A bispecific antibody against pd-l1 and hlla2 and preparation method and application thereof

Abstract

The present application relates to a kind of anti-PD-L1 and HHLA2 bispecific antibody and its preparation method and application.The bispecific antibody includes PD-L1 binding domain and HHLA2 binding domain, the PD-L1 binding domain includes anti-PD-L1 monoclonal antibody, the HHLA2 binding domain includes the variable region of anti-HHLA2 nanobody;The variable region of anti-HHLA2 nanobody is connected with the N terminal or C terminal of the light chain or heavy chain of anti-PD-L1 monoclonal antibody by flexible linker.The present application constructs specific structure anti-HHLA2 and PD-L1 bispecific antibody, with high specificity and affinity, can up-regulate cell-mediated immune response, and enhance the function of T cell and NK cell in tumor microenvironment.In addition, bispecific antibody drug conjugate can be further developed, with higher efficient tumor cell specific killing ability, provide new method, new idea for treating tumor and infectious disease.

Description

Technical Field

[0001] This invention belongs to the fields of tumor treatment and molecular immunology technology, and relates to a bispecific antibody against PD-L1 and HHLA2, its preparation method and application. Background Technology

[0002] To date, immunotherapy is considered one of the most promising systemic cancer treatments compared to traditional anti-cancer strategies. Immune checkpoint molecules are often highly expressed in the tumor microenvironment, and by inhibiting T cell activation and inducing T cell exhaustion, tumors evade the immune system's attack. Immune checkpoint inhibition is one of the most effective and cutting-edge treatment methods in current anti-tumor immunotherapy. Among them, monoclonal antibodies, due to their ability to specifically target molecules, have become a key and effective treatment in cancer treatment. For example, anti-PD-1, PD-L1, and CTLA-4 immune checkpoint inhibitors have shown potent anti-tumor activity in clinical practice.

[0003] Human endogenous retrovirus-H long terminal repeat-associating 2 (HHLA2), also known as B7-H7 or B7y, is a newly discovered B7 family molecule. Currently, 10 members of the B7 family have been identified: CD80 (B7-1), CD86 (B7-2), B7-H1 (PD-L1, CD274), B7-DC (CD273 or PD-L2), B7-H2 (CD275), B7-H3 (CD276), B7-H4 (B7S1, B7x or VTCN1), B7-H5 (Vista, GI24 or PD-1H), B7-H6 (NCR3LG1), and HHLA2 (B7-H7 or B7y).

[0004] HHLA2 is mainly distributed and highly expressed in various tumor tissues, with only small amounts expressed in epithelial cells of the intestine, kidney, gallbladder, and breast, as well as placental trophoblastic tissue. HHLA2 is closely related to the growth, metastasis, pathological grade, and prognosis of various tumors, including pancreatic cancer, lung cancer, breast cancer, osteosarcoma, colorectal cancer, and renal cancer. On the one hand, HHLA2 can co-stimulate T cells in the context of TCR-mediated activation by interacting with TMIGD2, enhancing T cell proliferation and cytokine production through an AKT-dependent signaling cascade. On the other hand, HHLA2 can recruit SHP-1 and SHP-2 by interacting with KIR3DL3 on T / NK immune cells, thereby attenuating downstream Vav1, ERK1 / 2, AKT, and NF-κB signaling, thus exhibiting immunosuppressive activity. Existing preclinical studies have shown that HHLA2 can promote tumor immune escape, while drugs that block the HHLA2 / KIR3DL3 pathway (especially antibody drugs) can inhibit tumor development and metastasis. Therefore, anti-tumor therapy targeting HHLA2 / KIR3DL3 is currently receiving much attention.

[0005] However, due to the complex pathogenesis of tumors, monoclonal antibodies targeting a single target often fail to demonstrate sufficient therapeutic efficacy. Therefore, bispecific monoclonal antibodies (BsAbs) targeting two targets simultaneously have emerged. A bispecific antibody is a synthetically produced antibody with two specific antigen-binding sites. This antibody can simultaneously bind to and recognize two different antigens, or two different epitopes of a single antigen. This gives bispecific antibodies biological activities not found in combination therapies, demonstrating significant therapeutic potential and thus broad application value in biomedical research. For example, in tumor treatment, they can simultaneously target tumor cells and immune cells. Bifunctional modulators bind to two different immune co-stimulatory or co-inhibitory molecules, inducing functional changes in target cells to enhance therapeutic effects. Most bifunctional modulators target PD-1 / PD-L1 and other immunosuppressive molecules such as CTLA-4 (CD152), TIM-3, LAG-3, or TIGIT. Given the multifunctionality of bispecific antibodies and their potential to mediate novel MOAs, the field of bispecific antibodies is expected to see more emerging methods and drug candidates enter clinical practice, potentially providing key data for oncology and non-oncology indications in the coming years, including applications in infectious diseases, virology, autoimmune diseases, metabolism, neurology, and ophthalmology.

[0006] Currently, publicly reported antibodies targeting the HHLA2 / KIR3DL3 signaling pathway (such as CN114729052A, CN112153977A, WO2023138579A1, and US20210198366A1) have entered clinical trials, including anti-HHLA2 monoclonal antibodies from Nextpoint and Harbour BioMed. However, there are no reports of bispecific antibodies against PD-L1 and HLLA2 or their drug conjugates (ADCs) or other antibody derivatives. Therefore, developing novel antibodies targeting the HHLA2 / KIR3DL3 signaling pathway is of great significance in the field of cancer treatment, providing new methods and ideas for cancer therapy. Summary of the Invention

[0007] To address the shortcomings of existing technologies and practical needs, this invention provides a bispecific antibody against PD-L1 and HHLA2, its preparation method, and its applications. It also develops novel antibodies targeting the HHLA2 / KIR3DL3 signaling pathway, providing new methods and ideas for tumor treatment.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a bispecific antibody against PD-L1 and HHLA2, the bispecific antibody comprising a PD-L1 binding domain and an HHLA2 binding domain, the PD-L1 binding domain comprising a variable region of an anti-PD-L1 monoclonal antibody, and the HHLA2 binding domain comprising a variable region of an anti-HHLA2 nanobody; the variable region of the anti-HHLA2 nanobody is connected to the N-terminus or C-terminus of the heavy chain of the anti-PD-L1 monoclonal antibody via a flexible linker, or the variable region of the anti-HHLA2 nanobody is connected to the N-terminus or C-terminus of the light chain of the anti-PD-L1 monoclonal antibody via a flexible linker; the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the anti-PD-L1 monoclonal antibody respectively comprise the sequences shown in SEQ ID NO. 1 to SEQ ID NO. 3, and the amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 respectively comprise the sequences shown in SEQ ID NO. 4 to SEQ ID NO. 5. The amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of the anti-HHLA2 nanobody include the sequences shown in SEQ ID NO.8 to SEQ ID NO.10, respectively.

[0010] In this invention, a bispecific antibody against PD-L1 and HHLA2 with a specific structure is designed. It has high specificity and affinity, and can bind to PD-L1 and HHLA2 antigens simultaneously and efficiently. It can enhance the function of T cells and NK cells, upregulate cell-mediated immune responses, and can be applied to treat diseases with T cell and NK cell dysfunction, including tumors and infectious diseases.

[0011] It is understood that the amino acid sequences of the CDRs listed above in this invention are represented according to the Kabat numbering system based on sequence variability (Kabat E A. Sequences of proteins of immunological interest [M]. USDapartment of Health and Human Services, Public Health Service, National Institutes of Health, 1991). Those skilled in the art will know that antibody CDRs can be represented using various numbering systems (e.g., IMGT, Chothia, Honegger, AbM, and Contact). Although the range of CDR sequences claimed in this invention is based on sequences represented by the Kabat numbering system, amino acid sequences defined according to other CDR numbering schemes should also fall within the scope of protection of this invention.

[0012] Preferably, the amino acid sequence of the flexible connector includes the sequence shown in SEQ ID NO.7.

[0013] Preferably, the amino acid sequence of the heavy chain variable region of the anti-PD-L1 monoclonal antibody includes the sequence shown in SEQ ID NO.11, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.14.

[0014] Preferably, the amino acid sequence of the variable region of the anti-HHLA2 nanobody includes the sequence shown in SEQ ID NO.16.

