Anti-PDL1 antibodies and uses thereof

Abstract

The present disclosure relates to the biomedicine field, specifically to an anti-PDL1 antibody or antigen-binding fragment thereof and uses. The anti-PDL1 antibody or antigen-binding fragment thereof according to the present disclosure comprises at least one CDR sequence selected from heavy chain variable region CDR sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and light chain variable region CDR sequences: SEQ ID NO:4, WAS, and SEQ ID NO:5, or an amino acid sequence having at least 80% identity thereto. The antibody has high binding affinity to PDL1, and the present disclosure further provides a CD3 and PDL1 bispecific antibody, which binds more strongly to tumor cells and promotes the anti-cancer function of T cells.

Description

[Technical Field]

[0001] The present disclosure relates to the biomedical field, and specifically to anti-PDL1 antibodies or antigen-binding fragments thereof, and uses. [Background technology]

[0002] Globally, cancer is a serious disease that threatens the life and health of humankind, and its incidence rate is increasing year by year due to the worsening of environmental pollution. At the same time, multiple cancer treatment methods have been developed, including traditional surgical resection, radiation therapy, chemotherapy, and new targeted therapies, including small molecule targeted drug therapy, macromolecular targeted drug therapy, immune checkpoint therapy, bispecific antibody therapy, and cell and gene therapy.

[0003] In recent years, immune checkpoint antibodies and bispecific antibodies have been the most promising therapeutic approaches. With the approval of CTLA4, PD-1 / L1, and LAG3 antibodies, immune checkpoint antibodies have become a trend in recent years, achieving remarkable therapeutic effects in a variety of cancer types, including melanoma and non-small cell lung cancer. The approval process for bispecific antibodies has also accelerated significantly. To date, approvals have included Eemovab (CD3×EPCAM), CD3×CD19 (Blincyto), Rybrevant (EGFR×cMet), Lunsumio (CD3×CD20), and Tecvayli (CD3×BCMA). Rybrevant (EGFR×cMet), Lunsumio (CD3×CD20), and Tecvayli (CD3×BCMA) were approved last year and this year, demonstrating the potential of these innovative drugs to treat a greater number of patients.

[0004] Radiation therapy and chemotherapy generally involve strong side effects and suppress the patient's immune system, which can lead to tumor recurrence. Unlike traditional radiation therapy and chemotherapy, which rely on cytotoxic effects, immune checkpoint therapy and bispecific antibody drugs rely on the patient's own immune system to fight cancer, resulting in relatively minimal side effects and not suppressing the patient's immune system.

[0005] PDL1 expression is significantly elevated in many tumors. By binding to the PD-1 receptor on the surface of T cells, it transmits immunosuppressive signals, promoting T cell exhaustion and reducing the patient's anti-cancer immune function. Using antibodies to block the interaction between PD-1 and PDL1 can reverse T cell immune exhaustion and restore anti-cancer function. To date, PD-1 antibodies such as Opdivo and Keytruda, and PDL1 antibodies such as Tecentirq (Atezolizumab), have benefited a significant number of patients. However, not all patients respond to PD-1 / L1 monoclonal antibody therapy, with an overall efficacy rate of around 30%, meaning many more patients are unable to benefit from it.

[0006] The extracellular portion of the PD-1 receptor and its mutants are limited by the diversity of glycosylation modifications, making them unsuitable for drug discovery. Although there are currently reports of PDL1 antibodies and related products, there is a problem in that they do not have sufficient binding ability to PDL1 on the surface of tumor cells.

[0007] Therefore, there is an urgent need to develop anti-PDL1 antibodies with higher affinity to PDL1. Summary of the Invention [Problem to be solved by the invention]

[0008] The present disclosure aims to solve at least to some extent one of the technical problems in the related art. [Means for solving the problem]

[0009] Thus, a first aspect of the disclosure provides an antibody or antigen-binding fragment thereof, Heavy chain variable region CDR sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and It comprises at least one CDR sequence selected from the light chain variable region CDR sequences of SEQ ID NO: 4, WAS, and SEQ ID NO: 5, or an amino acid sequence having at least 80% identity thereto.

[0010] The antibody or antigen-binding fragment according to the present disclosure comprises the above-mentioned specific CDR sequence, and can specifically bind to PDL1 highly expressed on the surface of tumor cells with high binding affinity, thereby inhibiting the interaction between PD-1 and PDL1 on the surface of immune cells and promoting the anti-cancer function of immune cells.

[0011] In some embodiments of the present disclosure, the antibody or antigen-binding fragment comprises: It comprises a heavy chain variable region CDR1 sequence shown in SEQ ID NO: 1, a heavy chain variable region CDR2 sequence shown in SEQ ID NO: 2, a heavy chain variable region CDR3 sequence shown in SEQ ID NO: 3, a light chain variable region CDR1 sequence shown in SEQ ID NO: 4, a light chain variable region CDR2 sequence shown in WAS, and a light chain variable region CDR3 sequence shown in SEQ ID NO: 5.

[0012] In some embodiments of the present disclosure, the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region set forth in SEQ ID NO:6 and a light chain variable region set forth in SEQ ID NO:7; (a) comprises an amino acid sequence that has at least 80% sequence identity compared to (a); (b) a heavy chain variable region set forth in SEQ ID NO:8 and a light chain variable region set forth in SEQ ID NO:9; (b) contains an amino acid sequence that has at least 80% sequence identity with respect to (b).

[0013] In some embodiments of the disclosure, the antibody or antigen-binding fragment comprises a heavy chain variable region set forth in SEQ ID NO:6 or SEQ ID NO:8, and / or a light chain variable region set forth in SEQ ID NO:7 or SEQ ID NO:9.

[0014] In some embodiments of the present disclosure, the antibody or antigen-binding fragment comprises a heavy chain variable region set forth in SEQ ID NO:6 and a light chain variable region set forth in SEQ ID NO:7, or a heavy chain variable region set forth in SEQ ID NO:8 and a light chain variable region set forth in SEQ ID NO:9.

[0015] In some embodiments of the present disclosure, the antibody or antigen-binding fragment comprises at least one of a heavy chain constant region and a light chain constant region, and at least a portion of the heavy chain constant region and / or the light chain constant region is derived from at least one of a primate-derived antibody and a mouse-derived antibody or a variant thereof.

[0016] In some embodiments of the present disclosure, the light chain constant region and the heavy chain constant region are both derived from a mouse-derived IgG antibody or a mutant thereof, or a human-derived IgG antibody or a mutant thereof.

[0017] In some embodiments of the present disclosure, the light chain constant region and the heavy chain constant region are both derived from a mouse-derived IgG1 antibody or a mutant thereof, or a human-derived IgG1 antibody or a mutant thereof.

[0018] In some embodiments of the present disclosure, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

[0019] In some embodiments of the present disclosure, the antibody or antigen-binding fragment has a heavy chain having the amino acid sequence set forth in either SEQ ID NO: 10 or SEQ ID NO: 12 and a light chain having the amino acid sequence set forth in either SEQ ID NO: 11 or SEQ ID NO: 13.

[0020] In some embodiments of the present disclosure, the antibody or antigen-binding fragment has a heavy chain having the amino acid sequence set forth in SEQ ID NO:10 and a light chain having the amino acid sequence set forth in SEQ ID NO:11, and the antibody or antigen-binding fragment has a heavy chain having the amino acid sequence set forth in SEQ ID NO:12 and a light chain having the amino acid sequence set forth in SEQ ID NO:13.

[0021] In some embodiments of the present disclosure, the antibody or antigen-binding fragment comprises a monoclonal antibody or a polyclonal antibody, the monoclonal antibody comprising at least one of a full-length antibody, an Fv, a single-chain antibody, an Fab, a single-domain antibody, and a minimal recognition unit, and the antibody or antigen-binding fragment is capable of binding to the amino acid sequence set forth in SEQ ID NO: 14.

[0022] A second aspect of the present disclosure provides a bispecific binding molecule, comprising a first binding region comprising an antibody or antigen-binding fragment according to the first aspect, and a second binding region having CD3 binding activity.

[0023] In some embodiments of the present disclosure, the bispecific binding molecule comprises a symmetric bispecific binding molecule or an asymmetric bispecific binding molecule, wherein the bispecific binding molecule is an asymmetric bispecific binding molecule.

[0024] In some embodiments of the present disclosure, the first binding region of the bispecific binding molecule comprises peptide chain 1 and peptide chain 2, wherein peptide chain 1 comprises the heavy chain variable region SEQ ID NO:6 or SEQ ID NO:8, and peptide chain 2 comprises the light chain variable region SEQ ID NO:7 or SEQ ID NO:9.

[0025] In some embodiments of the present disclosure, the second binding domain comprises at least one of a full-length antibody, Fv, single-chain antibody, Fab, single-domain antibody, and minimal recognition unit having CD3 binding activity.

[0026] In some embodiments of the present disclosure, the second binding domain comprises an anti-CD3 single chain antibody.

[0027] The CD3×PDL1 bispecific antibody of the present disclosure can bind more strongly to tumor cells and promote the anti-cancer function of T cells.

[0028] In some embodiments of the present disclosure, the anti-CD3 single-chain antibody comprises an anti-CD3 antibody heavy chain variable region and an anti-CD3 antibody light chain variable region, wherein the anti-CD3 antibody heavy chain variable region comprises a heavy chain variable region CDR1 sequence shown in SEQ ID NO: 15, a heavy chain variable region CDR2 sequence shown in SEQ ID NO: 16, and a heavy chain variable region CDR3 sequence shown in SEQ ID NO: 17, and the anti-CD3 antibody light chain variable region has a light chain variable region CDR1 sequence shown in SEQ ID NO: 18, a light chain variable region CDR2 sequence shown in GTN, and a light chain variable region CDR3 sequence shown in SEQ ID NO: 19.

[0029] In some embodiments of the present disclosure, the anti-CD3 single chain antibody comprises a heavy chain variable region set forth in SEQ ID NO:20 and a light chain variable region set forth in SEQ ID NO:21.

[0030] In some embodiments of the present disclosure, the anti-CD3 single-chain antibody further comprises a connecting peptide, wherein the N-terminus of the connecting peptide is connected to the C-terminus of the anti-CD3 antibody heavy chain variable region and the C-terminus of the connecting peptide is connected to the N-terminus of the anti-CD3 antibody light chain variable region; or the N-terminus of the connecting peptide is connected to the C-terminus of the anti-CD3 antibody light chain variable region and the C-terminus of the connecting peptide is connected to the N-terminus of the anti-CD3 antibody heavy chain variable region.

