids such as colEl, pCRl, pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include derivatives of phage DNA such as M13 and other filamentous single-stranded DNA phages. An example of a vector that can be used in yeast is the 2μ plasmid. Suitable vectors for expression in mammalian cells include well-known derivatives such as DNA sequences derived from SV-40, adenovirus, and retrovirus, and shuttle vectors derived from functional mammalian vectors (as described above) with functional plasmids and phage DNA.

[0064] Other eukaryotic expression vectors are known in the art (e.g., P. J. Southern & P. ​​Berg, J. Mol. Appl. Genet. 1:327-341 (1982); Subramani et al., Mol. Cell. Biol. 1:854-864 (1981); Kaufhiann & Sharp, "Amplification and Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene," J. Mol. Biol. 159:601-621 (1982); Kaufhiann & Sharp, Mol. Cell. Biol. 159:601-664 (1982); Scahill et al., "Expression and Characterization of the Product of a Human Immune Interferon DNA Gene in Chinese Hamster Ovary Cells," Proc. Nat'l Acad. Sci. USA, 80:4654-4659 (1983); Urlaub & Chasin, Proc. Nat'l Acad. Sci USA, 77:4216-4220 (1980), all of which are incorporated herein by reference).

[0065] Expression vectors usable in the present invention contain at least one expression control sequence operably linked to the DNA sequence or fragment to be expressed. The control sequence is inserted into the vector to control and regulate expression of the cloned DNA sequence. Examples of useful expression control sequences include the lac system, trp system, tac system, trc system, the phage lambda major operon and promoter region, the fd coat protein control region, yeast glycolytic promoters such as the 3-phosphoglycerate kinase promoter, yeast acid phosphatase promoters such as Pho5, the yeast α-mating factor promoter, and promoters from polyoma virus, adenovirus, retrovirus, and simian virus, such as the SV40 early and late promoters, as well as other sequences known to control gene expression in prokaryotic or eukaryotic cells and their viruses, or combinations thereof.

[0066] According to a fourth aspect, the present invention provides a host cell comprising a nucleic acid according to the second aspect or an expression vector according to the third aspect.

[0067] In some embodiments, the host cell is a mammalian cell. Mammalian cells can include, but are not limited to, CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, and PER.C6 cells. Those skilled in the art can select appropriate host cells as needed.

[0068] According to a fifth aspect, the present invention provides a method for producing a cellular membrane comprising: (a) culturing a host cell according to the fourth aspect; (b) recovering said antibody from said host cell or from the supernatant of a culture of said host cell.

[0069] According to a sixth aspect, the present invention provides a pharmaceutical composition comprising an antibody according to the first aspect, a nucleic acid according to the second aspect, an expression vector according to the third aspect, or a host cell according to the fourth aspect, and a pharmaceutically acceptable carrier.

[0070] The pharmaceutical composition of the sixth aspect can be prepared into a desired dosage form by conventional methods in the pharmaceutical field. In some embodiments, the pharmaceutical composition is preferably in the form of a liquid or suspension.

[0071] In some embodiments, the pharmaceutically acceptable carrier is a carrier that does not weaken the vitality and function of immune cells or affect the specific binding between an antibody or its antigen-binding fragment and an antigen, and includes, but is not limited to, cell culture medium, buffer, saline, balanced salt solution, etc. Examples of buffers include isotonic phosphate, acetate, citrate, borate, carbonate, etc. In a specific embodiment, the pharmaceutically acceptable carrier is phosphate buffer containing 1% serum.

[0072] The antibodies and pharmaceutical compositions thereof disclosed herein can be used to treat, ameliorate, or prevent tumors, autoimmune diseases, or infectious diseases in individuals.

[0073] According to a seventh aspect, the present invention provides the use of an antibody according to the first aspect, a nucleic acid according to the second aspect, an expression vector according to the third aspect, or a host cell according to the fourth aspect in the manufacture of a medicament for treating, ameliorating or preventing a tumor, an autoimmune disease or an infectious disease.

[0074] According to an eighth aspect, the present invention provides a method for treating, ameliorating or preventing a tumour, an autoimmune disease or an infectious disease in an individual comprising administering to the individual a therapeutically effective amount of an antibody according to the first aspect, a nucleic acid according to the second aspect, an expression vector according to the third aspect or a host cell according to the fourth aspect.

[0075] According to a ninth aspect, the present invention provides an antibody according to the first aspect, a nucleic acid according to the second aspect, an expression vector according to the third aspect, or a host cell according to the fourth aspect, for use in treating, ameliorating or preventing a tumour, an autoimmune disease or an infectious disease in an individual.

[0076] "Treatment" refers not only to therapeutic treatment, but also to prophylactic or preventative measures, the purpose of which is to prevent or alleviate (reduce) the targeted pathological condition or disease. Individuals in need of treatment include those in whom the disease is already present, as well as those who are about to develop the disease or those in whom the disease is being prevented. Thus, individuals to be treated herein may already be suffering from the disease, or may be prone to or have been diagnosed as being susceptible to the disease.