[0015] Preferably, the non-CDR region of the bispecific antibody includes the non-CDR region of a non-mouse species antibody, and more preferably the non-CDR region of a human antibody.

[0016] Preferably, the heavy chain type of the anti-PD-L1 monoclonal antibody is at least one of IgG1, IgG2, IgG3 or IgG4.

[0017] Preferably, the light chain type of the anti-PD-L1 monoclonal antibody may be a κ chain or a λ chain.

[0018] Preferably, the amino acid sequence of the heavy chain constant region of the anti-PD-L1 monoclonal antibody includes the sequence shown in SEQ ID NO.12 or SEQ ID NO.13, and the amino acid sequence of the light chain constant region includes the sequence shown in SEQ ID NO.15.

[0019] Preferably, the amino acid sequence of the heavy chain of the bispecific antibody includes the sequence shown in SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.20, and the amino acid sequence of the light chain includes the sequence shown in SEQ ID NO.21.

[0020] Preferably, the amino acid sequence of the heavy chain of the bispecific antibody includes the sequence shown in SEQ ID NO.24 or SEQ ID NO.25, and the amino acid sequence of the light chain includes the sequence shown in SEQ ID NO.22; or, the amino acid sequence of the heavy chain of the bispecific antibody includes the sequence shown in SEQ ID NO.24 or SEQ ID NO.25, and the amino acid sequence of the light chain includes the sequence shown in SEQ ID NO.23.

[0021] Preferably, the buffer solution for the bispecific antibody includes at least one of histidine buffer, phosphate buffer, citrate buffer, acetate buffer, succinate buffer, or tromethamine buffer.

[0022] Preferably, the buffer solution comprises a phosphate buffer with added sodium chloride and arginine; more preferably, it comprises a 5-50 mM phosphate buffer with added 10-150 mM sodium chloride and a pH of 5.5-6.5. More preferably, the buffer solution comprises a phosphate buffer with added sodium chloride and arginine; even more preferably, it comprises a 5-50 mM phosphate buffer with added 10-150 mM sodium chloride and 5-20 mM arginine and a pH of 5.5-6.5.

[0023] The above numerical range indicates that any value can be selected within this range. For example, 10–150 mM can be 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 146, 147, 148, or 149 mM, etc. 5–50 mM can be 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 46, 47, 48, or 49 mM, etc.; 5.5–6.5 can be 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, or 6.4, etc.; 5–20 mM can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 mM, etc.

[0024] In this invention, a buffer solution with a specific composition is designed for bispecific antibodies to further improve the solubility of bispecific antibodies and subsequent drug conjugate formulations prepared from them, enabling the preparation of high-concentration reagents and further facilitating their widespread application.

[0025] In a second aspect, the present invention provides a nucleic acid molecule that encodes the anti-PD-L1 and HHLA2 bispecific antibody described in the first aspect.

[0026] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecule described in the second aspect.

[0027] Fourthly, the present invention provides a recombinant cell containing the nucleic acid molecules described in the second aspect.

[0028] Fifthly, the present invention provides a method for preparing the anti-PD-L1 and HHLA2 bispecific antibody as described in the first aspect, the method comprising:

[0029] The nucleic acid described in the second aspect is inserted into the expression vector to obtain a recombinant vector. The recombinant vector is introduced into a host cell, the host cell is cultured, and the bispecific antibody is isolated and purified.

[0030] Preferably, the host cell comprises a mammalian cell, which includes any one of CHO, HEK293, NSO, or Per 6.

[0031] Preferably, the separation and purification methods include affinity chromatography and ultrafiltration concentration.

[0032] Preferably, the buffer solution used in the ultrafiltration concentration includes at least one of histidine buffer, phosphate buffer, citrate buffer, acetate buffer, succinate buffer, or tromethamine buffer.

[0033] Preferably, the buffer solution comprises a phosphate buffer with added sodium chloride; more preferably, it comprises a 10 mM phosphate buffer with added 50 mM sodium chloride and a pH of 6.0.

[0034] Preferably, the buffer solution comprises a phosphate buffer with added sodium chloride and arginine; more preferably, it is a 10mM phosphate buffer with added 50mM sodium chloride and 10mM arginine, at a pH of 6.0.

[0035] This invention incorporates a specific buffer solution that can further improve the solubility of bispecific antibodies, enabling the preparation of high-concentration bispecific antibodies.

[0036] In a sixth aspect, the present invention provides a bispecific antibody conjugate, the bispecific antibody conjugate comprising the anti-PD-L1 and HHLA2 bispecific antibodies described in the first aspect and conjugates thereto.

[0037] Preferably, the conjugate includes a detectable marker or a drug.

[0038] Preferably, the detectable marker includes at least one of a radioactive isotope, a luminescent substance, a colored substance, or an enzyme.

[0039] Preferably, the conjugate includes at least one of FITC, PE, APC, CY-5, CY-7, RF488, Alexa Fluor 488, PerCP-Cy5.5, APC-Cy7, maytansine derivatives (DM1, DM4 or Auristatin (MMAE / MMAF) etc.), carlecamycin, docamycin derivatives, PBD or camptothecin derivatives (SN38 or Dxd etc.), 131I, 90Y, 177Lu, 188Re, alkaline phosphatase, horseradish peroxidase or biotin.

[0040] In a seventh aspect, the present invention provides a kit, characterized in that the kit comprises the anti-PD-L1 and HHLA2 bispecific antibody as described in the first aspect or the bispecific antibody conjugate as described in the sixth aspect.

[0041] Preferably, the kit further includes a second antibody that specifically recognizes the bispecific antibody.

[0042] Preferably, the second antibody further includes a detectable marker, which includes at least one of a radioactive isotope, a luminescent substance, a colored substance, or an enzyme.

[0043] Eighthly, the present invention provides the use of the anti-PD-L1 and HHLA2 bispecific antibody described in the first aspect or the bispecific antibody conjugate described in the sixth aspect in the preparation of a reagent targeting tumors.

[0044] Preferably, the tumor-targeting reagent includes drugs for the prevention or treatment of tumors or tumor antigen detection reagents.

[0045] Preferably, the tumor antigen includes PD-L1 and / or HHLA2.

[0046] Preferably, the tumor includes tumors expressing PD-L1 and / or HHLA2, such as at least one of non-small cell lung cancer, ovarian cancer, melanoma, kidney tumor, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, or liver cancer.

[0047] In a ninth aspect, the present invention provides a pharmaceutical composition comprising the anti-PD-L1 and HHLA2 bispecific antibody as described in the first aspect and / or the bispecific antibody conjugate as described in the sixth aspect.

[0048] Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0049] Compared with the prior art, the present invention has at least the following beneficial effects:

[0050] This invention constructs a specific structured bispecific antibody against HHLA2 and PD-L1. This bispecific antibody exhibits high specificity and affinity for both HHLA2 and PD-L1 antigens, possesses NK cell-dependent tumor cell killing ability, can upregulate cell-mediated immune responses, activates T cell function in human peripheral blood in the Mixed Lymplocyte Response, and demonstrates highly efficient in vivo anti-tumor effects. Furthermore, bispecific antibody-drug conjugates with highly efficient tumor cell killing capabilities can be further developed, providing new methods and ideas for the treatment of tumors and infectious diseases. Attached Figure Description

[0051] Figure 1 A schematic diagram of the molecular structure of the anti-PD-L1 and HHLA2 bispecific antibody (I);

[0052] Figure 2 Schematic diagram of the molecular structure of the anti-PD-L1 and HHLA2 bispecific antibody (II);

[0053] Figure 3 This is an SDS-PAGE electrophoresis image of a bispecific antibody.

[0054] Figure 4 This is a chromatography image of the bispecific antibody Superdex-200;

[0055] Figure 5A The image shows the results of ELISA detection of bispecific antibodies (Ab01-01~Ab01-04) binding to recombinant human PD-L1 protein.

[0056] Figure 5B The image shows the results of ELISA detection of bispecific antibodies (Ab01-01~Ab01-04) binding to recombinant human HHLA2 protein.

[0057] Figure 5C The image shows the results of ELISA detection of bispecific antibodies (Ab01-05~Ab01-08) binding to recombinant human PD-L1 protein.

[0058] Figure 5D The image shows the results of ELISA detection of bispecific antibodies (Ab01-05~Ab01-08) binding to recombinant human HHLA2 protein.