[0031] In some embodiments of the present disclosure, the connecting peptide has the amino acid sequence (GGGGS)n, where n is an integer greater than or equal to 1, and preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0032] In some embodiments of the present disclosure, the anti-CD3 single chain antibody has the amino acid sequence shown in SEQ ID NO:22.

[0033] In some embodiments of the present disclosure, the first binding region further comprises at least one of a first heavy chain constant region and a first light chain constant region, and at least a portion of at least one of the first heavy chain constant region and the first light chain constant region is derived from at least one of a human-derived antibody, a primate-derived antibody, and a mouse-derived antibody or a variant thereof.

[0034] In some embodiments of the present disclosure, the first heavy chain constant region and the light chain constant region are both derived from a human IgG antibody or a variant thereof.

[0035] In some embodiments of the present disclosure, the first heavy chain constant region and the light chain constant region are both derived from a human IgG1 antibody or a variant thereof.

[0036] In some embodiments of the present disclosure, the N-terminus of the first heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

[0037] In some embodiments of the present disclosure, the peptide chain 1 has the amino acid sequence shown in SEQ ID NO:12, and the peptide chain 2 has the amino acid sequence shown in SEQ ID NO:13.

[0038] In some embodiments of the present disclosure, the peptide chain 1 and the peptide chain 2 are connected by a disulfide bond.

[0039] In some embodiments of the present disclosure, the second binding region further comprises a second heavy chain constant region, at least a portion of which is derived from at least one of a human-derived antibody, a primate-derived antibody, and a mouse-derived antibody or a variant thereof.

[0040] In some embodiments of the present disclosure, the second heavy chain constant region is derived from a human IgG antibody or a variant thereof.

[0041] In some embodiments of the present disclosure, the second heavy chain constant region is derived from a human IgG1 antibody or a variant thereof.

[0042] In some embodiments of the present disclosure, the N-terminus of the second heavy chain constant region is connected to the C-terminus of the anti-CD3 single-chain antibody.

[0043] In some embodiments of the present disclosure, the first heavy chain constant region and the second heavy chain constant region are connected by a knob-into-hole structure.

[0044] A third aspect of the present disclosure provides an isolated polynucleotide encoding the antibody or antigen-binding fragment according to the first aspect, or encoding the bispecific binding molecule according to the second aspect.

[0045] A fourth aspect of the present disclosure provides an expression vector carrying the polynucleotide according to the third aspect.

[0046] A fifth aspect of the present disclosure provides a recombinant cell carrying a polynucleotide according to the third aspect, an expression vector according to the fourth aspect, or capable of expressing an antibody or antigen-binding fragment according to the first aspect, or encoding a bispecific binding molecule according to the second aspect.

[0047] In some embodiments of the present disclosure, the recombinant cell is obtained by introducing the expression vector of the fourth aspect into a host cell.

[0048] In some embodiments of the present disclosure, the recombinant cell is a eukaryotic cell.

[0049] In some embodiments of the present disclosure, the recombinant cell is a mammalian cell.

[0050] A sixth aspect of the present disclosure provides a composition comprising the antibody or antigen-binding fragment according to the first aspect, the bispecific binding molecule according to the second aspect, the polynucleotide according to the third aspect, the expression vector according to the fourth aspect, and the recombinant cell according to the fifth aspect.

[0051] A seventh aspect of the present disclosure provides a method for preparing an antibody or antigen-binding fragment according to the first aspect, or a bispecific binding molecule according to the second aspect, comprising culturing a recombinant cell according to the fifth aspect.

[0052] An eighth aspect of the present disclosure provides a drug comprising the antibody or antigen-binding fragment according to the first aspect, the bispecific binding molecule according to the second aspect, the polynucleotide according to the third invention, the expression vector according to the fourth aspect, the recombinant cell according to the fifth aspect, or the composition according to the sixth aspect.

[0053] A ninth aspect of the present disclosure provides a kit containing an antibody or antigen-binding fragment according to the first aspect, a bispecific binding molecule according to the second aspect, a polynucleotide according to the third aspect, an expression vector according to the fourth aspect, and a recombinant cell according to the fifth aspect.

[0054] A tenth aspect of the present disclosure provides use of the antibody or antigen-binding fragment according to the first aspect, the bispecific antibody according to the second aspect, the polynucleotide according to the third aspect, the expression vector according to the fourth aspect, the recombinant cell according to the fifth aspect or the composition according to the sixth aspect in the preparation of a medicament for preventing and / or treating a PDL1-mediated associated disease, including cancer.

[0055] In some embodiments of the present disclosure, the cancer is: The cancer includes at least one selected from lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0056] An eleventh aspect of the present disclosure provides the use of a bispecific binding molecule according to the second aspect, a polynucleotide according to the third aspect, an expression vector according to the fourth aspect, a recombinant cell according to the fifth aspect or a composition according to the sixth aspect in the preparation of a medicament for preventing and / or treating a PDL1 and CD3-mediated associated disease.

[0057] In some embodiments of the present disclosure, the PDL1 and CD3-mediated associated disease includes cancer.

[0058] In some embodiments of the present disclosure, the cancer is: The cancer includes at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0059] A twelfth aspect of the present disclosure provides use of the antibody or antigen-binding fragment according to the first aspect, the bispecific antibody according to the second aspect, the polynucleotide according to the third aspect, the expression vector according to the fourth aspect, or the recombinant cell according to the fifth aspect in the preparation of a kit for detecting PDL1.

[0060] A thirteenth aspect of the present disclosure provides the use of a bispecific binding molecule according to the second aspect, a polynucleotide according to the third aspect, an expression vector according to the fourth aspect, or a recombinant cell according to the fifth aspect in the preparation of a kit for detecting PDL1 and / or CD3.

[0061] By constructing an antibody-fusion protein using the CD3 antibody and the extracellular portion of the PD-1 receptor provided in the present disclosure and bridging T cells with PDL1-positive tumors, T cells can promote tumor killing, and even if tumor cells express low levels of PDL1, T cells can still promote complete tumor killing.

[0062] A fourteenth aspect of the present disclosure provides use of the antibody or antigen-binding fragment according to the first aspect, the bispecific antibody according to the second aspect, the polynucleotide according to the third aspect, the expression vector according to the fourth aspect, the recombinant cell according to the fifth aspect, the composition according to the sixth aspect, or the drug according to the eighth aspect in the prevention or treatment of a disease including a cancer positive for PDL1 on the cancer cell surface.

[0063] According to a specific implementation of the present disclosure, the cancer is selected from at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0064] A fifteenth aspect of the present disclosure provides a method for administering to a subject: There is provided a method of treating a tumor comprising the step of administering at least one of the antibody or antigen-binding fragment according to the first aspect, the bispecific antibody according to the second aspect, the polynucleotide according to the third aspect, the expression vector according to the fourth aspect, the recombinant cell according to the fifth aspect, the composition according to the sixth aspect or the drug according to the eighth aspect.

[0065] According to a specific implementation of the present disclosure, the tumor cell surface PDL1 is positive, and the tumor is selected from at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0066] Additional aspects and advantages of the disclosure will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the disclosure. [Brief explanation of the drawings]

[0067] The above and / or additional aspects and advantages of the present disclosure will become apparent and easier to understand from the following description of the embodiments taken in conjunction with the drawings. [Figure 1]FIG. 1 shows the results of ELISA showing that mouse-derived 19B8 antibody binds to human (left A) and cynomolgus monkey PDL1 (right B) proteins in one embodiment of the present disclosure. [Figure 2] FIG. 1 shows the results of ELISA showing that mouse-derived 19B8 antibody inhibits the binding of PD-1 to PDL1 in one embodiment of the present disclosure. [Figure 3] FIG. 1 shows the results of flow ceteometry of the binding of mouse-derived 19B8 antibody to human lung cancer A549 cells in one embodiment of the present disclosure. [Figure 4] FIG. 1 shows the results of ELISA showing that the 19B8 chimeric antibody according to one embodiment of the present disclosure binds to human (left A) and cynomolgus monkey PDL1 (right B) proteins. [Figure 5] FIG. 1 shows the results of ELISA showing that the 19B8 chimeric antibody inhibits the binding of PD-1 to PDL1 in one embodiment of the present disclosure. [Figure 6] FIG. 1 shows the results of flow ceteometry of the binding of the 19B8 chimeric antibody to human melanoma A375 cells (left A) and human lung cancer A549 cells (right B) in one embodiment of the present disclosure. [Figure 7] FIG. 1 shows the results of ELISA showing that humanized 19B8 antibody inhibits the binding of PD-1 to PDL1 in one embodiment of the present disclosure. [Figure 8] FIG. 1 shows the results of flow ceteometry showing the binding of humanized 19B8 antibody according to one embodiment of the present disclosure to human melanoma A375 cells (left A) and human lung cancer A549 cells (right B). [Figure 9] FIG. 1 shows the results of enhancing SEB-induced IL-2 secretion from human PBMCs by humanized 19B8 antibody in one embodiment of the present disclosure. [Figure 10] FIG. 1 is a schematic diagram of a CD3×PDL1 bispecific antibody according to one embodiment of the present disclosure. [Figure 11] FIG. 1 shows the results of binding of a CD3×PDL1 bispecific antibody to a PDL1 fusion protein in one embodiment of the present disclosure. [Figure 12] FIG. 1 shows the results of ELISA showing that a CD3×PDL1 bispecific antibody according to one embodiment of the present disclosure inhibits the binding of PD-1 to PDL1. [Figure 13] FIG. 1 shows the results of the CD3×PDL1 bispecific antibody promoting the killing of human melanoma A375 cells by PBMCs in one embodiment of the present disclosure. [Figure 14] FIG. 1 shows the results of flow ceteometry of the binding of a CD3×PDL1 bispecific antibody to human lung adenocarcinoma A549 cells in one embodiment of the present disclosure. [Figure 15] FIG. 1 shows the results of the CD3×PDL1 bispecific antibody promoting the killing of human melanoma A375 cells by PBMCs in one embodiment of the present disclosure. [Figure 16] FIG. 1 shows the results of promoting anti-cancer activity in PBMC-reconstituted mice by CD3×PDL1 antibody in one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0068] The following describes in detail the embodiments of the present disclosure. The embodiments described below are illustrative and are used only to interpret the present disclosure, and should not be understood as limitations on the present disclosure.

[0069] It should be noted that the terms "first," "second," etc. are for descriptive purposes only and cannot be considered to indicate or imply relative importance or the express number of technical features. Thus, a feature qualified as "first" or "second" may expressly or imply the inclusion of one or more of the feature. Furthermore, in the description of this disclosure, unless specifically stated otherwise, "plurality" means at least two, e.g., two, three, etc.