[0077] As used herein, the term "individual" refers to a mammal, including, but not limited to, primates, cows, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice. Preferably, the mammal is a non-human primate or a human. A particularly preferred mammal is a human.

[0078] In some embodiments, the tumor is a primary or metastatic cancer. In certain embodiments, the tumor is selected from lung cancer (e.g., non-small cell lung cancer), colorectal cancer, bladder cancer, hematopoietic cancer (e.g., leukemia), breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, B-lymphocyte non-Hodgkin's lymphoma, Hodgkin's lymphoma, anaplastic large cell lymphoma, head and neck cancer (e.g., head and neck squamous cell carcinoma), malignant glioma, kidney cancer, melanoma, prostate cancer, bone cancer, giant cell tumor of bone, pancreatic cancer, sarcoma, liver cancer, squamous cell carcinoma of the skin, thyroid cancer, cervical cancer, nasopharyngeal carcinoma, endometrial cancer, or metastases of the above tumors.

[0079] In some embodiments, the autoimmune disease can include systemic lupus erythematosus, rheumatoid arthritis, scleroderma, systemic vasculitis, dermatomyositis, autoimmune hemolytic anemia, and the like.

[0080] In certain embodiments, the infectious diseases include respiratory infections, gastrointestinal infections, blood infections, superficial body infections, sexually transmitted diseases, etc. In certain embodiments, the infectious diseases can include, but are not limited to, influenza, pulmonary tuberculosis, mumps, measles, whooping cough, ascariasis, bacterial dysentery, hepatitis A, hepatitis B, malaria, epidemic encephalitis B, filariasis, schistosomiasis, trachoma, rabies, tetanus, gonorrhea, syphilis, AIDS, etc.

[0081] The "therapeutically effective amount" used herein can be determined based on specific circumstances, and can be easily understood by those skilled in the art based on the actual required dosage, for example, based on the patient's weight, age, and pathological condition.

[0082] In this specification and claims, the terms "comprise," "comprises," and "containing" mean "including but not limited to" and are not intended to exclude other moieties, additives, ingredients, or steps.

[0083] It should be noted that any feature, characteristic, component, or step described in a particular aspect, embodiment, or example of the invention is applicable to any other aspect, embodiment, or example described herein, unless inconsistent.

[0084] The above disclosure generally describes the present invention, and the following examples further illustrate the present invention. These examples are used only to illustrate the present invention and do not limit the scope of the present invention. Although specific terms and values ​​are used in this specification, it is understood that these terms and values ​​are also exemplary and do not limit the scope of the present invention. Unless otherwise specified, the experimental methods and techniques used in this specification are conventional methods and techniques in the relevant technical field. Other materials, equipment, etc., for which manufacturers are not specifically specified, are generally commercially available.

[0085] <Example> In the examples and comparative examples of the present invention, unless otherwise specified, all raw materials and reagents used are ordinary and commercially available.

[0086] The vectors used in Examples 1 to 5, pQKD1101-TNFα, pQK1114-IL-17A, pUC57 IgG1 CH1-Hinge mut and pUC57 Kappa-Hinge mut, pUC57 human PD-L1 ECD, pQKZW106H IgG1 CH1-Hinge mut, pQKZW106L Kappa-Hinge mut, Triad5H, Triad5L, pUC57 PD-L1 VL-Kappa-Hinge mut, and the vectors pUC57 PD-L1 VH-IgG1 CH1 Hinge mut, pUC57 PD-L1 VL-Kappa-Hinge mut, and pUC57 anti-CD3 scFv, are all recombinant vectors used as amplification templates in each Example.