[0059] Figure 6A Binding curves of bispecific antibody and control antibody with HCC827 cell line;

[0060] Figure 6B This is a graph showing the binding curves of the bispecific antibody and the control antibody to the NCI-H820 cell line;

[0061] Figure 6C This is a graph showing the binding curves of the bispecific antibody and the control antibody to the K562-HHLA2 cell line;

[0062] Figure 6D This is a graph showing the binding curves of the bispecific antibody and the control antibody to the BxPC-3 cell line;

[0063] Figure 6E This is a graph showing the binding curves of the bispecific antibody and the control antibody to the K562 cell line;

[0064] Figure 7 The image shows the results of the detection of the NK cell-dependent tumor cell killing ability of bispecific antibodies.

[0065] Figure 8A This is a graph showing the results of bispecific antibody-mediated killing of NCI-H820 cells by NK cells.

[0066] Figure 8B This is a diagram showing the results of NK cell killing of HCC-827 cells mediated by bispecific antibodies.

[0067] Figure 8C This is a diagram showing the results of NK cell killing of MDA-MB-231 cells mediated by bispecific antibodies.

[0068] Figure 9The image shows the results of detecting T cell function in human peripheral blood activated by bispecific antibodies in the Mixed Lymplocyte Response.

[0069] Figure 10 The image shows the results of the tumor cell killing ability test of the bispecific antibody-drug conjugate.

[0070] Figure 11 This is a curve showing the binding of the bispecific antibody to cells expressing monkey HHLA2.

[0071] Figure 12 This is a graph showing the results of the specificity detection of the binding between the bispecific antibody and the B7 family recombinant protein;

[0072] Figure 13 The graph shows the T-cell-dependent in vivo antitumor efficacy of bispecific antibodies in an HCC827 tumor model that reconstructs the human PBMC immune system.

[0073] Figure 14 This figure shows the in vivo antitumor efficacy results of bispecific antibodies in an NK cell-dependent HCC827 tumor model. Detailed Implementation

[0074] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0075] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0076] Unless otherwise defined, scientific and technical terms and their abbreviations used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Some of the terms and abbreviations used in this invention are listed below.

[0077] Antibody: Ab.

[0078] Immunoglobulin: Ig.

[0079] Heavy chain: HC.

[0080] Light chain: LC.

[0081] Heavy chain variable domain (VH).

[0082] Heavy chain constant domain (CH).

[0083] Light chain variable domain (VL).

[0084] Light chain constant domain (CL).

[0085] Antigen-binding fragment (Fab).

[0086] Hinge region.

[0087] Framework region (FR).

[0088] Fc region: fragment crystallizable region, Fc region.

[0089] Monoclonal antibodies (mAbs)

[0090] Bispecific antibody: BsAb.

[0091] The complementarity determining region (CDR) is the antigen-complementary binding region of an antibody.

[0092] Nanobody (Nb) refers to an antibody that is naturally absent from the light chain and exists in the peripheral blood of animals such as alpacas. The variable domain of the heavy chain is called VHH (variable domain of heavy chain of heavy-chain antibody).

[0093] The operational steps involved in molecular cloning, cell culture, protein purification, and animal model experiments described in this invention are standard procedures widely used in this field. Unless otherwise defined, the related terms used in this invention have the same meaning as commonly understood in this technical field.

[0094] The term "amino acid" refers to one of the 20 naturally occurring amino acids or any non-natural analogue that may be present at a specific defined position. The three-letter abbreviations for amino acids and the single-letter abbreviations for nucleotides used in this invention are the forms generally accepted in this technical field, and the single-letter abbreviations for amino acids are the form recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0095] The term "monoclonal antibody" or "monoclonal antibody" refers to an antibody molecule that consists of a single molecule. Monoclonal antibodies have monovalent affinity and bind to the same epitope (the site at which the antibody recognizes the antigen).

[0096] The term "antigen binding site" refers to one or more amino acid residues that directly interact with an antigen in an antigen-binding molecule. The antigen binding site of an antibody is composed of the antigen complementarity-determining region (CDR). Natural immunoglobulin molecules typically contain two antigen binding sites, while Fab molecules typically contain one antigen binding site.

[0097] The terms “antigen complementarity-determining region (CDR)” or “hypervariant region (HVR)” refer to regions where the composition and sequence of amino acid residues at certain specific positions are highly variable.

[0098] The term "framework region (FR)" refers to the region in the V region other than the CDR. This part has a low amino acid substitution frequency, which helps stabilize the structure of the CDR. The CDR and FR in the variable region are arranged in the pattern "FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4".

[0099] The term "light chain" or "L-chain" refers to a polypeptide chain consisting of approximately 214 amino acid residues, with a molecular weight of about 24 kDa. Each light chain contains two cyclic peptides formed by intrachain disulfide bonds. L-chains can be classified into κ and λ types, and an antibody molecule can only contain one of these two types of light chains.

[0100] The term "heavy chain" or "H chain" refers to a polypeptide chain composed of approximately 450–550 amino acid residues, with a molecular weight of about 55 or 75 kDa. Based on differences in H chain antigenicity, they can be classified into five types: μ chain, γ chain, α chain, δ chain, and ε chain. Different H chains combined with L chains (κ or λ chains) to form complete Ig molecules are respectively called IgM, IgG, IgA, IgD, and IgE.

[0101] The terms "Fc," "Fc region," or "Fc fragment" refer to the effector region of an antibody, capable of inducing responses such as CDC, ADCC, ADCP, and cytokine release. Natural antibody Fcs are typically composed of two identical protein fragments, each containing two or three immunoglobulin constant domains. The Fc described in this invention includes both natural Fc and mutant Fc. Under experimental conditions, fragments generated from immunoglobulin monomers by papain cleavage are designated Fab and Fc, respectively.

[0102] The term "EC" 50 "The half-maximal effect concentration (50% of maximal effect) refers to the antibody concentration that produces a 50% maximal effect."

[0103] The term "pharmaceutical composition" generally refers to a mixture containing one or more of the compounds described in this invention or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients.

[0104] Example 1

[0105] This embodiment designs bispecific antibodies against PD-L1 and HHLA2.

[0106] The bispecific antibody comprises an anti-PD-L1 monoclonal antibody and a variable region (VHH) of a humanized alpaca antibody targeting HHLA2. The variable region of the humanized alpaca antibody (nanobody) targeting HHLA2 is linked via a flexible linker to the N-terminus of the heavy chain of the monoclonal antibody targeting PD-L1 or the C-terminus of the Fc region of its heavy chain. Figure 1The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the anti-PD-L1 monoclonal antibody are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively; the amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively; the amino acid sequence of the flexible linker is shown in SEQ ID NO.7; and the amino acid sequences of the variable regions CDR1, CDR2, and CDR3 of the humanized alpaca antibody targeting HHLA2 are shown in SEQ ID NO.8 to SEQ ID NO.10, respectively. More specifically, the amino acid sequence of the heavy chain variable region of the anti-PD-L1 monoclonal antibody is shown in SEQ ID NO.11, and the amino acid sequence of the constant region is shown in SEQ ID NO.12 (wild-type IgG1) or SEQ ID NO.13 (N297A mutation in wild-type IgG1); the amino acid sequence of the light chain variable region is shown in SEQ ID NO.14, and the amino acid sequence of the constant region is shown in SEQ ID NO.15; the amino acid sequence of the variable region of the humanized alpaca antibody targeting HHLA2 is shown in SEQ ID NO.16.

[0107] That is, when the variable region of the humanized alpaca antibody targeting HHLA2 is linked to the C-terminus of the heavy chain of the anti-PD-L1 monoclonal antibody, the heavy chain sequence of the bispecific antibody is obtained as shown in SEQ ID NO.17 (corresponding bispecific antibody name Ab01-01) or SEQ ID NO.18 (corresponding bispecific antibody name Ab01-02). When the variable region of the humanized alpaca antibody targeting HHLA2 is linked to the N-terminus of the heavy chain of the anti-PD-L1 monoclonal antibody, the heavy chain sequence of the bispecific antibody is obtained as shown in SEQ ID NO.19 (corresponding bispecific antibody name Ab01-03) or SEQ ID NO.20 (corresponding bispecific antibody name Ab01-04). The light chain amino acid sequences are all as shown in SEQ ID NO.21.