[0070] The endpoints of ranges and any value disclosed herein should be understood to be not limited to such exact ranges or values, but to include values ​​close to those ranges or values. In the case of ranges of numerical values, values ​​between the endpoints of each range, between the endpoints of each range and any single point value, and between any single point value can be combined with each other to create one or more new numerical ranges, and these numerical ranges are considered to be specifically disclosed in the specification.

[0071] To make this disclosure more readily understandable, certain technical and scientific terms are defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meaning commonly understood by those of ordinary skill in the art. Abbreviations for amino acid residues refer to the standard three-letter and / or one-letter codes used in the art to denote one of the 20 commonly used L-amino acids.

[0072] As used herein, the terms "comprises" or "including" are open-ended expressions, i.e., include the content set forth in the present disclosure but do not exclude the content of other embodiments.

[0073] As used herein, the terms "optionally," "optional," or "optional" generally mean that a described event or circumstance may occur, but does not necessarily occur, and the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur.

[0074] The antibodies or antigen-binding fragments described herein are typically prepared by biosynthesis. Based on the nucleotide sequences described herein, those skilled in the art can conveniently prepare the encoding nucleic acids of the present disclosure using various known methods. These methods include, but are not limited to, PCR and artificial DNA synthesis. For specific methods, see J. Sambrook, "A Laboratory Guide to Molecular Cloning." In one embodiment of the present disclosure, the encoding nucleic acid sequences of the present disclosure are constructed by segmented synthesis of nucleotide sequences followed by overlap extension PCR. The antibodies or antigen fragments are numbered according to the Kabat numbering system. As used herein, the term "antibody" refers to an immune globular protein molecule capable of binding to a specific antigen. It comprises two light chains and two heavy chains, with the heavy chains (H chains) and light chains (L chains) connected by disulfide bonds to form a tetrameric molecule. The amino acid sequence of the amino terminus (N-terminus) of the peptide chain is highly variable and called the variable region (V region), while the carboxyl terminus (C-terminus) is relatively stable and less variable and called the constant region (C region). The V regions of the L chain and H chain are called VL and VH, respectively. Some regions within the variable region have a higher degree of variation in amino acid composition and sequence order, and are called hypervariable regions (HVRs). Because these are the sites where antigens and antibodies bind, they are also called complementarity-determining regions (CDRs). Both the heavy chain variable region and the light chain variable region contain three CDR regions.

[0075] The antibodies of the present disclosure include mouse-derived antibodies, chimeric antibodies, and humanized antibodies, and preferably humanized antibodies.

[0076] The term "mouse-derived antibody" in this disclosure refers to a monoclonal antibody against human B7H6 prepared according to the knowledge and skill of the art. During preparation, a test subject is injected with an antigen, and then hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated. In a preferred embodiment of this disclosure, the mouse-derived B7H6 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a mouse-derived kappa or lambda chain or a variant thereof, or a heavy chain constant region of a mouse-derived IgG1, IgG2, or IgG3 chain or a variant thereof.

[0077] As used herein, the term "antigen-binding fragment" refers to an "antibody fragment," and an antibody fragment generally refers to an antigen-binding antibody fragment, which may include a portion of an intact antibody, generally the antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv or scFv, diabodies, linear antibodies, single-chain antibody molecules, etc.

[0078] As used herein, the term "monoclonal antibody" refers to an antibody having a single antigen-binding site.

[0079] As used herein, the term "double antibody" refers to an antibody that has two different antigen-binding sites.

[0080] As used herein, the term "mutant" or "variant" refers to a molecule obtained by making a mutation involving one or more nucleotides or amino acids to a naturally occurring or engineered molecule.

[0081] As used herein, the term "complementarity determining region" or "CDR" or "CDR sequence" refers to an amino acid sequence in an antibody that is responsible for antigen binding, typically consisting of amino acid residues 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and amino acid residues 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and / or "high-variability loops" (e.g., amino acid residues 26-32 (LI), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and amino acid residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987))

[0082] As used herein, the term "functional fragment" refers in particular to antibody fragments or diabodies, such as Fv, scFv (sc referring to single chain), Fab, F(ab')2, Fab', scFv-Fc fragments, or any fragment whose half-life can be extended by chemical modification or liposome encapsulation, said chemical modification being the addition of a poly(alkylene) glycol, such as polyethylene glycol ("pegylated, PEGylation") (referred to as pegylated fragments of Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG), where "PEG" is polyethylene glycol.

[0083] As used herein, the term "chimeric antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace the amino acid sequence of the constant region of a monoclonal antibody derived from one species (e.g., mouse) with the constant region of an antibody derived from another species (e.g., human).

[0084] As used herein, the term "humanized antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace all of the amino acid sequences of the constant and variable regions non-CDR (Fv framework region (FR)) of a monoclonal antibody derived from one species (e.g., mouse) with the amino acid sequences of the constant and variable regions non-CDR of an antibody derived from another species (e.g., human). In other words, an antibody in which the constant region has been humanized is called a chimeric antibody, and an antibody in which all of the non-CDR amino acid sequences of the constant and variable regions have been humanized is called a humanized antibody.

[0085] As used herein, the term "full antibody" refers to a tetrameric structure consisting of two identical light chains and two identical heavy chains connected by interchain disulfide bonds. Examples include immunosphere protein G (IgG), immunosphere protein A (IgA), immunosphere protein M (IgM), immunosphere protein D (IgD), and immunosphere protein E (IgE). The same immunosphere protein can also be classified into different subclasses, such as IgG1, IgG2, IgG3, and IgG4, based on amino acid composition. The light chains of immunosphere proteins are classified into κ chains and λ chains depending on the constant region.

[0086] As used herein, the term "knob-into-hole structure" refers to a knob-into-hole mutation formed in the CH3 region of an antibody heavy chain constant region, which facilitates heavy chain interdigitation to form heterodimers.

[0087] As used herein, the term "identity," when describing an amino acid sequence or a nucleic acid sequence relative to a reference sequence, refers to the percentage of identical amino acids or nucleotides between two amino acid or nucleic acid sequences determined by conventional methods, such as those described in Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York), and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5:Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). Many algorithms exist for aligning sequences and measuring sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443, the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444, the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997)), and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs utilizing these algorithms are also available, and may be used in conjunction with ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)), or GAP, BESTFIT, BLAST (Altschul et al., above), FASTA, TFASTA available from the Genetics Computing Group (GCG) Bag, Version 8, Madison, Wisconsin, USA, and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0088] Those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequences disclosed herein to obtain variants of the antibody or functional fragment thereof, provided that the antibody activity is not substantially affected (retaining at least 95% activity). These variants are considered to be within the scope of protection provided by the present disclosure. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences described in the present disclosure have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. Sequence identity as described in the present disclosure can be determined using sequence analysis software, such as the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. All amino acid sequences referred to in the present disclosure are presented in N- to C-terminal order.

[0089] Anti-PDL1 antibody or antigen-binding fragment

[0090] According to a specific implementation of the present disclosure, the present disclosure provides an antibody (anti-PDL1 antibody) or antigen-binding fragment that can specifically recognize PDL1, and the antibody or antigen-binding fragment thereof comprises at least one CDR sequence selected from heavy chain variable region CDR sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and light chain variable region CDR sequences: SEQ ID NO:4, WAS, SEQ ID NO:5, or an amino acid sequence having at least 80% identity thereto.

[0091] "WAS" in the CDR sequence refers to the amino acid sequence.

[0092] According to one specific embodiment of the present disclosure, the anti-PDL1 antibody or antigen-binding fragment thereof is It comprises a heavy chain variable region CDR1 sequence shown in SEQ ID NO: 1, a heavy chain variable region CDR2 sequence shown in SEQ ID NO: 2, a heavy chain variable region CDR3 sequence shown in SEQ ID NO: 3, a light chain variable region CDR1 sequence shown in SEQ ID NO: 4, a light chain variable region CDR2 sequence shown in WAS, and a light chain variable region CDR3 sequence shown in SEQ ID NO: 5.

[0093] According to a specific embodiment of the present disclosure, the anti-PDL1 antibody or antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO: 6 or SEQ ID NO: 8, and / or It comprises a light chain variable region shown in SEQ ID NO:7 or SEQ ID NO:9.

[0094] According to one specific embodiment of the present disclosure, the anti-PDL1 antibody or antigen-binding fragment thereof is a heavy chain variable region set forth in SEQ ID NO: 6 and a light chain variable region set forth in SEQ ID NO: 7, or It comprises a heavy chain variable region shown in SEQ ID NO:8 and a light chain variable region shown in SEQ ID NO:9.

[0095] According to specific implementations of the present disclosure, those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acids in the sequences disclosed herein to obtain variants of the antibody or functional fragment thereof, provided that the antibody activity is not substantially affected (retaining at least 95% activity). These variants are considered to be within the scope of protection provided by the present disclosure. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences described in the present disclosure have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. The sequence identity described in the present disclosure can be determined using sequence analysis software, such as the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. All amino acid sequences referred to in the present disclosure are presented in N- to C-terminal order. Those skilled in the art know that the CDR sequences analyzed by different databases may differ, and these variations should be included in the scope of protection of the present disclosure.

[0096] According to specific implementations of the present disclosure, the antibodies of the present disclosure may be full-length (e.g., IgG1 or IgG4 antibodies), may contain only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments), or may be modified to affect function. The present disclosure includes anti-PDL1 antibodies with modified glycosylation modes. In some applications, modifications to remove undesired glycosylation sites are useful, or antibodies without glycan moieties on the oligosaccharide chain are useful, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function. In other applications, galactosylation modifications are performed to alter complement-dependent cytotoxicity (CDC). After a series of modifications, the fragments described in the present disclosure still have PDL1-binding activity, particularly the amino acid sequence shown in SEQ ID NO: 10. Preferably, the functional fragment consists of or comprises a subsequence of the heavy or light chain variable region of the antibody from which it is derived, the subsequence being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least 1 / 100th, and in a more preferred embodiment at least 1 / 10th, of the affinity of the antibody from which it is derived. Such functional fragments comprise at least 5 amino acids, preferably 10, 15, 25, 50, and 100 consecutive amino acids of the antibody sequence from which it is derived.

[0097] According to a specific implementation of the present disclosure, in order to further improve the bioacceptability of the antibody, the present disclosure humanizes the antibody. The humanization method can be carried out by referring to conventional antibody engineering techniques. The sequence of the heavy chain variable region of the humanized antibody provided in the present disclosure is shown in SEQ ID NO: 8, and the sequence of the light chain variable region is shown in SEQ ID NO: 9.