[0087] Example 1: Production, expression and identification of anti-TNFα x anti-IL-17A bispecific antibodies material The VH and VL coding nucleic acid sequences of the anti-TNFα monoclonal antibody adalimumab and the anti-IL-17A monoclonal antibody secukinumab were all obtained by DNA synthesis (Tongyong Biosystems (Anhui) Co., Ltd.). Each coding sequence was inserted into the fully synthetic expression vectors pQKD1101 (Tongyong Biosystems (Anhui) Co., Ltd.) and pQK1114 (Tongyong Biosystems (Anhui) Co., Ltd.), respectively, and the resulting products were designated pQKD1101-TNFα and pQK1114-IL-17A, respectively. IgG1 CH1-Hinge mut (i.e., C239 The coding nucleic acid sequence of Kappa-Hinge mut (containing a deleted IgG1 hinge region) C239 The coding nucleic acid sequences of the deletion mutant IgG1 CH1-Hinge mut (containing the hinge region of the IgG1) were also obtained by DNA synthesis and cloned into the vector pUC57 (Common Biosystems (Anhui) Co., Ltd.), and the resulting products were designated pUC57 IgG1 CH1-Hinge mut (C239 deletion mutation) and pUC57 Kappa-Hinge mut ( C239 deletion mutation). The nucleotide sequences of IgG1 CH1-Hinge mut and Kappa-Hinge mut are as follows: [Table 1] 1.1 Construction of anti-TNFα x anti-IL-17A bispecific antibody expression vector 1.1.1 Construction of bispecific antibody heavy chain expression vector pQKE1H Using pQKD1101-TNFα, pQK1114-IL-17A, and pUC57 IgG1 CH1-Hinge mut as templates, anti-TNFα antibody VH, anti-IL-17A antibody VH, and IgG1 CH1-Hinge mut ( C239 The three PCR amplification products (TNFα antibody VH-CH1-Hinge mut-anti-IL-17A antibody VH) were each amplified, yielding amplification products of approximately 0.4 kb, 0.42 kb, and 0.4 kb, respectively. The synthetic vector pQKX1 (Common Biosystems (Anhui) Co., Ltd.) was enzymatically cleaved with the restriction endonucleases EcoRI (NEB, R3101S) and SapI (NEB, R0712S). The resulting three PCR amplification products (ligation order from 5' to 3': TNFα antibody VH-CH1-Hinge mut-anti-IL-17A antibody VH) and the enzymatically cleaved vector were recombined using a BM Seamless Cloning Kit (BioMed) according to the kit's instructions to obtain the heavy chain expression vector pQKE1H. The PCR amplification primer pair was as follows: [Table 2] 1.1.2 Construction of bispecific antibody light chain expression vector pQKE1L Using pQKD1101-TNFα, pQK1114-IL-17A, and pUC57 Kappa-Hinge mut as templates, anti-TNFα antibody VL, anti-IL-17A antibody VL, and Kappa-Hinge mut ( C239 The sizes of the amplified products were approximately 0.36 kb, 0.36 kb, and 0.42 kb, respectively. The synthetic vector pQKX2 (Common Biosystems (Anhui) Co., Ltd.) was also enzymatically cleaved with the restriction endonucleases EcoRI (NEB, R3101S) and SapI (NEB, R0712S). The resulting three PCR amplified products (ligation order from 5' to 3': TNFα antibody VL-Kappa-Hinge mut-anti-IL-17A antibody VL) and the enzymatically cleaved vector were recombined using the BM Seamless Cloning Kit (BioMed) according to the kit's instructions to obtain the light chain expression vector pQKE1L. The PCR amplification primer pair was as follows: [Table 3] 1.1.3 Amplification and production of recombinant plasmids The heavy chain expression vector pQKE1H and the light chain expression vector pQKE1L obtained as described above were each transformed into Escherichia coli (E. coli) TOP10. After selection and identification of monoclonal clones, they were cultured in LB medium containing ampicillin (final concentration 100 mg / L) at 37°C and 200 rpm for 16 hours with shaking. Bacteria were harvested by centrifugation at 8000 × g for 20 minutes. Plasmids were isolated and extracted using the NucleoBond Xtra Midi kit (Macherey-Nagel) according to the kit's instructions, eluted with 1 mL of sterile ultrapure water, and the plasmid concentration was measured using a Nanodrop microspectrophotometer. 1.2 Antibody expression The heavy chain expression vector pQKE1H and the light chain expression vector pQKE1L were co-transfected into HEK293 cells for expression. 24 hours before transfection, 1.5 × 106 HEK293 (ATCC, CRL-1573) cells were inoculated into 100 mL of OPM-293 CD05 serum-free medium (Okuura Co., Ltd., Cat. 81075-001) in a 500 mL flask and cultured in suspension at 36.5°C, 7.5% CO2, and 120 rpm. For transfection, the recombinant plasmids pQKE1H and pQKE1L were mixed in a 1:1 weight ratio (100 μg total DNA) with 10 mL of OPM-293 CD05 medium, followed by 100 μL of PEI (3 mg / mL). The mixture was rapidly mixed homogenously with a bodex and incubated at room temperature for 15 minutes. The mixture was then added to the cell culture. The cells were cultured at 36.5°C, 7.5% CO2, and 120 rpm for 7 days, and the expressed antibodies were isolated. This antibody is an anti-TNFα × anti-IL-17A bispecific antibody expressed in the plasmids pQKE1H and pQKE1L, and was named E1. Its structure is shown in Figure 1A. 1.3 Antibody purification The harvested cell culture was centrifuged at 3000 × g for 20 min, and the supernatant was collected and filtered through a 0.45 μm filter. A 5 mL Capto L affinity chromatography column (GE) was equilibrated with a 20 mM PB and 150 mM NaCl buffer (pH 7.4) at a flow rate of 5 mL / min and a volume of more than 5 CV. The filtered sample solution was injected at a flow rate of 5 mL / min. After the injection, the Capto L affinity column was washed with a 20 mM PB and 150 mM NaCl buffer (pH 7.4) at a flow rate of 5 mL / min. The complete elution peak was collected with 50 mM citrate buffer (pH 3.0) at a flow rate of 5 mL / min, and the pH of the collected eluate was adjusted to approximately 7.0 with 1 M Tris-HCl (pH 9.0) buffer (Figure 2A). The purified product was ultrafiltered using an ultrafiltration tube, and the Tris-citrate buffer was replaced with commercially available PBS buffer. The resulting protein was detected by SDS-PAGE and Coomassie Brilliant Blue staining (Figure 2B). The protein concentration was measured using a Nanodrop microspectrophotometer and calculated to be 55 mg / L. 1.4 Antibody Identification 1.4.1 Determining antibody purity by HPLC The purity of the antibody purified by Capto L was determined by HPLC (Agilent 1260 II) SEC. The column was a Sepax aqueous size-exclusion column, the mobile phase was 50 mM PB + 300 mM NaCl pH 7.0, the injection volume was 10 μg, the flow rate was 1 ml / min, and the isocratic elution time was 20 min. The results, shown in Figure 3, indicated that the monomer purity was ≥90%. 1.4.2 Antibody affinity measurement with Fortebio The affinity constant K of the purified antibody was measured using a molecular interaction analyzer, Fortebio Octet QK (Molecular Devices). D The E1 antibody and control antibodies, adalimumab (AbbVie) and secukinumab (Novartis), were immobilized on a Fab-Ch1 sensor at a concentration of 0.25 μM in a volume of 200 μL. The antigens human TNF-α (Sino Biological Inc., Cat: 10602-H01H) and human IL-17A (Sino Biological Inc., Cat: 12047-H07Y) were injected at concentrations of 600 nM, 300 nM, 150 nM, and 75 nM, respectively, in a total volume of 200 μL. The binding-dissociation curves are shown in Figure 4, and the affinity constants are shown in Figure 6.