[0108] Alternatively, the bispecific antibody comprises a variable region of a monoclonal antibody targeting PD-L1 and a variable region of a humanized alpaca antibody targeting HHLA2, wherein the variable region of the humanized alpaca antibody targeting HHLA2 is linked via a flexible linker to the N-terminus or the C-terminus of the light chain of the monoclonal antibody targeting PD-L1. Figure 2When the variable region of the humanized alpaca antibody targeting HHLA2 is linked to the N-terminus of the light chain of the anti-PD-L1 monoclonal antibody, the light chain sequence of the bispecific antibody is obtained as shown in SEQ ID NO.22 (the corresponding bispecific antibody is named Ab01-05, and the constant region of the heavy chain is wild-type SEQ ID NO.24; the constant region of the heavy chain of Ab01-06 is N297A mutant, SEQ ID NO.25). When the variable region of the humanized alpaca antibody targeting HHLA2 is linked to the C-terminus of the light chain of the anti-PD-L1 monoclonal antibody, the light chain sequence of the bispecific antibody is obtained as shown in SEQ ID NO.23 (the corresponding bispecific antibody is named Ab01-07, and the constant region of the heavy chain is wild-type SEQ ID NO.24; Ab01-08, and the constant region of the heavy chain is N297A mutant, SEQ ID NO.25).

[0109] SEQ ID NO.1: DSWIH.

[0110] SEQ ID NO. 2: WISPYGGSTYYADSVKG.

[0111] SEQ ID NO.3: RHWPGGFDY.

[0112] SEQ ID NO.4: RASQDVSTAVA.

[0113] SEQ ID NO.5: SASFLYS.

[0114] SEQ ID NO.6: QQYLYHPAT.

[0115] SEQ ID NO.7: GGGGSGGGGSGGGGS.

[0116] SEQ ID NO.8: YYVIG.

[0117] SEQ ID NO.9: CISSSTGSTYSAQSVKG.

[0118] SEQ ID NO. 10: RRSPGIEGWCALPFFFGS.

[0119] SEQ ID NO.11:

[0120] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS.

[0121] SEQ ID NO.12:

[0122] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0123] SEQ ID NO.13:

[0124] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0125] SEQ ID NO.14:

[0126] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK.

[0127] SEQ ID NO.15:

[0128] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0129] SEQ ID NO.16:

[0130] EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSTGSTYSAQSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARRSPGIEGWCALPFFFGSWGQGTLVTVSS。

[0131] SEQ ID NO.17:

[0132] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSTGSTYSAQSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARRSPGIEGWCALPFFFGSWGQGTLVTVSS。

[0133] SEQ ID NO.18:

[0134] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSTGSTYSAQSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARRSPGIEGWCALPFFFGSWGQGTLVTVSS。

[0135] SEQ ID NO.19:

[0136] EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSTGSTYSAQSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARRSPGIEGWCALPFFFGSWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0137] SEQ ID NO.20:

[0138] EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSTGSTYSAQSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARRSPGIEGWCALPFFFGSWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0139] SEQ ID NO.21:

[0140] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0141] SEQ ID NO.22:

[0142] EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSTGSTYSAQSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARRSPGIEGWCALPFFFGSWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0143] SEQ ID NO.23

[0144] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSTGSTYSAQSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARRSPGIEGWCALPFFFGSWGQGTLVTVSS。

[0145] SEQ ID NO.24:

[0146] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0147] SEQ ID NO.25:

[0148] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0149] Example 2

[0150] This embodiment describes the gene synthesis and expression vector construction of bispecific antibodies.

[0151] Based on the bispecific antibody sequence of Example 1, six heavy chain plasmids and three light chain plasmids were designed and constructed. The six heavy chain plasmids and their carried antibody sequences (in parentheses) are: Ab01-01 heavy chain (SEQ ID No. 17), Ab01-02 heavy chain (SEQ ID No. 18), Ab01-03 heavy chain (SEQ ID No. 19), Ab01-04 heavy chain (SEQ ID No. 20), Ab01-05 / Ab01-07 heavy chain (SEQ ID No. 24), and Ab01-06 / Ab01-08 heavy chain (SEQ ID No. 25); the three light chain plasmids and their carried antibody sequences are: Ab01-01 / Ab01-02 / Ab01-03 / Ab01-04 light chain (SEQ ID No. 21), Ab01-05 / Ab01-06 light chain (SEQ ID No. 22), and Ab01-07 / Ab01-08 light chain (SEQ ID No. 23).

[0152] Beijing Qingke Biotechnology Co., Ltd. was commissioned to optimize the amino acid sequences of the heavy and light chains of the aforementioned bispecific antibody to make them suitable for expression in CHO cells. Subsequently, the encoding gene was synthesized and cloned into the modified eukaryotic expression vector pcDNA3.1. Upon receiving the plasmid synthesized by Qingke Biotechnology, DH5α competent cells (Qingke Biotechnology) were heat-shocked at 42℃. Single colonies were picked from plates cultured overnight at 37℃ and cultured overnight in 200mL LB medium containing 100μg / mL at 37℃ using a shaker at 220rpm. The bacterial cells were collected, and an expression plasmid (OMEGA, catalog number D6926-03) was prepared using an endotoxin-free plasmid extraction kit. The plasmid concentration was determined using a Nanodrop One micro-spectrophotometer (Thermo Scientific).

[0153] Example 3

[0154] This example demonstrates the expression and purification of bispecific antibodies.

[0155] Use plants in their exponential growth phase (density approximately 3–5 × 10⁻⁶). 6 HEK293F cells (number of cells / mL) with a viability greater than 98% were transfected. Cells were passaged one day prior to transfection, and the cell density was adjusted to 2 × 10⁻⁶ cells / mL. 6Cells / mL, after 24 hours, transient transfection was performed (taking 100mL as an example). Two 15mL sterile centrifuge tubes were prepared. In tube A, 5mL of serum-free cell transfection buffer solution KPM (Zhuhai Kairui, catalog number K03125) and 100μg of plasmid DNA prepared in Example 2 were added, and the mixture was gently pipetted to mix. In tube B, 5mL of KPM buffer and 500μL of TA-293 cell suspension chemical transfection reagent (Zhuhai Kairui, catalog number K20001) were added, and the tube was capped and gently inverted several times to mix. The liquid in tube A was transferred to tube B, the tube was capped, and the mixture was gently inverted several times to mix. The mixture was then incubated at room temperature for 10 minutes to prepare the plasmid-transfection reagent mixture. Cells were removed from the constant temperature shaker, and the plasmid-transfection reagent mixture was added dropwise while gently shaking the flask to avoid excessive local concentrations that could cause cell toxicity. The cells were then returned to the CO2 constant temperature shaker for incubation. Twenty hours after transfection, 600 μL of 293 cell protein expression enhancer KE-293 (Zhuhai Kairui, catalog number K30001) and transient recombinant protein expression plant peptone nutrient additive KT-Feed 50× (Zhuhai Kairui, K40001) were added to increase antibody expression.

[0156] On day 5 post-transfection, the cell culture supernatant was collected by centrifugation at 11,000 rpm for 20 min. 1 / 4 volume of 5× equilibration buffer (0.1 M Na2HPO4, 0.75 M NaCl, pH 7.4) was added to the supernatant. The target antibody was purified by affinity chromatography using rProtein A Beads 4FF packing material (Tiandi Renhe, SA015025). The antibody solution was transferred to a dialysis bag (Solepro, catalog number YA1073) and dialyzed overnight. The next day, the solution was concentrated using an ultrafiltration tube (Merck, catalog number UFC8030) and the buffer was changed to four different antibody preparation buffers (Table 1). The concentration was determined by A280 assay, and the purity was checked by SDS-PAGE protein electrophoresis (GenScript, catalog number M00930) and molecular sieve chromatography (Cytiva HiLoad 16 / 600 Superdex 200, catalog number 28989335). The purified solution was then aliquoted and frozen for later use.