[0098] Nucleic acid molecules, recombinant vectors, recombinant cells, immunoconjugates

[0099] In the process of preparing or obtaining these antibodies, nucleic acid molecules that express these antibodies are utilized, connected to different vectors, and then expressed in different cells to obtain the corresponding antibodies.

[0100] Therefore, the present disclosure further provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof, as well as a recombinant vector and a transformant containing the nucleic acid. The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof described above, and preferably, the nucleic acid is an expression cassette obtained by genetic engineering means.

[0101] The recombinant vector may refer to a cloning vector or an expression vector, which is obtained by operably linking the nucleic acid to a commercially available vector (e.g., a plasmid or viral vector); commonly used plasmids include pSeTag2, PEE14, pMH3, etc.

[0102] In some preferred implementations, the nucleic acid molecules are species-specifically optimized for expression by mammalian cells.

[0103] The present disclosure further provides an expression vector, which comprises the isolated nucleic acid molecule. When the isolated polynucleotide is connected to a vector, the polynucleotide can be directly or indirectly connected to control elements on the vector, as long as these control elements are capable of controlling the translation and expression of the polynucleotide. Of course, these control elements may be derived directly from the vector itself or may be exogenous, i.e., not necessarily derived from the vector itself. Of course, the polynucleotide only needs to be operably connected to the control elements. As used herein, "operably connected" refers to connecting an exogenous gene to a vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can function to regulate the transcription and translation of the exogenous gene. Of course, the polynucleotides encoding the heavy and light chains of an antibody can be inserted independently into different vectors, but are generally inserted into the same vector. Commonly used vectors include plasmids, bacteriophages, and the like.

[0104] The present disclosure further provides recombinant cells containing the expression vector. The expression vector can be introduced into mammalian cells to construct recombinant cells, which can then be used to express the antibodies or antigen-binding fragments provided herein. The corresponding antibodies can be obtained by culturing the recombinant cells. The host cells described herein can be prokaryotic host cells, eukaryotic host cells, or bacteriophages. The prokaryotic host cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis. The eukaryotic host cells can be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Trichoderma; insect cells such as grass rotifers; plant cells such as tobacco; or mammalian cells such as BHK cells, CHO cells, COS cells, or myeloma cells. In some embodiments, the host cells described herein are preferably mammalian cells, more preferably BHK cells, CHO cells, NSO cells, or COS cells.

[0105] The immunoconjugates provided herein comprise a therapeutic agent and the aforementioned antibody or antigen-binding fragment thereof conjugated to the therapeutic agent, which can be conjugated to the antibody or antigen-binding fragment thereof in any conventional manner.

[0106] The compositions provided herein include the aforementioned antibody or antigen-binding fragment thereof, and / or the aforementioned immunoconjugate, and a pharmaceutically acceptable vector. In some embodiments, the compositions include temporally and / or spatially separated combinations, as long as they can act in concert to achieve the objectives of the present disclosure. For example, the components included in the composition can be administered to a subject as a whole, or separately to a subject. When the components included in the composition are administered to a subject separately, the components can be administered to the subject simultaneously or sequentially.

[0107] Use of drugs, kits and pharmaceuticals and their use in kit preparation

[0108] The present disclosure further provides a drug, which comprises the above-mentioned antibody or antigen-binding fragment thereof and a pharmaceutically acceptable vector, and may further include the above-mentioned immunoconjugate, nucleic acid molecule, expression vector, or recombinant cell.

[0109] In some embodiments, these drug compositions further comprise a pharmaceutically acceptable vector, including any solvents, solid excipients, diluents, binders, disintegrants, or other liquid excipients, dispersing agents, flavoring or suspending agents, surfactants, isotonicity agents, thickeners, emulsifiers, preservatives, solid binders, flow aids, or lubricants, etc., appropriate for the particular target dosage form. To the extent that any conventional adjuvant is incompatible with the compounds of the present disclosure, e.g., results in any adverse biological effect or interacts in a deleterious manner with any other component of the pharmaceutically acceptable composition, it is also contemplated by the present disclosure.

[0110] The compositions of the present disclosure can be administered in combination with each other or with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Thus, the compositions may further comprise a chemotherapeutic agent. The antibodies or antigen-binding fragments thereof, or immunoconjugates of the present disclosure may be combined with a second therapeutic agent, examples of which include, but are not limited to, other agents that inhibit PDL1 activity (such as other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and / or agents that interfere with PDL1 upstream or downstream signaling.

[0111] Typically, the antibody or antigen-binding fragment thereof is administered in an effective amount, i.e., an amount sufficient to achieve the desired therapeutic and / or prophylactic effect, e.g., an amount that causes prevention or alleviation of symptoms associated with the disease being treated, such as a disease associated with PDL1. The effective amount of the composition administered to a subject will depend on the type and severity of the disease and individual characteristics such as general health, age, sex, weight, and tolerance to drugs, as well as the severity and type of disease, and those skilled in the art will be able to determine the appropriate dose based on these and other factors.

[0112] In some embodiments of the present disclosure, a kit for detecting PDL1 in a sample provided herein includes the aforementioned antibody or antigen-binding fragment thereof, a pharmaceutically acceptable vector, an immunoconjugate, a nucleic acid molecule, an expression vector, or a recombinant cell. In some embodiments, the sample may be tissue from a patient with a PDL1-mediated disease (particularly a patient with transplant rejection, autoimmune disease, infectious disease, or cancer, more preferably a patient with at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer). The kit may further include a reagent for detecting normal PDL1, such as a coating solution.

[0113] The present disclosure further provides use of the antibody or antigen-binding fragment thereof, pharmaceutically acceptable vector, immunoconjugate, nucleic acid molecule, expression vector, or recombinant cell in the preparation of a medicament for preventing and / or treating a PDL1-mediated disease. Preferably, the PDL1-mediated disease is transplant rejection, autoimmune disease, infectious disease, or cancer. More preferably, the cancer is a PDL1-expressing cancer. Even more preferably, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer. Even more preferably, the infectious disease includes, but is not limited to, HIV infection and / or hepatitis B virus infection.

[0114] The present disclosure further relates to methods for preventing and / or treating PDL1-mediated diseases (as described above), comprising administering to a patient an effective amount of at least one of an antibody or antigen-binding fragment thereof, immunoconjugate, and composition according to the present disclosure, which can be administered orally, intranasally, intradermally, subcutaneously, intramuscularly, intravenously, or intraperitoneally.

[0115] Nucleic acids encoding the heavy and / or light chains of the antibodies according to the present disclosure are within the scope of the present disclosure, and depending on the amino acid sequence of the heavy and / or light chains, the corresponding nucleic acid sequences can be easily obtained by one skilled in the art and are shown in Table 1.

[0116] [Table 1] JPEG2026503979000003.jpg250156JPEG2026503979000004.jpg179156JPEG2026503979000005.jpg159156 JPEG2026503979000006.jpg234156JPEG2026503979000007.jpg189156JPEG2026503979000008.jpg113156

[0117] The following examples are used in combination to illustrate the present disclosure. Those skilled in the art can understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. If no specific techniques or conditions are shown in the examples, they should be carried out according to the techniques or conditions described in the literature of the field or according to the product specifications. If no manufacturer is shown for the reagents or equipment used, they are conventional products that can be purchased commercially.

[0118] Example 1 Preparation of antibodies

[0119] A mouse-derived monoclonal antibody against human PDL1 was generated, and purified recombinant PDL1 extracellular domain Strep tag fusion protein (PDL1-strep) was used as an antigen to immunize C57BL / 6 mice (9 weeks old, purchased from Shanghai Resc, weighing approximately 20 g).

[0120] Mice were immunized three times with purified antigen and complete Freund's adjuvant, and immune responses were detected after blood collection from the tail vein. Sera were screened by ELISA and flow cytometry to obtain mice with anti-human PDL1 immunoglobulin protein. Splenocytes were collected from mice with the highest anti-PDL1 immunoglobulin protein levels and fused with mouse myeloma cells SP2 / 0 (ATCC No. CRL-1581). Antibody screening was performed on the fused hybridoma cells to obtain mouse monoclonal antibodies.

[0121] A total of 10 candidate hybridoma cells 6 The cells were cultured until they reached 100 cells, and then centrifuged at 800 rpm for 10 minutes to harvest the cells. Total RNA was extracted using a Trizol kit (Invitrogen). A cDNA library (Invitrogen) was synthesized by reverse transcription using the total RNA as a template. The cDNA was then used as a template for PCR amplification of the variable region nucleic acid sequences corresponding to the hybridoma cells. Primer sequences used in the PCR amplification reaction were complementary to the first framework region or signal peptide region and constant region of the antibody variable region (Larrick, JW, et al., (1990) Scand. J. Immunol., 32, 121-128; Coloma, JJ, et al., (1991) BioTechniques, 11, 152-156). To a 50 μL reaction mixture, 2 μl of cDNA, 5 μl of 10× PCR buffer, 2 μl (5 μmol) of upstream and downstream primers, 2 μl of dNTPs, 1 μl of Taq enzyme (Takara, Ex Taq), and 38 μl of HO were added. The initial denaturation was performed at 95°C for 5 minutes, followed by temperature cycling and PCR amplification. The reaction conditions were: 30 seconds of denaturation at 94°C, 45 seconds of annealing at 58°C, and 50 seconds of extension at 72°C (for a total of 32 cycles), followed by 7 minutes of extension at 72°C. The sequences of the heavy and light chain variable regions of the mouse monoclonal antibody were obtained by sequencing the amplified products.