[0088] Example 2: Production, expression, and identification of anti-CD137 x PD-1 ECD protein bispecific antibodies 2.1 Construction of anti-CD137×PD-1 ECD protein bispecific antibody expression vector For specific procedures for constructing the expression vector and amplifying the plasmid, see Example 1. For the PD-1 ECD protein, the amplification template is pUC57 human PD-1 ECD, which contains a synthetic human PD-1 extracellular domain nucleic acid sequence inserted into the vector pUC57 (the human PD-1 extracellular domain nucleic acid sequence is found in the NCBI database NP_005009.2 and in the reference Eszter Lazar-Molnar et al., "Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy", EbioMedicine 17(2017)30-44) (General Biological Systems (Anhui) Co., Ltd.). The fully synthesized vector pQKZW106H IgG1 CH1-Hinge mut (Common Biosystems (Anhui) Co., Ltd.) contains the VH portion of the heavy chain of an anti-CD137 antibody (see patent number US-2019-0284292-A1 for the sequence) and IgG1 CH1-Hinge mut (hinge region). C239 The vector was digested with EcoRI and then ligated with the PCR product of the pUC57 human PD-1 ECD (5' to 3' ligation order: CD137 antibody VH-CH1 Hinge mut-PD-1 ECD) to obtain the final heavy chain expression vector, designated pQKE2H. Similarly, the fully synthesized vector pQKZW106L Kappa-Hinge mut (Common Biosystems (Anhui) Co., Ltd.) contained the VL portion of the anti-CD137 antibody light chain (sequence see patent number US-2019-0284292-A1) and Kappa-Hinge mut (hinge region). C239 This vector also contained a deletion mutation. It was then digested with EcoRI and recovered. After recovery, it was ligated with the pUC57 human PD-1 ECD PCR product (5' to 3' ligation order) using a recombinant enzyme to obtain the final light chain expression vector, designated pQKE2L. The primer pairs used are as follows: [Table 4] 2.2 Antibody Expression The transfection cells were HEK293 (ATCC, No. CRL-1573), and the transfection volume was 100 mL. Transfection was performed according to the procedure described in Example 1. The transfected cells were placed in a 500 mL flask and incubated at 36.5°C, 7.5% CO 2、 The cells were cultured in suspension at 120 rpm for 7 days to obtain the antibody. The resulting antibody, an anti-CD137 × PD-1 ECD protein bispecific antibody expressed in the pQKE2H and pQKE2L plasmids, was designated E2 and its structure is shown in Figure 1B. 2.3 Antibody purification Purification was carried out according to the procedure described in Example 1, and after buffer exchange, the protein concentration was measured using a Nanodrop microspectrophotometer and calculated to be 35 mg / L. 2.4 Antibody Identification 2.4.1 Measurement of antibody purity by HPLC Referring to Example 1, the purity of antibody E2 was determined by HPLC to be ≧90% monomeric. 2.4.2 Antibody affinity measurement by Fortebio The specific procedure was as described in Example 1. The E2 antibody was immobilized on the Fab-Ch1 sensor at a concentration of 0.25 μM. The antigens human PD-L1 (Sino Biological Inc., Cat: 10084-HNAH) and human CD137 (Sino Biological Inc., Cat: 10041-H002H) were injected at concentrations of 600 nM, 300 nM, 150 nM, and 75 nM, respectively, in a total volume of 200 μL. The affinity constants were measured and shown in Figure 6. 2.4.3 Detection of antibody binding activity to cells by flow cytometry 2.4.3.1 Binding activity of antibodies to MC38-PD-L1 cells Successfully resuscitated MC38 cells (Kyohwa Cell Resource Center, resource number: 3111C0001CCC000523) were subcultured for at least three generations and then passaged 24 h before transfection and seeded into 6-well plates. On the day of transfection, PEI (Sigma, Cat. No. 764647) and the synthetic plasmid pENTER PD-L1 (Common Biosystems (Anhui) Co., Ltd.) were resuscitated to room temperature. 5 μg of the plasmid was added to 500 μL of DMEM medium (Gibco, REF: 11965-092). 15 μg of PEI was added immediately after vortexing for 15 min. The mixture was then gently added dropwise to the cell culture medium and placed in an incubator for 24–48 h. After 48 h, the medium was replaced with DMEM containing 2 μg / mL purinmycin and 10% FBS. After three days, when significant cell death was observed, the flask wall was gently tapped and the supernatant discarded. Cells that adhered well to the wall were considered stably transfectable cell lines. After eight to ten days, depending on the cell growth status, the cells were digested and plated onto 96-well plates for screening of monoclonal cell lines. During this time, they were pressure-cultured in DMEM medium containing 2 μg / mL purinmycin and 10% FBS. The final cell line was named MC38-PD-L1. This cell line contained the human PD-L1 extracellular domain gene transfected into its genome, and was capable of stably expressing and displaying the human PD-L1 extracellular domain protein on the cell membrane. Resuscitated MC-38-PD-L1 cells cultured for more than three generations were washed once with 10 mL of PBS, digested with 1 mL of 0.05% pancreatin for 1 min, and then added with 4 mL of 1640 medium containing 10% FBS. After homogenous pipetting, the cells were collected and centrifuged at 1000 rpm / min for 5 min. The cells were suspended in a heavy fluid and counted to a cell density of 1 x 10 5The cell suspension was adjusted to 10 cells / mL. Groups were divided into blank, positive control, secondary antibody, and E2 groups. 10 μL of the above cell suspension was added to each tube. 100 ng of E2 BsAb was added to the E2 group tube and incubated at room temperature for 30 minutes. The tube was washed with 3 mL of PBS buffer containing 4% FBS and centrifuged at 1000 rpm / min for 5 minutes. The cells were then resuspended in 50 μL of PBS buffer containing 4% FBS. 0.5 μL of water was added to the blank group, 0.5 μL of PE anti-human PD-L1 (BioLegend, clone number: 29E-2A3) was added to the positive control group, and 0.5 μL of APC anti-human Ig light chain K (BioLegend, clone number: TB28-2) was added to the E2 and secondary antibody groups. The mixture was mixed uniformly and incubated at room temperature for 30 minutes in the dark. After incubation, each tube was washed with PBS containing 4% FBS, centrifuged at 1000 rpm / min for 5 min, and resuspended in 100 μL of PBS containing 4% FBS. The cells were then injected into a flow cytometer (ACEA, model number: NovoCyte) for detection. The results are shown in Figure 5A. 2.4.3.2 Binding activity of antibodies to MC38-CD137 cells MC38-CD137 cells are a cell line developed by stably transfecting MC38 cells (Kyohwa Cell Resource Center, resource number: 3111C0001CCC000523) with human CD137 stably integrated into the genome, resulting in the expression and presentation of human CD137 protein on the cell membrane. This cell line was constructed using the MC38-PD-L1 construction procedure described in 2.4.3.1, with the pENTER CD137 plasmid (Common Biosystems (Anhui) Co., Ltd.). The specific experimental procedures for the cells are described in 3.4.3.1. The following groups were used: blank group, positive control group, secondary antibody group, and E2 group. The positive control group contained 0.5 μL of APC anti-human 4-1BB. The secondary antibody group contained 0.5 μL of APC anti-human Ig light chain K (BioLegend, clone number TB28-2). The E2 group contained 100 ng of E2 BsAb and incubated at room temperature for 30 min. After washing, 0.5 μL of APC anti-human Ig light chain K was added. All samples were incubated, washed, resuspended in 100 μL of PBS buffer containing 4% FBS, and injected into a flow cytometer for detection. The results are shown in Figure 5B.