[0157] The experimental results are shown in Table 1. The bispecific antibody showed good solubility in all four buffers, and could be concentrated to concentrations above 10 mg / mL without precipitation. Specifically, the 10 mM PB buffer (containing 50 mM NaCl and 50 mM arginine (Arg)) was concentrated to 18.5 mg / mL. The sample was subjected to SDS-PAGE electrophoresis (reduced and non-reduced), and the results are as follows... Figure 3As shown, lane 1 contains protein molecular weight standards, lane 2 contains Ab01-05, lane 3 contains Ab01-06, lane 4 contains Ab01-07, lane 5 contains Ab01-08, lane 6 contains Ab01-03, lane 7 contains Ab01-04, lane 8 contains Ab01-01, and lane 9 contains Ab01-02. The non-reduced SDS-PAGE electrophoresis pattern shows that the molecular weight of the bispecific antibody is as expected, and the reduced SDS-PAGE electrophoresis pattern shows that the molecular weight and ratio of the light and heavy chains of the bispecific antibody are as expected. As controls, lane 10 contains PCC3 antibody (heavy chain variable region as shown in SEQ ID NO. 16), and lane 11 contains Atezolizumab antibody (Ate, Roche Pharmaceuticals). The molecular weight of the control antibody is also as expected. Superdex-200 chromatography results are shown below. Figure 4 As shown, no obvious impurity peaks were observed in the bispecific antibody Ab01-01 purified by protein A in one step.

[0158] Table 1

[0159] Buffer Components pH Antibody concentration (mg / mL) Buffer preparation 1 10mM PB*+25mM NaCl 6 10.3 Buffer preparation 2 10mM PB + 50mM NaCl 6 13.9 Buffer preparation 3 10mM PB + 50mM NaCl + 10mM Arg 6 18.5 Buffer preparation 4 10mM citrate buffer + 50mM NaCl 5.5 10.8

[0160] *: PB, phosphate buffer.

[0161] Example 4

[0162] To further improve the solubility of the bispecific antibody Ab01-01 and subsequent drug conjugate formulations prepared from it, we selected four basic buffers, including two salt concentrations and two pH values, as screening targets: buffer 1: PBS buffer pH 6.0 + 150mM Arg; buffer 2: PBS buffer pH 6.5 + 150mM Arg; buffer 3: PB buffer pH 6.0 + 150mM Arg; and buffer 4: PB buffer pH 6.2 + 150mM Arg. During the purification and dialysis process, the bispecific antibody Ab01-01 exhibited significant precipitation in PBS buffer at pH 6.5 + 150mM Arg. During ultrafiltration concentration, precipitation occurred at a concentration of 0.56 mg / mL in PBS buffer at pH 6.0 + 150mM Arg, and at a concentration of 0.5 mg / mL in PB buffer at pH 6.2 + 150mM Arg. However, the antibody remained relatively stable at a concentration of 1.2 mg / mL in PB buffer at pH 6.0 + 150mM Arg. This indicates that the antibody's solubility decreases above pH 6.0; therefore, we chose PB buffer at pH 6.0 + 150mM Arg. Arg was used as a buffer for the bispecific antibody Ab01-01. However, during subsequent concentration, it was found that the antibody precipitated at a concentration of 1.6 mg / mL in this buffer. We then tried arginine at final concentrations of 10 mM and 50 mM. We found that the antibody also precipitated at a concentration of 1.6 mg / mL in the 50 mM arginine buffer. However, after adding sodium chloride to the 10 mM arginine solution, the antibody could be concentrated to 18.5 mg / mL. Therefore, the bispecific antibody Ab01-01 is relatively stable in the 10 mM arginine buffer.

[0163] To facilitate subsequent ADC preparation, a low-salt-concentration buffer was designed, and low-concentration sodium chloride was used for screening: final concentrations of 25 mM and 50 mM sodium chloride. It was found that in PB buffer with added 25 mM and 50 mM sodium chloride at pH 6.0, the antibody Ab01-01 remained relatively stable even when concentrated to over 10 mg / mL during dialysis. In particular, after adding 10 mM arginine to PB buffer at pH 6.0 + 50 mM NaCl, the antibody Ab01-01 concentration in this buffer could reach 18.5 mg / mL.

[0164] Example 5

[0165] This embodiment uses the ELISA method to detect the binding ability of bispecific antibodies to recombinant human PD-L1 protein and recombinant human HHLA2 protein.

[0166] Dilute recombinant human PD-L1 protein (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., 10084-H05H) or recombinant human HHLA2 protein (ARCO biosystems, B77-H52H6) to 0.25 or 0.5 μg / mL with carbonate buffer, add 100 μL / well to an ELISA plate (Thermo, 468667), and incubate overnight at 4°C. Wash the ELISA plate wells twice with 0.05% PBST, add 300 μL / well of 3% skim milk powder-PBS solution, and incubate at 37°C for 1 h; wash the ELISA plate wells three times with 0.05% PBST, add bispecific antibody solution diluted with PBS (100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM and 0.00128 nM or 300 nM, 60 nM, 12 nM, 2.4 nM, 0.48 nM, 0.096 nM, 0.0192 nM, 0.00384 nM, 0.000768 nM and 0.0001 nM). 100 μL of 536 nM antibody (536 nM) per well was added to each well, with Atezolizumab antibody, PCC3 antibody, and Isotype antibody used as controls. The plates were incubated at 37°C for 1 hour. The plates were washed three times with 0.05% PBST, and 100 μL of diluted secondary antibody (Invitrogen, A18817) per well was added. The plates were incubated at 37°C for 30 minutes. The plates were washed five times with 0.05% PBST, and 100 μL of TMB chromogenic buffer (Solepro, PR1200) per well was added. After appropriate incubation time, 50 μL of ELISA stop buffer (Solepro, C1058) per well was added. The OD was immediately measured using a microplate reader. 450 reading.

[0167] Figure 5A and 5C The graph shows the binding curves of the bispecific antibody to the PD-L1 protein, indicating that the bispecific antibody has good binding activity with the recombinant human PD-L1 protein (Table 2).

[0168] Figure 5B and 5D The graph shows the binding curves of the bispecific antibody to the HHLA2 protein, indicating that the bispecific antibody has good binding activity to the HHLA2 protein (Table 2).

[0169] Table 2

[0170]

[0171] Example 6

[0172] This embodiment uses flow cytometry to detect the binding ability of bispecific antibodies to human HHLA2.

[0173] Antibody binding experiments were performed at the cellular level using the K562 cell line (Pronos), the K562 cell line overexpressing human HHLA2 (K562-hHHLA2, Kehuizhi Pharmaceutical), the HEK293 cell line overexpressing cynomolgus monkey HHLA2 (HEK293-cyHHLA2, Kehuizhi Pharmaceutical), the tumor cell lines HCC827 (Pronos) and NCI-HCC820 (Nanjing Kebai) that are positive for both human HHLA2 and PD-L1, and the PD-L1 positive tumor cell line BxPC-3 (Pronos).

[0174] Cells were collected using 1×PBS, centrifuged at 300×g for 5 min, and resuspended in ice-cold blocking buffer (PBS containing 5% FBS) to adjust the cell density to 10-1. 6 Cells / mL; Add 50 μL of cells and 50 μL of serially diluted test antibody to each well of a 96-well V plate (Corning, 3894), and incubate on ice in the dark for 2 h; Add 200 μL of pre-chilled washing buffer (PBS containing 0.1% BSA), centrifuge at 300×g for 5 min, carefully discard the supernatant, and repeat once; Resuspend cells in a solution containing 400-fold diluted fluorescent secondary antibody (APC anti-human IgG Fc, BioLegend, 409306; or Alexa). Cells were incubated for 30 min on ice in blocking buffer (PBS containing 5% FBS) for 30 min in the dark. Then, 200 μL of pre-chilled washing buffer (PBS containing 0.1% BSA) was added, and the cells were centrifuged at 300 × g for 5 min. The supernatant was discarded, and the process was repeated once. Cells were resuspended in 100 μL of PBS and placed on ice. Samples were transferred to flow cytometry tubes, and the corresponding fluorescence signals were detected using a flow cytometer (Wellgrow Easy Cell 206A1). The above tests were repeated with PCC3 antibody, Atezolizumab antibody, and Isotype as controls.