[0122] The heavy chain variable region amino acid sequence is shown in SEQ ID NO:6: EVQLQQSGPDLVKPGASVKISKASGYSFTGYYMHWVKQSHVKSLEWIGRINPYNGATTYSQNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCVPTGMGYFLMDYWGQGTSVTVSS The light chain variable region amino acid sequence is shown in SEQ ID NO:7: DIVMTQSHKFMSASVGDRVSITCKASQDVGSAVVWYRQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQFTSYPTFGSGTRLEIK

[0123] Example 2 ELISA binding experiment of PDL1 antibody

[0124] ELISA experiments were used to detect the binding properties of PDL1 antibodies. PDL1 extracellular domain protein was coated onto a 96-well plate, and the signal intensity after antibody loading was used to determine the binding properties of the antibody and PDL1. Antibody production: Antibodies were prepared by transiently transfecting ExpiCHO-S cells (Gibco, Item A29127) with the pcDNA3.4 vector (Nanjing Jinsirui Synthetic, mouse-derived 19B8 heavy chain 19B8-mIgG1 (amino acid sequence shown in SEQ ID NO: 10) and mouse-derived h19B8 light chain 19B8-mK (amino acid sequence shown in SEQ ID NO: 11) containing the nucleotide sequences of the heavy and light chains of the above antibodies. EVQLQQSGPDLVKPGASVKISCKASGYSFTGYYMHWVKQSHVKSLEWIGRINPYNGATTYSQNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCVPTGMGYFLMDYWGQ GTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGC KPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPMHQDWLNGKEFKCRVNSAAFPAPIEKTISKT KGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:10) DIVMTQSHKFMSASVGDRVSITCKASQDVGSAVVWYRQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQFTSYPTFGSGTRLEIKRA DAAPTVSIFPPSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:11)

[0125] The day before transfection, ExpiCHO-S cells were cultured at a cell density of (3–4) × 10 6 The solution was adjusted to a concentration of 7 × 10 cells / mL and cultured overnight at 37°C, 8% CO2, and 95 rpm with shaking. 6 ~1×10 7 Grow cells to 6 × 10 / mL and prepare for transfection when viability is greater than 95%. Discard cells using fresh, prewarmed ExpiCHO medium (Gibco, item A2910002). 6The pcDNA3.4 plasmids carrying the heavy and light chains were diluted to 1 mL / mL and transfected into ExpiCHO-S cells with the above heavy and light chain plasmids (1:1 light-heavy chain plasmid ratio) and ExpiFectamine CHO Transfection Reagent (Gibco, Item A29129). The cells were then cultured at 37°C, 8% CO2, and 95 rpm with shaking. 18-22 h after transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were mixed uniformly and immediately added to the transfected cells. The mixture was then cultured at 32°C, 5% CO2, and 95 rpm with shaking. Five days after transfection, 8 mL of ExpiCHO Feed was added to the cells, mixed uniformly, and the culture was continued. Cell count and viability were monitored daily. Cells were harvested by centrifugation when cell viability fell below 80% or after 10-14 days of culture. The expression supernatant was filtered through a 0.45 μm filter, and the Fc domain-containing antibody was captured from the expression supernatant using a Mabselect prism Aprotein A affinity chromatography column (purchased from Suzhou Nawei). After equilibrating the chromatography column with phosphate buffer at pH 7.2, the supernatant was passed through the affinity chromatography column and eluted with elution buffer (100 mM citric acid, pH 2.7). Finally, the column was concentrated and replaced with PBS buffer. The purified antibody was identified as having a purity of over 95% by SDS-PAGE, and the results showed that the above antibody was finally obtained.

[0126] Human PDL1-Fc fusion protein (purchased from Acro) or monkey PDL1-His tag protein (purchased from Acro) was diluted to 1 μg / ml in PBS buffer and added to a 96-well plate at a volume of 100 μL per well and incubated overnight at 4°C. The PBS buffer was removed from the 96-well plate, and the plate was washed six times with PBST (pH 7.2, 0.1% Tween 20). 200 μL of PBS / 10% BSA was added per well and incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed six times with PBST. 100 μL of the mouse-derived PDL1 antibody 19B8, diluted to the appropriate concentration in PBST / 0.05% BSA, was added per well and incubated at 37°C for 1 hour. The reaction mixture was removed, and the plate was washed six times with PBST. Then, 100 μL / well of HRP (horseradish peroxidase)-conjugated anti-mouse IgG secondary antibody was diluted in PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added, followed by incubation at room temperature for 3 minutes. To stop the reaction, 80 μL / well of 4M sulfuric acid was added. The absorbance at 450 mm was read using a microplate reader. The results are shown in Figure 1. The PDL1 antibody of the present disclosure can bind to human and monkey PDL1 proteins.

[0127] Example 3: Experiment to detect the inhibitory activity of mouse-derived PDL1 antibodies

[0128] ELISA experiments were used to detect the effect of the antibody on the binding of PDL1 to its receptor PD-1, and the specific experiments are as follows:

[0129] PDL1-Fc fusion protein (purchased from Acro) was diluted to 5 μg / mL in PBS buffer and added to a 96-well plate at a volume of 100 μL per well and incubated overnight at 4°C. The PBS buffer was removed from the 96-well plate, and the plate was washed six times with PBST (pH 7.2, 0.1% Tween 20). 200 μL of PBS / 10% BSA was added per well and incubated at 37°C for 2 hours for blocking. After removing the blocking solution and washing the plate six times with PBST, 19B8 antibody diluted to different concentrations in PBST / 0.05% BSA or PD-1-Avi tag (purchased from Acro) diluted to 2 μg / mL was added at 100 μL per well and incubated at 37°C for 1 hour. After removing the reaction mixture and washing the plate six times with PBST, 100 μL / well of HRP (horseradish peroxidase)-conjugated streptavidin secondary antibody (purchased from Southern Biotech) was diluted in PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 minutes. 80 μL / well of 4M sulfuric acid was added to stop the reaction. mIgG (purchased from Biolegend) was used as a control. The absorbance was read at 450 mm using a microplate reader. The results are shown in Figure 2, demonstrating that the mouse-derived PDL1 antibody (19B8) disclosed herein can inhibit the binding of PDL1 to its receptor PD-1.

[0130] Example 4: Flow cerebrospinal fluid binding experiments of mouse-derived PDL1 antibodies

[0131] 1 x 10 human lung adenocarcinoma A549 cells in PBS 6The cells were diluted to 1 mL / mL and added to 1.5 mL EP tubes at a volume of 100 μL per tube. 10 μL of goat serum was added per tube and blocked at 4°C for 30 minutes. Different concentrations of 19B8 antibody (heavy chain amino acid sequence shown in SEQ ID NO:10, light chain amino acid sequence shown in SEQ ID NO:11) were added and incubated at 4°C for 30 minutes. 1 mL of PBS was added to the EP tubes, and the cells were centrifuged at 3500 rpm for 5 minutes at 4°C. The supernatant was completely removed, and the cells were washed once with PBS. After centrifugation, the supernatant was completely removed, and the cells were resuspended in 100 μL per tube of PBS. 1 μL of Alexa-647-labeled goat anti-mouse secondary antibody (Biolegend) was added per tube and incubated at 4°C for 30 minutes in the dark. The cells were washed twice with PBS, centrifuged, and the supernatant was completely removed. The cells were resuspended in 200 μL / tube of PBS and detected by a flow cytometer, and the results are shown in FIG. 3, which further demonstrates that the mouse-derived PDL1 antibody according to the present disclosure can bind to PDL1 on the surface of tumor cells.

[0132] Example 5: ELISA binding experiments of human-mouse chimeric PDL1 antibodies

[0133] ELISA experiments are used to detect the binding properties of PDL1 antibodies. PDL1 extracellular domain protein is coated onto a 96-well plate, and the signal intensity after antibody loading is used to determine the binding properties of the antibody and PDL1.

[0134] Antibody production: Antibodies were prepared by transiently transfecting ExpiCHO-S cells (Gibco, Item A29127) with the pcDNA3.4 vectors (Nanjing Jinsirui Synthetic; mouse-derived 19B8 heavy chain hIgG1LALA, 19B8-hIgG1LALA (amino acid sequence shown in SEQ ID NO: 31), mouse-derived h19B8 light chain hK, 19B8-hK (amino acid sequence shown in SEQ ID NO: 32), fully human-derived 12A4 antibody heavy chain 12A4-hIgG1LALA (amino acid sequence shown in SEQ ID NO: 28), and fully human-derived 12A4 antibody light chain 12A4-hK (amino acid sequence shown in SEQ ID NO: 30) containing the nucleotide sequences of the heavy and light chains of the above antibodies. EVQLQQSGPDLVKPGASVKISKASGYSFTGYYMHWVKQSHVKSLEWIGRINPYNGATTYSQNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCVPTGMGYFLMDYWGQGT SVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:31) DIVMTQSHKFMSASVGDRVSITCKASQDVGSAVVWYRQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQFTSYPTFGSGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:32) QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:28) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:30)

[0135] The day before transfection, ExpiCHO-S cells were cultured at a cell density of (3–4) × 10 6 The cells were adjusted to a concentration of 7 × 10 / mL and cultured overnight at 37°C, 8% CO2, and 95 rpm with shaking. On the day of transfection, the cells were cultured at a concentration of 7 × 10 6 ~1×10 7 Grow cells to 6 × 10 / mL and prepare for transfection when viability is greater than 95%. Discard cells using fresh, prewarmed ExpiCHO medium (Gibco, item A2910002). 6 The pcDNA3.4 plasmids carrying the heavy and light chains were diluted to 1 mL / mL and transfected into ExpiCHO-S cells with the above heavy and light chain plasmids (1:1 light-heavy chain plasmid ratio) and ExpiFectamine CHO Transfection Reagent (Gibco, Item A29129). The cells were then cultured at 37°C, 8% CO2, and 95 rpm with shaking. 18-22 h after transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were mixed uniformly and immediately added to the transfected cells. The mixture was then cultured at 32°C, 5% CO2, and 95 rpm with shaking. Five days after transfection, 8 mL of ExpiCHO Feed was added to the cells, mixed uniformly, and the culture was continued. Cell count and viability were monitored daily. Cells were harvested by centrifugation when cell viability fell below 80% or after 10-14 days of culture. The expression supernatant was filtered through a 0.45 μm filter, and the Fc domain-containing antibody was captured from the expression supernatant using a Mabselect prism Aprotein A affinity chromatography column (purchased from Suzhou Nawei). After equilibrating the chromatography column with phosphate buffer at pH 7.2, the supernatant was passed through the affinity chromatography column and eluted with elution buffer (100 mM citric acid, pH 2.7). Finally, the column was concentrated and replaced with PBS buffer. The purified antibody was identified as having a purity of over 95% by SDS-PAGE, and the results showed that the above antibody was finally obtained.