[0089] Example 3: Production, expression and identification of anti-CD3 x anti-CD19 bispecific antibodies 3.1 Construction of anti-CD3 x anti-CD19 bispecific antibody expression vector The specific procedures for constructing the expression vector and amplifying the plasmid are described in Example 1, where the heavy chain amplification templates are Triad5H and pUC57 IgG1 CH1-Hinge mut. Triad5H is a fully synthesized plasmid containing the anti-CD19 VH-encoding nucleic acid sequence of Blincyto (Amgen) and the VH-encoding nucleic acid sequence of the anti-CD3 monoclonal antibody Pasotuxizumab (Bayer) (Common Biosystems (Anhui) Co., Ltd.), synthesized by DNA synthesis. Triad5H was used as a template to amplify the anti-CD19 VH fragment and the anti-CD3 VH fragment, and pUC57 IgG1 CH1-Hinge mut was used as a template to amplify the IgG1 CH1-Hinge mut ( C239The deletion and mutation fragments were amplified and subjected to double digestion with SapI and EcoRI in the synthetic vector pQKX1 (Common Biosystems (Anhui) Co., Ltd.). The corresponding PCR products and digestion products were collected and ligated using recombinase (ligation order: 5' to 3', anti-CD19 antibody VH-CH1-Hinge mut-anti-CD3 antibody VH). The final recombinant plasmid, designated pQKE3H, was obtained. The light chain amplification template was Triad5L and pUC57 Kappa-Hinge mut. Triad5L is a synthetic plasmid containing the DNA-synthesized anti-CD19 VL coding nucleic acid sequence of the BiTE dual antibody and the VL coding nucleic acid sequence of the anti-CD3 monoclonal antibody Pasotuximab (Bayer) (Common Biosystems (Anhui) Co., Ltd.). Anti-CD19 VL fragments and anti-CD3 VL fragments were amplified using Triad5L as a template, and Kappa-Hinge mut ( C239 The deletion mutation fragment was amplified, and the total synthesis vector pQKX2 was subjected to double enzyme digestion with SapI and EcoRI. The corresponding PCR product and enzyme digestion product were recovered and subjected to recombination enzyme ligation (ligation order: anti-CD19 antibody VL-Kappa-Hinge mut-anti-CD3 antibody VL, from 5' to 3'). The final recombinant plasmid was obtained and named pQKE3L. The primer pairs used are as follows: [Table 5] [Table 6] 3.2 Antibody Expression The transfection cells were HEK293 (ATCC, No. CRL-1573), and the transfection volume was 100 mL. Transfection was performed according to the procedure described in Example 1. The transfected cells were placed in a 500 mL flask and incubated at 36.5°C, 7.5% CO 2、The cells were cultured in suspension at 120 rpm for 7 days to obtain the antibody. The resulting antibody, an anti-CD3 × anti-CD19 bispecific antibody expressed in the pQKE3H and pQKE3L plasmids, was designated E3 and its structure is shown in Figure 1C. 3.3 Antibody purification Purification was carried out according to the procedure described in Example 1, and after buffer exchange, the protein concentration was measured using a Nanodrop microspectrophotometer and calculated to give a protein yield of 59 mg / L. 3.4 Antibody Identification 3.4.1 Measurement of antibody purity by HPLC Referring to Example 1, the purity of antibody E3 was determined by HPLC to be ≧90% monomeric. 3.4.2 Antibody affinity measurement by Fortebio The specific procedure was as described in Example 1. The E3 antibody was immobilized at a concentration of 0.25 μM using a Fab-Ch1 sensor. The antigens human CD3 (Sino Biological Inc., Cat: 10977-H02H) and human CD19 (Sino Biological Inc., Cat: 11880-H02H) were injected at concentrations of 600 nM, 300 nM, 150 nM, and 75 nM, respectively, in a volume of 200 μL. The affinity constant measurements are shown in Figure 6.