[0175] The result of bispecific antibody molecules specifically binding to tumor cells is as follows: Figure 6A , Figure 6B and Figure 6D As shown, the binding results of various antibodies with HHLA2-overexpressing K562-HHLA2 and K562 cells are as follows: Figure 6C and Figure 6EAs shown. Bispecific antibody molecules and PCC3 exhibited specific high-affinity binding to HHLA2-overexpressing K562 cells (e.g., Figure 6C (and Table 3), no binding signal was observed in K562 cells within the experimentally tested concentration range (e.g., ...). Figure 6E (and Table 3). Figure 6A , Figure 6B , Figure 6D As shown in Table 3, the bispecific antibody molecules exhibited high affinity binding to the HCC827, NCI-H820, and BxPC-3 cancer cells tested in the experiment. The affinity or the number of maximum binding sites on cells of the bispecific antibodies were superior to or similar to those of the anti-PD-L1 antibody Atezolizumab and the anti-hHHLA2 antibody (PCC3). It is evident that the bispecific antibody molecules constructed in this invention exhibit specific high affinity binding to various HHLA2 and PD-L1 positive tumor cells and overexpressing cell lines.

[0176] Table 3

[0177]

[0178] *The value in parentheses represents the average error of two or more experiments.

[0179] Example 7

[0180] This embodiment tests the NK cell-dependent tumor cell killing ability of bispecific antibodies.

[0181] Collect NCI-H820 cells, adjust the density to 1.5E+05 cells / mL with RPMI 1640 + 10% FBS medium, add 100 μL to each well of a 96-well plate, and incubate overnight at 37°C. On the second day, the culture medium in the 96-well plates was removed, and the wells were washed once with N500 serum-free medium (Dakeway, 6113031). 50 μL of N500 serum-free medium was added per well, followed by 50 μL of serially diluted test antibody per well. Pre-incubation was performed for 30 min. Effector cells NK92MI (Pricella, CL-0533; KIR3DL3 positive effector cells) were collected and their density adjusted to 3E+05 cells / mL with N500 medium. 100 μL of this solution was added to the corresponding wells of the 96-well plates, centrifuged at 250×g for 3 min, and incubated at 37℃ for 6 h. After centrifugation at 250×g at room temperature for 5 min, 50 μL of the supernatant was transferred to the 96-well plates. Cell killing was determined using an LDH cytotoxicity assay kit (Roche, 11644793001). The killing rate was defined as: (OD value of experimental group - lowest release well) / (maximum release well - lowest release well) × 100%.

[0182] LDH cell killing results showed ( Figure 7(See Table 4). Bispecific antibodies Ab01-01, Ab01-02, and PCC3 all significantly enhanced the killing effect of NK92MI cells on target cells K562-hHHLA2. The killing effect of the bispecific antibodies on target cells NCI-H820 was superior to that of the anti-hHHLA2 antibody (PCC3). The anti-PD-L1 antibody Atezolizumab could not activate the killing effect of NK92MI cells on target cells NCI-H820.

[0183] Table 4

[0184] Antibody <![CDATA[EC 50 (nM)]]> PCC3 0.16(0.07)* Atezolizumab No casualties Ab01-01 0.05(0.01) Ab01-02 0.09(0.04) Ab01-03 0.05(0.03) Ab01-04 0.08(0.03) Isotype No casualties

[0185] *The value in parentheses represents the average error of two or more experiments.

[0186] Example 8

[0187] This embodiment tests the in vitro antibody-dependent cell-mediated cytotoxicity of bispecific antibodies.

[0188] Cryopreserved PBMCs were purchased from Shanghai Saili Biotechnology Co., Ltd. Donors with strong ADCC activity were selected. The cell cryovials were quickly transferred from the liquid nitrogen tank to a 37°C water bath, and the cells were thawed until only a small ice crystal remained. Cells were transferred from the cryovials to 15mL test tubes. The cryovials were rinsed with 1mL of cell culture medium and transferred to another 15mL test tube. Cell culture medium was added to bring the volume to 10mL. The cells were centrifuged at 2000rpm for 7 minutes, the supernatant was discarded, and the test tube was gently tapped to loosen the cell pellet. The cells were resuspended in 1mL of cell culture medium and transferred slowly up and down. Cell culture medium was added to bring the total volume to 10mL, and the cells were centrifuged at 2000rpm for 7 minutes, the supernatant was discarded, and the cells were resuspended in 4mL of cell culture medium. Trypan blue staining was used to count the cells and determine cell viability. The PBMC cell density was adjusted to 1×10⁶ cells / mL. 6 Cells / mL were added to IL-2 cytokine (200 IU / mL, Biolegend, #589102) and placed in a CO2 incubator for overnight culture.

[0189] On the second day, cell counts were performed to determine viability. Cells were centrifuged, resuspended, and the cell density was adjusted to 2 × 10⁻⁶. 6Cells were added at a density of 100 μL / mL to each well of a 96-well plate, followed by 50 μL of prepared bispecific antibody solutions of different concentrations. NCI-H820 cells (CBP61114), HCC827 cells (SCSP-538), and MDA-MB-231 cells (SCSP-5043) were prepared into PBS suspensions after trypsin digestion. Cell counts were performed to determine viability, and cells were centrifuged, resuspended, and adjusted to a cell density of 2 × 10⁻⁶ cells / mL. 5 Add 50 μL of target cells per well at a concentration of 20:1 (target-effect ratio 20:1) and incubate for 6 hours. Centrifuge at 200 × g for 10 minutes. Use an LDH assay kit (11644793001, Merck) to detect cytotoxicity. Take a new 96-well plate, add 50 μL of cell culture supernatant and 50 μL of the prepared assay solution to each well, incubate at room temperature for 25 minutes, and read the value at 490 nM. Cytotoxicity or mortality (%) = (absorbance of treated sample - absorbance of sample control well) / (absorbance of maximum enzyme activity of cells - absorbance of sample control well) × 100%. Based on this, the EC50 at a specific time of drug treatment can be calculated. 50 .

[0190] In the ADCC activity assay, bispecific antibodies AB01-01 and AB01-03 and the positive control antibody Ave (Avelumab; jointly developed by Pfizer and Merck; purchased from Shanghai Sanyou Biotechnology Co., Ltd. #CHA-081) demonstrated their effectiveness against NCI-H820 cells (…). Figure 8A HCC827 cells ( Figure 8B ) and MDA-MB-231 cells ( Figure 8C The antibody showed a significant in vitro killing effect (even at a concentration of 0.01 mg / mL, it still exhibited a strong killing effect). PCC3, Atezolizumab, and the negative control antibody did not show any killing effect.

[0191] Example 9

[0192] This embodiment tests the activation of T cell function in human peripheral blood by bispecific antibodies in in vitro PBMC MLR (Mixed Lymphocyte Response).

[0193] Cryopreserved PBMCs were purchased from Shanghai Saili Biotechnology Co., Ltd. Donors with significant HLA typing differences (donor 1 and donor 2) were selected. The cell cryovials were quickly transferred from liquid nitrogen tubes to a 37°C water bath and the cells were thawed until only a small ice crystal remained. The cells were then transferred from the cryovials to 15mL test tubes. The cryovials were rinsed with 1mL of cell culture medium and transferred to the 15mL test tubes. Cell culture medium was added to bring the volume to 10mL, and the tubes were centrifuged at 2000rpm for 7min. The supernatant was discarded, and the test tubes were gently tapped to loosen the cell pellet. The cells were resuspended in 1mL of cell culture medium and transferred slowly up and down. Cell culture medium was added to bring the total volume to 10mL, and the tubes were centrifuged at 2000rpm for 7min. The supernatant was discarded, and the cells were resuspended in 4mL of cell culture medium. Trypan blue staining was performed to count the cells and determine cell viability. The PBMC cell density was adjusted to 2×10⁶ cells / mL. 6 Cells / mL were seeded from donor 1 and donor 2 into 48-well plates, with 2 × 10⁶ cells per well. 5 The cell ratio of donor 1 to donor 2 was 1:1. The cells were incubated for 5 days in a cell culture incubator with prepared bispecific antibodies (10 μg / mL, 1 μg / mL, 0.1 μg / mL, 0.01 μg / mL). The cytokine content in the cell supernatant was detected using a human IFN-γ cytokine assay kit (KIT11725A).

[0194] like Figure 9 As shown, in the MLR activity assay, the bispecific antibody and the positive control antibody Atezolizumab, compared with the MLR without antibody control and the negative control, exhibited stronger T cell activation ability and significantly upregulated IFN-γ expression. The PCC3 monoclonal antibody showed very weak T cell activation ability.

[0195] Example 10

[0196] This embodiment tests the tumor cell killing ability of a bispecific antibody conjugated with a drug-dependent cytotoxic conjugate (ADC).