[0136] Human PDL1-Fc fusion protein (purchased from Acro) or monkey PDL1-His tag fusion protein (purchased from Acro) was diluted to 1 μg / ml in PBS buffer and added to a 96-well plate at a volume of 100 μL per well and incubated overnight at 4°C. The PBS buffer was removed from the 96-well plate, and the plate was washed six times with PBST (pH 7.2, 0.1% Tween 20). 200 μL of PBS / 10% BSA was added per well and incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed six times with PBST. The test chimeric antibody 19B8-hIgGLALA or the control antibody 12A4-hIgG1LALA, diluted to the appropriate concentration in PBST / 0.05% BSA, was added at 100 μL per well and incubated at 37°C for 1 hour. The reaction mixture was removed, and the plate was washed six times with PBST. Then, 100 μL / well of HRP (horseradish peroxidase)-conjugated anti-human IgG-Fab secondary antibody was diluted in PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added, followed by incubation at room temperature for 3 minutes. To stop the reaction, 80 μL / well of 4M sulfuric acid was added. The absorbance was read at 450 mm using a microplate reader. The results are shown in Figure 4, demonstrating that the chimeric antibody 19B8-hIgG1LALA of the present disclosure binds to both human and monkey PDL1 proteins.

[0137] Example 6: Experiment to detect the inhibitory activity of human-mouse chimeric PDL1 antibodies

[0138] ELISA experiments were used to detect the binding effect of the chimeric antibody on PDL1 and its receptor PD-1, and the specific experiments are as follows:

[0139] PDL1-Fc fusion protein (purchased from Acro) was diluted to 5 μg / mL in PBS buffer and added to a 96-well plate at a volume of 100 μL / well and incubated overnight at 4°C. The PBS buffer was removed from the 96-well plate, and the plate was washed six times with PBST (pH 7.2, 0.1% Tween 20). 200 μL / well of PBS / 10% BSA was added and incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed six times with PBST. Then, 100 μL / well of 19B8-hIgG1LALA antibody or 12A4-hIgG1LALA antibody diluted to different concentrations in PBST / 0.05% BSA, 2 μg / mL of PD-1-Avi tag protein (purchased from Acro), and 100 μL / well of PBST / 0.05% BSA were added and incubated at 37°C for 1 hour. After removing the reaction mixture and washing the plate six times with PBST, 100 μL / well of HRP (horseradish peroxidase)-conjugated streptavidin secondary antibody (purchased from Southern Biotech) was diluted in PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 minutes. 80 μL / well of 4M sulfuric acid was added to stop the reaction. mIgG (purchased from Biolegend) was used as a control. The absorbance was read at 450 mm using a microplate reader. The results are shown in Figure 5, demonstrating that the chimeric antibody 19B8-hIgG1LALA disclosed herein can inhibit the binding of PDL1 to its receptor PD-1.

[0140] Example 7: Flow cetocemometry binding experiments of human-mouse chimeric PDL1 antibodies

[0141] 1 × 10 human melanoma A375 cells and human lung adenocarcinoma A549 cells were cultured in PBS. 6The cells were diluted to 1 mL / mL and added to 1.5 mL EP tubes at a volume of 100 μL per tube. 10 μL of rat serum was added per tube and blocked at 4°C for 30 minutes. Different concentrations of the chimeric antibody 19B8-hIgG1LALA or the control antibody 12A4-hIgG1LALA were added and incubated at 4°C for 30 minutes. 1 mL of PBS was added to the EP tubes, and the cells were centrifuged at 3500 rpm for 5 minutes at 4°C. The supernatant was completely removed and the cells were washed once with PBS. After centrifugation, the supernatant was completely removed and the cells were resuspended in 100 μL of PBS per tube. 1 μL of Alexa-647-labeled rat anti-human secondary antibody (Biolegend) was added per tube and incubated at 4°C for 30 minutes in the dark. The cells were washed twice with PBS, centrifuged, and the supernatant was completely removed. The cells were resuspended in 200 μL / tube of PBS and detected using a flow cytometer. The results are shown in Figure 6, which further demonstrates that the chimeric antibody disclosed herein can bind to the PDL1 protein on the surface of tumor cells, and its binding is stronger than that of the control antibody 12A4.

[0142] Example 8: Humanization experiment of mouse antibody

[0143] The humanization template that best matched the non-CDR regions of the PDL1 antibody was selected based on the light chain variable region and heavy chain variable region sequences. The CDR regions of the mouse antibody were grafted onto the selected humanization template, replacing the CDR regions of the human template, to obtain a humanized antibody. Based on the three-dimensional structure of the mouse antibody, buried residues, residues that directly interact with the CDR regions, and residues that significantly affect the conformation of the VL and VH were backmutated to obtain a humanized antibody. The sequence of the humanized PDL1 antibody heavy chain variable region is shown in SEQ ID NO: 8, and the sequence of the light chain variable region is shown in SEQ ID NO: 9.

[0144] Example 9: Experiment to detect the inhibitory activity of humanized PDL1 antibodies

[0145] ELISA experiments were used to detect the effects of the antibodies on the binding of PDL1 and its receptor PD-1, and the specific experiments were as follows:

[0146] Antibody production: Antibodies were prepared by transiently transfecting ExpiCHO-S cells (Gibco, Item A29127) with the pcDNA3.4 vector (Nanjing Jinsirui Synthetic, humanized 19B8 heavy chain hIgG1LALA, h19B8-hIgG1LALA (amino acid sequence shown in SEQ ID NO: 12), and humanized h19B8 light chain hK, h19B8-hK (amino acid sequence shown in SEQ ID NO: 13), containing the nucleotide sequences of the heavy and light chains of the above antibodies, respectively. QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYMHWVRQAPGQGLEWIGRINPYNGATTYSQNFKDRATMTVDTSISTAYMELSRLRSDDTAVYYCVPTGMGYFLMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:12) DIQLTQSPSFLSASVGDRVTITCKASQDVGSAVVWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFTSYPTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:13)

[0147] The day before transfection, ExpiCHO-S cells were cultured at a cell density of (3–4) × 10 6 The cells were adjusted to a concentration of 7 × 10 / mL and cultured overnight at 37°C, 8% CO2, and 95 rpm with shaking. On the day of transfection, the cells were cultured at a concentration of 7 × 10 6 ~1×10 7 Grow cells to 6 × 10 / mL and prepare for transfection when viability is greater than 95%. Discard cells using fresh, prewarmed ExpiCHO medium (Gibco, item A2910002). 6 The pcDNA3.4 plasmids carrying the heavy and light chains were diluted to 1 mL / mL and transfected into ExpiCHO-S cells with the above heavy and light chain plasmids (1:1 ratio of light to heavy chain plasmid) and ExpiFectamine CHO Transfection Reagent (Gibco, Item A29129). The cells were then cultured at 37°C, 8% CO2, and 95 rpm with shaking. 18-22 h after transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were mixed uniformly and immediately added to the transfected cells. The mixture was then cultured at 32°C, 5% CO2, and 95 rpm with shaking. Five days after transfection, 8 mL of ExpiCHO Feed was added to the cells, mixed uniformly, and the culture was continued. Cell count and viability were monitored daily. Cells were harvested by centrifugation when cell viability fell below 80% or after 10-14 days of culture. The expression supernatant was filtered through a 0.45 μm filter, and the Fc domain-containing antibody was captured from the expression supernatant using a Mabselect prism Aprotein A affinity chromatography column (purchased from Suzhou Nawei). After equilibrating the chromatography column with phosphate buffer at pH 7.2, the supernatant was passed through the affinity chromatography column and eluted with elution buffer (100 mM citric acid, pH 2.7). Finally, the column was concentrated and replaced with PBS buffer. The purified antibody was identified by SDS-PAGE to have a purity of over 95%. The results showed that the above antibody was finally obtained.

[0148] PDL1-Fc fusion protein (purchased from Acro) was diluted to 5 μg / mL in PBS buffer and added to a 96-well plate at a volume of 100 μL per well and incubated overnight at 4°C. The PBS buffer was removed from the 96-well plate, and the plate was washed six times with PBST (pH 7.2, 0.1% Tween 20). 200 μL of PBS / 10% BSA was added per well and incubated at 37°C for 2 hours for blocking. After removing the blocking solution and washing the plate six times with PBST, 100 μL of the target humanized PDL1 antibody h19B8-hIgG1LALA or control antibody 12A4-hIgG1LALA, an atezulizumab analog (purchased from Hyakuei Bio), or 2 μg / mL PD-1-Avi tag (purchased from Acro) diluted to the appropriate concentration in PBST / 0.05% BSA was added per well and incubated at 37°C for 1 hour. After removing the reaction mixture and washing the plate six times with PBST, 100 μL / well of HRP (horseradish peroxidase)-conjugated streptavidin secondary antibody (purchased from Southern Biotech) was diluted in PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 minutes. 80 μL / well of 4M sulfuric acid was added to stop the reaction. mIgG (purchased from Biolegend) was used as a control. The absorbance was read at 450 mm using a microplate reader. The results are shown in Figure 7. The humanized PDL1 antibody h19B8 disclosed herein was able to inhibit the binding of PDL1 to its receptor PD-1, with an inhibitory activity comparable to that of 12A4, an atezulizumab analog.

[0149] Example 10: Flow cerebrospinal fluid binding experiments of humanized PDL1 antibodies

[0150] 1 × 10 human melanoma A375 cells and human lung adenocarcinoma A549 cells were cultured in PBS. 6The cells were diluted to 1 mL / mL and added to 1.5 mL EP tubes at a volume of 100 μL per tube. 10 μL of rat serum was added per tube and blocked at 4°C for 30 minutes. Different concentrations of humanized antibody h19B8-hIgG1LALA (heavy and light chains shown in SEQ ID NOs: 12-13) or control antibody 12A4-hIgG1LALA, an atezulizumab analog (purchased from Hyakuei Bio), were added and incubated at 4°C for 30 minutes. 1 mL of PBS was added to the EP tube, and the tubes were centrifuged at 3500 rpm for 5 minutes at 4°C. The supernatant was completely removed, and the tubes were washed once with PBS. After centrifugation, the supernatant was completely removed, and the cells were resuspended in 100 μL per tube of PBS. 1 μL of Alexa-647-labeled rat anti-human secondary antibody (Biolegend) was added per tube and incubated at 4°C for 30 minutes in the dark. The tubes were washed twice with PBS, centrifuged, and the supernatant was completely removed. The cells were resuspended in 200 μL / tube of PBS and detected using a flow cytometer. The results are shown in Figure 8, which shows that the chimeric antibody disclosed herein can bind to the tumor cell surface PDL1 protein, and its binding is higher than that of the control antibody 12A4, an Atezulizumab analog.