[0090] Example 4: Production, expression and identification of anti-PD-L1 x anti-CD137 bispecific antibodies 4.1 Construction of anti-PD-L1 × anti-CD137 bispecific antibody expression vector The specific procedures for constructing the expression vector and amplifying the plasmid are described in Example 1. The synthetic vector pUC57 PD-L1 VH-IgG1 CH1 Hinge mut (which contains the anti-PD-L1 VH coding nucleic acid sequence of atezolizumab (Roche) synthesized by DNA synthesis) and IgG1 CH1 Hinge mut ( C239 Deletion mutation and hinge region D234 - S252The anti-PD-L1 VH-IgG1 CH1 Hinge mut and anti-CD137 VH fragments were amplified using the coding nucleic acid sequence (manufactured by General Biosystems (Anhui) Co., Ltd.) and the vector pQKZW106H IgG1 CH1-Hinge mut (manufactured by General Biosystems (Anhui) Co., Ltd.), respectively. The pQKX1 vector (manufactured by General Biosystems (Anhui) Co., Ltd.) was then double-cleaved with SapI and EcoRI, and the corresponding PCR products and cleavage products were collected and ligated (ligation order from 5' to 3': anti-PD-L1 antibody VH-CH1-Hinge mut-anti-CD137 antibody VH) to obtain the final recombinant plasmid, designated pQKE4H. The vector pUC57 PD-L1 VL-Kappa-Hinge mut (which contains the anti-PD-L1 VL encoding nucleic acid sequence of atezolizumab synthesized by DNA synthesis) and Kappa-Hinge mut ( C239 Deletion mutation and hinge region D234 - S252 The coding nucleic acid sequence for the anti-PD-L1 VL-Kappa-Hinge mut (reverse fragment) (Common Biosystems (Anhui) Co., Ltd.) and the vector pQKZW106L Kappa-Hinge mut were used to amplify the anti-PD-L1 VL-Kappa-Hinge mut and anti-CD137 VL fragments, respectively. The pQKX2 vector (Common Biosystems (Anhui) Co., Ltd.) was double-cleaved with SapI and EcoRI, and the corresponding PCR and cleavage products were collected and ligated using recombinant enzymes (ligation order: 5' to 3', anti-PD-L1 antibody VL-Kappa-Hinge mut-anti-CD137 antibody VL). The final recombinant plasmid was named pQKE4L. IgG1 CH1-Hinge mut and Kappa-Hinge mut are as follows: [Table 7] The primer pairs used are as follows: [Table 8] [Table 9] 4.2 Antibody Expression HEK293 (ATCC, CRL-1573) cells were transfected in a transfection volume of 100 mL, and transfection was performed according to the procedure described in Example 1. The transfected cells were cultured in suspension in a 500 mL flask at 36.5°C, 7.5% CO2, and 120 rpm / min for 7 days to obtain the antibody. The resulting antibody was an anti-PD-L1 x anti-CD137 bispecific antibody expressed in the pQKE4H and pQKE4L plasmids, designated E4, and its structure is shown in Figure 1D. 4.3 Antibody purification Purification was carried out according to the procedure described in Example 1, and after buffer exchange, the protein concentration was measured using a Nanodrop microspectrophotometer and calculated to give a protein yield of 38 mg / L. 4.4 Antibody Identification 4.4.1 Measurement of antibody purity by HPLC Referring to Example 1, the purity of antibody E4 was determined by HPLC to be ≧90% monomeric. 4.4.2 Antibody affinity measurement by Fortebio The specific procedure was as described in Example 1. The E4 antibody was immobilized at a concentration of 0.25 μM using a Fab-Ch1 sensor. The antigens human PD-L1 (Sino Biological Inc., Cat: 10084-HNAH) and human CD137 (Sino Biological Inc., Cat: 10041-H002H) were injected at concentrations of 600 nM, 300 nM, 150 nM, and 75 nM, respectively, in a total volume of 200 μL. The affinity constants were measured and shown in Figure 6.