[0197] DT3C is a recombinant fusion protein composed of fragment A (toxin only) of diphtheria toxin and the 3C fragment (IgG binding portion) of group G streptococcus. This protein exhibits high affinity for the Fc terminus of antibodies. DT3C molecules bound to the antibody enter the cell along with the antibody during endocytosis. Under the action of intracellular furin protease, DT3C releases toxic DT, which inhibits EF2-ADP ribosylation activity, blocking protein translation and ultimately leading to cell death. DT3C molecules that do not enter the cell do not possess cytotoxic activity. Therefore, the internalization efficiency of the antibody can be evaluated based on the cell-killing effect. This example uses DT3C to verify the internalization efficiency of a bispecific antibody.

[0198] Logarithmic growth phase NCI-H820 cells were harvested, digested with Trypsin, counted, centrifuged, and resuspended in RPMI 1640 + 20% FBS medium to a density of 3E+05 cells / mL. 100 μL / well of the cell suspension was injected into each 96-well plate and cultured overnight. The next day, DT3C (Jiman Biotechnology, GM-046001RP-1) and antibody were diluted to appropriate concentrations in serum-free RPMI 1640 medium, mixed proportionally, filtered sterilized, and incubated at 37°C for 30 min. The mixture was then serially diluted 5-fold with serum-free RPMI 1640 medium. 100 μL / well of each serially diluted mixture was added to each 96-well plate. The plates were incubated for 60 h. 20 μL of CCK8 assay reagent was added to each well, and OD values ​​were read after 2 h. 450 Value. According to OD 450 The cell viability of NCI-H820 cells at each concentration was calculated numerically. Cell viability = (OD of experimental group - OD of blank group) / (OD of target cell group alone - OD of blank group) × 100%.

[0199] The results show ( Figure 10 (See Table 5) The bispecific antibodies Ab01-01 and Ab01-02, along with Atezolizumab, exhibited significant NCI-H820 cell-killing function associated with antibody internalization.

[0200] Table 5

[0201]

[0202]

[0203] Example 11

[0204] This embodiment examines the binding ability of bispecific antibodies to cells expressing monkey HHLA2.

[0205] Flow cytometry was used to detect the binding of bispecific antibodies to HEK293 cell lines (HEK293-cyHHLA2, Kehui Zhizhi Pharmaceutical) overexpressing cynomolgus monkey HHLA2. An appropriate amount of HEK293-cyHHLA2 cells were collected, centrifuged at 300×g for 5 min, resuspended in washing buffer (PBS containing 0.1% BSA), centrifuged at 300×g for 5 min, and resuspended in ice-cold blocking buffer (PBS containing 5% FBS). The cell density was adjusted to 10T. 6Cells / mL; Add 50 μL of cells and 50 μL of serially diluted test antibody (maximum concentration 100 nM, 3-fold serial dilution, 8 concentrations) to each well of a 96-well V plate (Corning, 3894), and incubate on ice in the dark for 2 h; Add 200 μL of ice-chilled washing buffer (PBS containing 0.1% BSA), centrifuge at 300×g for 5 min, carefully discard the supernatant, repeat once; Resuspend cells in blocking buffer (PBS containing 5% FBS) containing 400-fold diluted fluorescent secondary antibody (APC anti-human IgG Fc, BioLegend, 410712), and incubate on ice in the dark for 30 min; Add 200 μL of ice-chilled washing buffer (PBS containing 0.1% BSA), centrifuge at 300×g for 5 min, discard the supernatant, repeat once, and resuspend cells in 100 μL of... Place the sample in PBS on ice; transfer it to a flow cytometry tube and detect the corresponding fluorescence signal using a flow cytometer (Wellgrow Easy Cell206A1).

[0206] The results are as follows Figure 11 As shown in Table 6, the binding activity of the bispecific antibody to the HEK293-cyHHLA2 cell line was comparable to that of the PCC3 monoclonal antibody, EC 50 The difference is not significant.

[0207] Table 6

[0208]

[0209]

[0210] Example 12

[0211] This embodiment performs antigen-specific analysis of bispecific antibodies.

[0212] HHLA2 belongs to the B7 family of proteins, of which 10 members have been identified, with each protein sharing less than 40% homology with HHLA2. To verify the antigen specificity of the bispecific antibody, recombinant human PD-L1 protein (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., 10084-H05H) or recombinant human HHLA2 protein (ARCO biosystems, B77-H52H6), as well as B7H3 recombinant human protein (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., 11188-H08H-B) and B7H6 recombinant human protein (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., 16140-H08H), which have the highest homology with HHLA2, were purchased. The proteins were diluted to 0.5 μg / mL with carbonate buffer, and 100 μL / well was added to an ELISA plate (Thermo, 468667) and incubated overnight at 4°C. Wash the ELISA plate wells twice with 0.05% PBST, add 300 μL / well of 3% skim milk powder-PBS solution, and incubate at 37°C for 1 h; wash the ELISA plate wells three times with 0.05% PBST, add 100 μL / well of antibody solution diluted with PBS (10 nM, 1 nM), and incubate at 37°C for 1 h; wash the ELISA plate wells three times with 0.05% PBST, add 100 μL / well of diluted secondary antibody (Invitrogen, A18817), and incubate at 37°C for 30 min; wash the ELISA plate wells five times with 0.05% PBST, add 100 μL / well of TMB chromogenic solution (Solepro, PR1200), and after an appropriate development time, add 100 μL / well of ELISA stop solution (Solepro, C1058); immediately use a microplate reader to complete the OD. 450 reading.

[0213] The results showed that the bispecific antibody antigens exhibited good specificity, binding to their corresponding target proteins PD-L1 (B7H1) and HLLA2, respectively, but not to the family-related proteins B7H3 and B7H6 (B7H1 and B7H2). Figure 12 ).

[0214] Example 13

[0215] This embodiment mainly studies the in vivo antitumor efficacy of bispecific antibodies in a T-cell-dependent HCC827 tumor model that reconstructs the human PBMC immune system.

[0216] HCC827 tumor cells endogenously express HHLA2 and PD-L1. This example mainly focuses on the T-cell-dependent in vivo pharmacodynamics study of bispecific antibodies in an HCC827 tumor model that reconstructs the human PBMC immune system. The specific experimental methods are as follows: HCC827 cells (provided by Wuhan Pronosai Life Science Technology Co., Ltd.) were resuscitated and expanded (mycoplasma detection negative), and subcutaneously seeded on D-6 at a seeding condition of 5 × 10⁶ cells / year. 6Cell / mouse (100μL cells + 100μL matrix gel), tumor growth in mice was continuously observed after inoculation. At day 0 (the day of PBMC inoculation was designated as day 0), the tumor volume was approximately 100 mm². 3 Randomized groups were formed, and PBMC (1×10) was performed on the same day. 7 (Mice / cells, provided by Shanghai Saili Biotechnology Co., Ltd.) were inoculated, and drug administration began on day 1 (D1) at a dose of 12.5 mpk, administered every three days for a total of four times. The administration route was intraperitoneal. An equal volume of solvent was used as a control group. After the start of drug administration, mouse weight and tumor volume were recorded every three days. Tumor volume was calculated as follows: mouse tumor volume (mm²) 3 = 0.5 × tumor long diameter × tumor short diameter 2 The experiment ended on day 12 after drug administration, and all animals were subsequently euthanized according to ethical requirements. The T-cell-dependent in vivo antitumor efficacy of the bispecific antibody in an HCC827 tumor model reconstructing the human PBMC immune system is shown in [reference needed]. Figure 13 The experimental results showed that the average tumor volume of mice in the PBMC + solvent control group was 356.2 mm on day 12 after drug administration. 3 The average tumor volume of mice in the bispecific antibody group on day 12 after drug administration was 264.4 mm. 3 Compared with the solvent control group, there was a significant difference (P < 0.01), with a tumor inhibition rate (TGI) of 37.0%.

[0217] Example 14

[0218] This embodiment mainly studies the in vivo antitumor efficacy of bispecific antibodies in an NK cell-dependent HCC827 tumor model.