[0151] Example 11: Humanized PDL1 antibody enhances SEB-induced IL-2 secretion from human PBMCs

[0152] 2 x 10 human PBMC cells in complete RPMI-1640 medium 6The cells were diluted to 1 / mL and added to a 96-well plate at a volume of 100 μL per well. 50 μL per well of humanized antibody h19B8-hIgG1LALA or Atezulizumab analogues at different concentrations were added, followed by blocking at 4°C for 30 minutes. Different concentrations of humanized antibody h19B8-hIgG1LALA (shown in SEQ ID NOs: 12-13) or the control antibody Atezulizumab analogue (purchased from Hyakuei Bio) were added, followed by 50 μL per well of 0.4 μg / mL SEB. The cells were then cultured at 37°C in a 5% CO2 incubator for 72 hours. IL-2 content in the culture supernatant was detected using a human IL-2 ELISA kit. The results are shown in Figure 9, which further demonstrates that the humanized PDL1 antibody of the present disclosure can promote IL-2 secretion by PBMCs (wherein the PBMC cells in Figures A and B are derived from different human individuals), and that the promotion ability is comparable to or slightly higher than that of Atezulizumab analogs.

[0153] Example 12: ELISA binding experiment of CD3xPDL1 antibody

[0154] ELISA experiments were used to detect the binding properties of PDL1 antibodies. PDL1 extracellular domain fusion proteins were coated onto 96-well plates, and the signal intensity after antibody loading was used to determine the binding properties of the antibody to PDL1.

[0155] PDL1-Fc fusion protein was diluted to 1 μg / ml in PBS buffer and added to a 96-well plate at a volume of 100 μL / well and incubated overnight at 4°C. The PBS buffer was removed from the 96-well plate, and the plate was washed six times with PBST (pH 7.2, 0.1% Tween 20). 200 μL / well of PBS / 10% BSA was added and incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed six times with PBST. The humanized antibody h19B8-hIgG1LALA or the bispecific antibody CD3×h19B8, diluted to the appropriate concentration in PBST / 0.05% BSA, was added at 100 μL / well and incubated at 37°C for 1 hour. After removing the reaction mixture and washing the plate six times with PBST, 100 μL / well of HRP (horseradish peroxidase)-conjugated anti-human IgG-Fab secondary antibody was added in PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 minutes. 80 μL / well of 4M sulfuric acid was added to stop the reaction. The absorbance was read at 450 mm using a microplate reader. Figure 10 shows several connection methods for the CD3×PDL1 bispecific antibody described herein. Figure 11 shows that the CD3×h19B8 antibody described herein can bind to PDL1 protein, although its binding ability is slightly lower than that of the h19B8 monoclonal antibody.

[0156] The specific sequence information of the CD3×h19B8 antibody (schematically shown in FIG. 10) and CD3×12A4 antibody used in the examples of the present disclosure is shown in Table 2.

[0157] [Table 2]

[0158] Example 13: Experiment to detect the inhibitory activity of CD3×PDL1 antibody

[0159] ELISA experiments were used to detect the effects of the antibodies on the binding of PDL1 and its receptor PD-1, and the specific experiments were as follows:

[0160] PDL1-Fc fusion protein (purchased from Acro) was diluted to 5 μg / mL in PBS buffer and added to a 96-well plate at a volume of 100 μL per well. The plate was then incubated overnight at 4°C. The PBS buffer was removed from the 96-well plate, washed six times with PBST (pH 7.2, 0.1% Tween 20). 200 μL of PBS / 10% BSA was added per well and incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed six times with PBST. Then, 100 μL of the target humanized antibody h19B8-hIgG1LALA or bispecific antibody CD3×h19B8, 2 μg / mL PD-1-Avi tag (purchased from Acro) diluted to the appropriate concentration in PBST / 0.05% BSA was added per well and incubated at 37°C for 1 hour. After removing the reaction mixture and washing the plate six times with PBST, 100 μL / well of HRP (horseradish peroxidase)-conjugated streptavidin secondary antibody (purchased from Southern Biotech) was diluted in PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 minutes. 80 μL / well of 4M sulfuric acid was added to stop the reaction. The absorbance was read at 450 mm using a microplate reader. The results are shown in Figure 12, demonstrating that the bispecific antibody CD3×h19B8 of the present disclosure was able to inhibit the binding of PDL1 to its receptor PD-1, with the inhibitory activity being slightly lower than that of the control atezulizumab.

[0161] Example 14: CD3xPDL1 antibody enhances tumor cell killing by PBMCs

[0162] This example detects PBMC killing of bispecific antibodies. (1) Complete RPMI-1640 medium was added to a 96-well RTCA plate at a volume of 50 μL / well to perform instrument calibration. (2) 2 × 10 human lung adenocarcinoma A375 cells in complete RPMI-1640 medium 5 The cells were diluted to 1 mL / mL and added singly to the RTCA plate obtained in step (1) at a volume of 50 μL / well, and then the cell count was detected for 24 hours using an xCELLigence RTCA MP instrument at 37°C and 5% CO2. (3) Dilute the bispecific antibody in complete RPMI-1640 medium to a series of concentration gradients and add it to the RTCA plate obtained in step (2), with an addition volume of 20 μL / well. (4) 1.25 × 10 PBMCs (Daigasa organisms) in complete RPMI-1640 medium. 6 The solution was diluted to 80 μL / mL and added to the RTCA plate obtained in step (3), with an addition volume of 80 μL / well. (5) The reaction system obtained in step (4) was subjected to cell count detection for 24 hours using an xCELLigence RTCA MP instrument at 37°C and 5% CO2.

[0163] Specific experimental results are shown in Figure 13, which further demonstrate that the CD3xh19B8(a) bispecific antibody disclosed herein can promote the killing of A375 tumor cells by PBMCs, and its killing-promoting ability is superior to that of the (b) and (c) configurations.

[0164] Example 15: Flow cetocemometry binding experiments of CD3xPDL1 antibodies

[0165] 1 x 10 human lung adenocarcinoma A549 cells in PBS 6The cells were diluted to 1 mL / mL and added to 1.5 mL EP tubes at a volume of 100 μL per tube. 10 μL of rat serum was added per tube and blocked at 4°C for 30 minutes. Different concentrations of the bispecific antibody CD3×h19B8 or the control antibody CD3×12A4 were added and incubated at 4°C for 30 minutes. 1 mL of PBS was added to the EP tubes, and the cells were centrifuged at 3500 rpm for 5 minutes at 4°C. The supernatant was completely removed and the cells were washed once with PBS. After centrifugation, the supernatant was completely removed and the cells were resuspended in 100 μL per tube of PBS. 1 μL per tube of Alexa-647-labeled rat anti-human secondary antibody (Biolegend) was added and incubated at 4°C for 30 minutes in the dark. The cells were washed twice with PBS, centrifuged, and the supernatant was completely removed. The cells were resuspended in 200 μL / tube of PBS and detected by flow cytometer. The results are shown in FIG. 14, which further demonstrates that the bispecific antibody CD3×h19B8 according to the present disclosure can bind to the tumor cell surface PDL1 protein, and its binding is stronger than that of the control antibody CD3×12A4.

[0166] Example 16: CD3xPDL1 antibody enhances tumor cell killing by PBMCs

[0167] This example detects the killing of PBMCs by bispecific antibodies. (1) Complete RPMI-1640 medium was added to a 96-well RTCA plate at a volume of 50 μL / well to perform instrument calibration. (2) 2 × 10 human lung adenocarcinoma A375 cells in complete RPMI-1640 medium 5 The cells were diluted to 1 mL / mL and added individually to the RTCA plate obtained in step (1) at a volume of 50 μL / well, and then the cell count was detected for 24 hours using an xCELLigence RTCA MP instrument at 37°C and 5% CO2. (3) Dilute the bispecific antibody in complete RPMI-1640 medium to a series of concentration gradients and add it to the RTCA plate obtained in step (2), with an addition volume of 20 μL / well. (4) 1.25 × 10 PBMCs (Daigasa organisms) in complete RPMI-1640 medium. 6The solution was diluted to 80 μL / mL and added to the RTCA plate obtained in step (3), with an addition volume of 80 μL / well. (5) The reaction system obtained in step (4) is subjected to cell count detection for 24 hours using the xCELLigence RTCA MP instrument at 37°C and 5% CO2.

[0168] Specific experimental results are shown in Figure 15, which further demonstrate that the CD3xh19B8 bispecific antibody of the present disclosure can promote the killing of A375 tumor cells by PBMCs, and its killing-promoting ability is comparable to or slightly superior to that of the control antibody CD3x12A4.

[0169] Example 17: Anti-cancer effect of CD3×PDL1 antibody in mice

[0170] In vivo efficacy experiments were used to detect the promotion of anti-cancer function of the bispecific antibody CD3×h19B8 in immune-reconstituted mice. (1) On day 4, human PBMCs were transplanted via the tail vein into NCG mice (purchased from Chubu Electric) at a rate of 1E7 / mouse. (2) On day 0, NCG mice were subcutaneously implanted with tumors on the right flank, with 1 × 10 6 Inject human melanoma A375 cells. (3) On days 8, 11, 14, and 17, the mice were injected with the CD3×h19B8(a) antibody via the tail vein, at 25 μg per mouse. (4) After the injection of the above antibodies, the tumor volume was measured once every three days.

[0171] The results are shown in FIG. 16, which demonstrate that the CD3×h19B8 bispecific antibody of the present disclosure can effectively inhibit tumor growth and has anti-cancer function.

[0172] The above experimental results demonstrate that the PDL1 antibody disclosed herein has the following properties: it has stronger binding ability to tumor cells, can bind to human and monkey PDL1, can inhibit the binding of PDL1 to its receptor PD-1, and can promote the anti-cancer function of immune cells.

[0173] The present disclosure provides a PDL1 antibody (h19B8) that binds more strongly to PDL1 on tumor cell surfaces than antibodies 12A4 and atezolizumab. Furthermore, after recombinantly expressing the CD3×PDL1 bispecific antibody, the CD3×h19B8 antibody of the present disclosure exhibits stronger binding ability than the CD3×12A4 antibody, demonstrating that the bispecific antibody exhibits stronger tumor recognition and induces stronger T cell killing. The present disclosure also compares the functional activities of CD3×PDL1 antibodies with different structures and identifies bispecific antibodies with stronger functional activity.

[0174] In the description herein, a statement referring to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples" means that a particular feature, structure, material, or characteristic described with reference to that embodiment or example is included in at least one embodiment or example of the present disclosure. In this specification, general references to such terms do not necessarily refer to the same embodiment or example. In addition, a particular feature, structure, material, or characteristic described may be incorporated in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art can combine and combine the various embodiments or examples described herein and the features of the various embodiments or examples without mutual contradiction.

[0175] Although the embodiments of the present disclosure have been shown and described, the above embodiments are merely illustrative and should not be construed as limiting the present disclosure, and it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to the above embodiments within the scope of the present disclosure.