[0091] Example 5: Production, expression, and identification of anti-CD3 x anti-CD137 x PD-1 ECD protein trispecific antibodies 5.1 Construction of anti-CD3 x anti-CD137 x PD-1 ECD protein trispecific antibody expression vector The specific procedures for constructing the expression vector and amplifying the plasmid were as described in Example 1. Using pQKE2H from Example 2 as the heavy chain expression vector, pUC57 anti-CD3 scFv (containing the VH-VL coding nucleic acid sequence of anti-CD3 monoclonal antibody (Pasotuxizumab, Bayer), obtained by DNA synthesis (Common Biosystems (Anhui) Co., Ltd.), the coding sequence was inserted into the expression vector pUC57), and pQKE2L from Example 2 as a template, the anti-CD3 scFv fragment and the anti-CD137 VL-PD-1 ECD fragment were amplified by PCR, respectively. Simultaneously, the pQKX2 vector (Common Biosystems (Anhui) Co., Ltd.) was double-cleaved with SapI and EcoRI. The two PCR products and the cleavage products were then recombined (the ligation order was from 5' to 3', anti-CD3 scFv-anti-CD137 VL-Kappa-Hinge mut-PD-1 ECD) to obtain the final recombinant plasmid, designated pQKE5L. The primer pairs used are as follows: [Table 10] 5.2 Antibody Expression The transfection cells were HEK293 (ATCC, No. CRL-1573), and the transfection volume was 100 mL. Transfection was performed according to the procedure described in Example 1. The transfected cells were placed in a 500 mL flask and incubated at 36.5°C, 7.5% CO 2、 The cells were cultured in suspension at 120 rpm for 7 days to obtain the antibody. The resulting antibody, an anti-CD3 × anti-CD137 × PD-1 ECD protein trispecific antibody expressed in the pQKE5H and pQKE5L plasmids, was designated E5 and its structure is shown in Figure 1E. 5.3 Antibody purification Purification was carried out according to the procedure described in Example 1, and after buffer exchange, the protein concentration was measured using a Nanodrop microspectrophotometer and calculated to give a protein yield of 11 mg / L. 5.4 Antibody Identification 5.4.1 Determining antibody purity by HPLC Referring to Example 1, the purity of antibody E5 was determined by HPLC and was found to be ≧90% monomeric. 5.4.2 Antibody affinity measurement by Fortebio The specific procedure was as described in Example 1. The E5 antibody was immobilized at a concentration of 0.25 μM using a Fab-Ch1 sensor. The antigens human CD3 (Sino Biological Inc., Cat: 10977-H02H), human CD137 (Sino Biological Inc., Cat: 10041-H002H), and human PD-L1 (Sino Biological Inc., Cat: 10084-HNAH) were injected at concentrations of 600 nM, 300 nM, 150 nM, and 75 nM, respectively, in a total volume of 200 μL. The affinity constants were measured and shown in Figure 6.

[0092] Although the technical solutions of the present invention have been described in detail above using general descriptions and specific embodiments, based on these technical solutions, any modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention belong to the protection scope of the present invention.