[0219] The specific experimental method is as follows: HCC827 cells (provided by Wuhan Pronosai Life Science Technology Co., Ltd.) and NK92MI cells (provided by Wuhan Pronosai Life Science Technology Co., Ltd.) were revived and cultured extensively (mycoplasma test negative). On D-6, HCC827 cells were subcutaneously seeded at a density of 5 × 10⁶ cells / year. 6 Cell / mouse (100 μL cells + 100 μL matrix gel). Tumor growth in mice was continuously monitored after inoculation; by day 1, the tumor volume was approximately 150 mm². 3 Students were randomly assigned to groups and began administration on the same day. The dosage was 10 mpk, administered once every three days for a total of five administrations. The administration was intravenous, with an equal volume of solvent used as a control group. Within 2 hours of bispecific antibody administration, expanded and cultured NK92MI cells were collected and processed at a rate of 1×10⁻⁶. 7NK cells / mouse (50 μL total volume / mouse) were administered intratumorally using an insulin injector. Intratumoral injection of NK cells was performed every six days for a total of three administrations. After the start of administration, body weight and tumor volume were recorded every three days. Tumor volume was calculated as follows: mouse tumor volume (mm²). 3 = 0.5 × tumor long diameter × tumor short diameter 2 The experimental observation ended on day 16 after drug administration, and all animals were subsequently euthanized according to ethical requirements. The NK cell-dependent in vivo antitumor efficacy of the bispecific antibody in the HCC827 tumor model is shown in [reference needed]. Figure 14 The experimental results showed that the average tumor volume of mice in the PBS + solvent control group was 638.2 mmHg on day 16 after drug administration. 3 In the NK92MI+ solvent control group mice, the average tumor volume on day 16 after drug administration was 400.07 mm. 3 The tumor inhibition rate (TGI%) was 48.6%, and the average tumor volume on day 16 after administration in the bispecific antibody group was 292.9 mm. 3 The tumor inhibition rate (TGI) was 70.5%, and there was a significant difference in tumor volume compared with the NK92MI+ solvent control group (P < 0.05), with the tumor inhibition rate (TGI) increasing by 21.9%.

[0220] In summary, this invention constructs a specific structured bispecific antibody against HHLA2 and PD-L1. This bispecific antibody exhibits high specificity and affinity for both HHLA2 and PD-L1 antigens, possesses NK cell-dependent tumor cell killing ability, upregulates cell-mediated immune responses, and can activate T cell function in human peripheral blood in a mixed lymplocyte response, demonstrating highly efficient in vivo anti-tumor effects. Furthermore, bispecific antibody-drug conjugates with highly efficient tumor cell killing capabilities can be further developed, providing new methods and ideas for the treatment of tumors and infectious diseases.

[0221] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A bispecific antibody against PD-L1 and HHLA2, characterized in that, The bispecific antibody includes a PD-L1 binding domain and an HHLA2 binding domain, wherein the PD-L1 binding domain includes an anti-PD-L1 monoclonal antibody and the HHLA2 binding domain includes a variable region of an anti-HHLA2 nanobody. The variable region of the anti-HHLA2 nanobody is connected to the N-terminus or C-terminus of the heavy chain of the anti-PD-L1 monoclonal antibody via a flexible connector, or the variable region of the anti-HHLA2 nanobody is connected to the N-terminus or C-terminus of the light chain of the anti-PD-L1 monoclonal antibody via a flexible connector. The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the anti-PD-L1 monoclonal antibody include the sequences shown in SEQ ID NO.1 to SEQ ID NO.3, respectively, and the amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 include the sequences shown in SEQ ID NO.4 to SEQ ID NO.6, respectively. The amino acid sequences of the variable regions CDR1, CDR2, and CDR3 of the anti-HHLA2 nanobody include the sequences shown in SEQ ID NO. 8 to SEQ ID NO. 10, respectively.

2. The anti-PD-L1 and HHLA2 bispecific antibody according to claim 1, characterized in that, The amino acid sequence of the flexible connector includes the sequence shown in SEQ ID NO.7; Preferably, the amino acid sequence of the heavy chain variable region of the anti-PD-L1 monoclonal antibody includes the sequence shown in SEQ ID NO.11, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.14; Preferably, the amino acid sequence of the variable region of the anti-HHLA2 nanobody includes the sequence shown in SEQ ID NO.16; Preferably, the non-CDR region of the bispecific antibody includes the non-CDR region of a non-mouse species antibody, and more preferably the non-CDR region of a human antibody; Preferably, the heavy chain type of the anti-PD-L1 monoclonal antibody is any one of IgG1, IgG2, IgG3 or IgG4; Preferably, the light chain type of the anti-PD-L1 monoclonal antibody may be a κ chain or a λ chain; Preferably, the amino acid sequence of the heavy chain constant region of the anti-PD-L1 monoclonal antibody includes the sequence shown in SEQ ID NO.12 or SEQ ID NO.13, and the amino acid sequence of the light chain constant region includes the sequence shown in SEQ ID NO.15; Preferably, the amino acid sequence of the heavy chain of the bispecific antibody includes the sequence shown in SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.20, and the amino acid sequence of the light chain includes the sequence shown in SEQ ID NO.21; Preferably, the amino acid sequence of the heavy chain of the bispecific antibody includes the sequence shown in SEQ ID NO.24 or SEQ ID NO.25, and the amino acid sequence of the light chain includes the sequence shown in SEQ ID NO.22; or, the amino acid sequence of the heavy chain of the bispecific antibody includes the sequence shown in SEQ ID NO.24 or SEQ ID NO.25, and the amino acid sequence of the light chain includes the sequence shown in SEQ ID NO.

23.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-PD-L1 and HHLA2 bispecific antibody as described in claim 1 or 2.

4. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 3.

5. A recombinant cell, characterized in that, The recombinant cells contain the nucleic acid molecules as described in claim 3.

6. The method for preparing the anti-PD-L1 and HHLA2 bispecific antibody according to claim 1 or 2, characterized in that, The preparation method includes: The nucleic acid described in claim 3 is inserted into the expression vector to obtain a recombinant vector. The recombinant vector is introduced into a host cell, the host cell is cultured, and the bispecific antibody is isolated and purified. Preferably, the host cell comprises a mammalian cell, and the mammalian cell comprises any one of CHO, HEK293, NSO or Per 6; Preferably, the separation and purification method includes affinity chromatography and ultrafiltration concentration; Preferably, the buffer solution used in the ultrafiltration concentration includes at least one of histidine buffer, phosphate buffer, citrate buffer, acetate buffer, succinate buffer, or tromethamine buffer. Preferably, the buffer solution comprises a phosphate buffer solution with added sodium chloride and arginine.

7. A bispecific antibody conjugate, characterized in that, The bispecific antibody conjugate includes the anti-PD-L1 and HHLA2 bispecific antibody as described in claim 1 or 2 and its conjugate. Preferably, the conjugate includes a detectable marker or a drug; Preferably, the detectable marker includes at least one of a radioactive isotope, a luminescent substance, a colored substance, or an enzyme; Preferably, the conjugate includes at least one of FITC, PE, APC, CY-5, CY-7, RF488, Alexa Fluor 488, PerCP-Cy5.5, APC-Cy7, maytansine derivative, carlecamycin, docamycin derivative, PBD, camptothecin derivative, 131I, 90Y, 177Lu, 188Re, alkaline phosphatase, horseradish peroxidase, or biotin.

8. A reagent kit, characterized in that, The kit comprises the anti-PD-L1 and HHLA2 bispecific antibody as described in claim 1 or 2 and / or the bispecific antibody conjugate as described in claim 7; Preferably, the kit further includes a second antibody that specifically recognizes the bispecific antibody; Preferably, the second antibody further includes a detectable marker, which includes at least one of a radioactive isotope, a luminescent substance, a colored substance, or an enzyme.

9. The use of the anti-PD-L1 and HHLA2 bispecific antibody as described in claim 1 or 2, or the bispecific antibody conjugate as described in claim 7, in the preparation of reagents targeting tumors; Preferably, the tumor-targeting reagent includes drugs for the prevention or treatment of tumors or tumor antigen detection reagents; Preferably, the tumor antigen includes PD-L1 and / or HHLA2; Preferably, the tumor includes at least one of non-small cell lung cancer, ovarian cancer, melanoma, kidney tumor, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, or liver cancer.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the anti-PD-L1 and HHLA2 bispecific antibody as described in claim 1 or 2 and / or the bispecific antibody conjugate as described in claim 7.

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