Claims

1. (a) a Fab fragment capable of specifically binding to a first antigen, the Fab fragment consisting of one light chain and one heavy chain CH1 and variable region; (b) a first peptide linker N-terminally fused to the heavy chain; (c) a second peptide linker fused at its N-terminus to the light chain, an antibody in which only one disulfide bond can be formed between the first peptide linker and the second peptide linker, the first peptide linker and the second peptide linker being each independently selected from the group consisting of peptide linkers comprising any of the sequences set forth in Seq ID NOs. 1-2 (wherein X represents any amino acid except Cys, or is deleted); optionally, the first peptide linker and / or the second peptide linker is a hinge region of a native antibody that can be subjected to a deletion mutation that retains only one cysteine, and optionally, the first peptide linker and / or the second peptide linker is an IgG1 hinge region with a C229 deletion mutation.

2. 2. The antibody of claim 1, further comprising a first binding moiety fused to the C-terminus of the first peptide linker, and optionally further comprising a second binding moiety fused to the C-terminus of the second peptide linker.

3. 3. The antibody of claim 2, further comprising a third binding moiety that binds to the N-terminus of the light chain or heavy chain of the Fab fragment, preferably wherein the third binding moiety binds to the N-terminus of the light chain of the Fab fragment.

4. 4. The antibody of claim 2 or 3, wherein the first binding moiety is selected from the group consisting of an antibody or antigen-binding fragment thereof, a ligand, and a receptor capable of specifically binding to a second antigen; optionally, the second binding moiety is selected from the group consisting of an antibody or antigen-binding fragment thereof, a ligand, and a receptor capable of specifically binding to a third antigen; optionally, the third binding moiety is selected from the group consisting of an antibody or antigen-binding fragment thereof, a ligand, and a receptor capable of specifically binding to a fourth antigen; optionally, the antigen-binding fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a diabody, and a single-chain antibody molecule such as an sc-Fv; optionally, the first peptide linker and second peptide linker are the same or different; and optionally, the first binding moiety, the second binding moiety, and the third binding moiety are the same or different.

5. 5. The antibody of claim 4, wherein the first binding moiety is a heavy chain variable region (VH) of an antibody capable of specifically binding to the second antigen, and the second binding moiety is a light chain variable region (VL) of an antibody capable of specifically binding to the second antigen.

6. The antibody of claim 4 or 5, wherein the third binding moiety is a single-chain antibody molecule such as an sc-Fv.

7. 7. The antibody of any one of claims 1 to 6, which is bispecific or multispecific, optionally wherein the antibody is bivalent or multivalent, optionally wherein the first antigen, second antigen, third antigen, and fourth antigen are independently selected from the group consisting of TNFα, IL17, CD137, CD3, CD19, and PD-L1, optionally wherein the ligand is selected from the group consisting of PD-L1, EphrinA1, VEGF, and EGF, and optionally wherein the receptor is correspondingly selected from PD-1, EphA2, VEGFR1, and EGFR.

8. A nucleic acid encoding the antibody according to any one of claims 1 to 7.

9. An expression vector comprising the nucleic acid of claim 8.

10. 10. A host cell comprising the nucleic acid of claim 8 or the expression vector of claim 9, wherein the host cell is optionally a mammalian cell selected from the group consisting of a CHO cell, an NS0 cell, an SP2 / 0 cell, a HEK293 cell, a COS cell, and a PER.C6 cell.

11. A method for producing the antibody according to any one of claims 1 to 7, comprising: (a) culturing the host cell of claim 10; and (b) recovering the antibody from the host cell or the supernatant of a culture of the host cell.

12. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 7, the nucleic acid according to claim 8, the expression vector according to claim 9, or the host cell according to claim 10, and a pharmaceutically acceptable carrier.

13. Use of the antibody of any one of claims 1 to 7, the nucleic acid of claim 8, the expression vector of claim 9 or the host cell of claim 10 in the manufacture of a medicament for treating, ameliorating or preventing a tumor, an autoimmune disease or an infectious disease in an individual.

14. A method for treating, ameliorating, or preventing a tumor, an autoimmune disease, or an infectious disease in an individual, the method comprising administering to the individual the antibody of any one of claims 1 to 7, the nucleic acid of claim 8, the expression vector of claim 9, or the host cell of claim 10.

15. 11. An antibody according to any one of claims 1 to 7, a nucleic acid according to claim 8, an expression vector according to claim 9 or a host cell according to claim 10, for use in treating, ameliorating or preventing a tumor, an autoimmune disease or an infectious disease in an individual.

16. The tumor is selected from the group consisting of lung cancer, colorectal cancer, bladder cancer, leukemia, breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, B-lymphocyte non-Hodgkin's lymphoma, Hodgkin's lymphoma, anaplastic large cell lymphoma, head and neck cancer, malignant glioma, kidney cancer, melanoma, prostate cancer, bone cancer, pancreatic cancer, sarcoma, liver cancer, squamous cell carcinoma of the skin, cervical cancer, nasopharyngeal cancer, endometrial cancer, or metastases of the above tumors; and optionally, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, and autoimmune 16. The antibody of any one of claims 1 to 7, the nucleic acid of claim 8, the expression vector of claim 9 or the host cell of claim 10 in the use of claim 13 or the method of claim 14 or the use of claim 15, wherein the infectious disease is selected from the group consisting of influenza, hepatitis B, rabies, syphilis and AIDS, and optionally the individual is a mammal, preferably the mammal is a human.

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