[0176] This application claims priority to and benefits from a patent application with patent application number 202211737491.9, filed with the State Intellectual Property Office of China on December 31, 2022, the entire contents of which are incorporated herein by reference.

Claims

1. An antibody or antigen-binding fragment, Heavy chain variable region CDR sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and An antibody or antigen-binding fragment comprising at least one CDR sequence selected from the light chain variable region CDR sequences of SEQ ID NO: 4, WAS, and SEQ ID NO: 5, or an amino acid sequence having at least 80% identity thereto.

2. 2. The antibody or antigen-binding fragment of claim 1, comprising a heavy chain variable region CDR1 sequence shown in SEQ ID NO: 1, a heavy chain variable region CDR2 sequence shown in SEQ ID NO: 2, a heavy chain variable region CDR3 sequence shown in SEQ ID NO: 3, a light chain variable region CDR1 sequence shown in SEQ ID NO: 4, a light chain variable region CDR2 sequence shown in WAS, and a light chain variable region CDR3 sequence shown in SEQ ID NO:

5.

3. (a) a heavy chain variable region as set forth in SEQ ID NO: 6 and a light chain variable region as set forth in SEQ ID NO: 7; 2. The antibody or antigen-binding fragment of claim 1, comprising an amino acid sequence that has at least 80% sequence identity compared to (a).

4. (b) a heavy chain variable region set forth in SEQ ID NO: 8 and a light chain variable region set forth in SEQ ID NO: 9; The antibody or antigen-binding fragment of claim 1, comprising an amino acid sequence that has at least 80% sequence identity compared to (b).

5. a heavy chain variable region as set forth in SEQ ID NO: 6 or SEQ ID NO: 8, and / or 2. The antibody or antigen-binding fragment of claim 1, comprising a light chain variable region as shown in SEQ ID NO: 7 or SEQ ID NO:

9.

6. a heavy chain variable region set forth in SEQ ID NO: 6 and a light chain variable region set forth in SEQ ID NO: 7, or 6. The antibody or antigen-binding fragment of claim 5, comprising a heavy chain variable region set forth in SEQ ID NO:8 and a light chain variable region set forth in SEQ ID NO:

9.

7. The antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment comprises at least one of a heavy chain constant region and a light chain constant region, and at least a portion of the heavy chain constant region and at least one of the light chain constant regions is derived from at least one of a primate-derived antibody and a mouse-derived antibody or a mutant thereof.

8. The antibody or antigen-binding fragment of any one of claims 1 to 7, wherein the light chain constant region and the heavy chain constant region are both derived from a mouse-derived IgG antibody or a mutant thereof, or a human-derived IgG antibody or a mutant thereof.

9. The antibody or antigen-binding fragment of any one of claims 1 to 7, wherein the light chain constant region and the heavy chain constant region are both derived from a mouse-derived IgG1 antibody or a mutant thereof, or a human-derived IgG1 antibody or a mutant thereof.

10. The antibody or antigen-binding fragment of any one of claims 1 to 8, wherein the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

11. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment has a heavy chain having the amino acid sequence set forth in either SEQ ID NO: 10 or SEQ ID NO: 12 and a light chain having the amino acid sequence set forth in either SEQ ID NO: 11 or SEQ ID NO:

13.

12. the antibody or antigen-binding fragment has a heavy chain having the amino acid sequence set forth in SEQ ID NO: 10 and a light chain having the amino acid sequence set forth in SEQ ID NO: 11; 12. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment has a heavy chain having the amino acid sequence set forth in SEQ ID NO: 12 and a light chain having the amino acid sequence set forth in SEQ ID NO:

13.

13. The antibody or antigen-binding fragment of any one of claims 1 to 12, wherein the antibody or antigen-binding fragment comprises a monoclonal antibody or a polyclonal antibody.

14. The antibody or antigen-binding fragment of any one of claims 1 to 13, wherein the monoclonal antibody comprises at least one of a full-length antibody, an Fv, a single-chain antibody, an Fab, a single-domain antibody, and a minimal recognition unit.

15. The antibody or antigen-binding fragment of any one of claims 1 to 13, wherein the antibody or antigen-binding fragment is capable of binding to the amino acid sequence shown in SEQ ID NO:

14.

16. 1. A bispecific binding molecule comprising: a first binding region comprising the antibody or antigen-binding fragment of any one of claims 1 to 15; and a second binding region having CD3 binding activity.

17. The bispecific binding molecule of claim 16 , wherein the bispecific binding molecule comprises a symmetric bispecific binding molecule or an asymmetric bispecific binding molecule.

18. The bispecific binding molecule of claim 16 or 17, wherein the bispecific binding molecule is an asymmetric bispecific binding molecule.

19. 19. The bispecific binding molecule of any of claims 16 to 18, wherein the first binding region comprises peptide chain 1 and peptide chain 2, wherein peptide chain 1 comprises a heavy chain variable region of any of claims 1 to 15, and peptide chain 2 comprises a light chain variable region of any of claims 1 to 15.

20. The bispecific binding molecule of any one of claims 16 to 19, wherein the second binding region comprises at least one of a full-length antibody, an Fv, a single-chain antibody, an Fab, a single-domain antibody, and a minimal recognition unit having CD3 binding activity.

21. The bispecific binding molecule of any one of claims 16 to 20, wherein the second binding region comprises an anti-CD3 single chain antibody.

22. The anti-CD3 single-chain antibody comprises an anti-CD3 antibody heavy chain variable region and an anti-CD3 antibody light chain variable region, wherein the anti-CD3 antibody heavy chain variable region comprises a heavy chain variable region CDR1 sequence shown in SEQ ID NO: 15, a heavy chain variable region CDR2 sequence shown in SEQ ID NO: 16, and a heavy chain variable region CDR3 sequence shown in SEQ ID NO: 17; The bispecific binding molecule of any one of claims 16 to 21, wherein the anti-CD3 antibody light chain variable region has a light chain variable region CDR1 shown in SEQ ID NO: 18, a light chain variable region CDR2 shown in GTN, and a light chain variable region CDR3 shown in SEQ ID NO:

19.

23. 23. The bispecific binding molecule of any one of claims 16 to 22, wherein the anti-CD3 single chain antibody comprises a heavy chain variable region as shown in SEQ ID NO: 20 and a light chain variable region as shown in SEQ ID NO:

21.

24. The bispecific binding molecule of any one of claims 16 to 23, wherein the anti-CD3 single-chain antibody further comprises a connecting peptide, wherein the N-terminus of the connecting peptide is connected to the C-terminus of the anti-CD3 antibody heavy chain variable region and the C-terminus of the anti-CD3 antibody light chain variable region, or the N-terminus of the connecting peptide is connected to the C-terminus of the anti-CD3 antibody light chain variable region and the C-terminus of the connecting peptide is connected to the N-terminus of the anti-CD3 antibody heavy chain variable region.

25. 25. The bispecific binding molecule of any one of claims 16 to 24, wherein the connecting peptide has the amino acid sequence (GGGGS)n, where n is an integer equal to or greater than 1, and preferably is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

26. The bispecific binding molecule of any one of claims 16 to 25, wherein said anti-CD3 single chain antibody has the amino acid sequence shown in SEQ ID NO:

22.

27. The bispecific binding molecule of any one of claims 16 to 26, wherein the first binding region further comprises at least one of a first heavy chain constant region and a first light chain constant region, and at least a portion of at least one of the first heavy chain constant region and the first light chain constant region is derived from at least one of a human-derived antibody, a primate-derived antibody, and a mouse-derived antibody, or a mutant thereof.

28. The bispecific binding molecule of any one of claims 16 to 27, wherein the first heavy chain constant region and the light chain constant region are both derived from a human IgG antibody or a variant thereof.

29. The bispecific binding molecule of any one of claims 16 to 28, wherein the first heavy chain constant region and the light chain constant region are both derived from a human IgG1 antibody or a variant thereof.

30. The bispecific binding molecule of any one of claims 16 to 29, wherein the N-terminus of the first heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

31. 31. The bispecific binding molecule of any one of claims 16 to 30, wherein said peptide chain 1 has the amino acid sequence shown in SEQ ID NO: 12 and said peptide chain 2 has the amino acid sequence shown in SEQ ID NO:

13.

32. The bispecific binding molecule of any one of claims 16 to 31, wherein said peptide chain 1 and said peptide chain 2 are connected by a disulfide bond.

33. 33. The bispecific binding molecule of any one of claims 16 to 32, wherein the second binding region further comprises a second heavy chain constant region, and at least a portion of the second heavy chain constant region is derived from at least one of a human-derived antibody, a primate-derived antibody, and a mouse-derived antibody or a variant thereof.

34. The bispecific binding molecule of any one of claims 16 to 33, wherein the second heavy chain constant region is derived from a human IgG antibody or a variant thereof.

35. The bispecific binding molecule of any one of claims 16 to 34, wherein the second heavy chain constant region is derived from a human IgG1 antibody or a variant thereof.

36. The bispecific binding molecule of any one of claims 16 to 35, wherein the N-terminus of the second heavy chain constant region is connected to the C-terminus of the anti-CD3 single-chain antibody.

37. The bispecific binding molecule of any one of claims 16 to 36, wherein the first heavy chain constant region and the second heavy chain constant region are connected by a knob-into-hole structure.

38. 38. An isolated polynucleotide encoding the antibody or antigen-binding fragment of any one of claims 1 to 15, or encoding the bispecific binding molecule of any one of claims 16 to 37.

39. An expression vector carrying the polynucleotide of claim 38.

40. 39. A recombinant cell carrying the polynucleotide of claim 38, the expression vector of claim 39, or capable of expressing the antibody or antigen-binding fragment of any one of claims 1 to 15, or encoding the bispecific binding molecule of any one of claims 16 to 37.

41. 41. The recombinant cell of claim 40, wherein the recombinant cell is obtained by introducing the expression vector of claim 39 into a host cell.

42. 42. The recombinant cell of claim 41, wherein the recombinant cell is a eukaryotic cell.

43. 42. The recombinant cell of claim 41, wherein the recombinant cell is a mammalian cell.

44. 42. A composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 15, the bispecific binding molecule of any one of claims 16 to 37, the polynucleotide of claim 38, the expression vector of claim 39, or the recombinant cell of any one of claims 40 to 43.

45. 48. A method for preparing an antibody or antigen-binding fragment of any one of claims 1 to 15 or a bispecific binding molecule of any one of claims 16 to 37, comprising the step of culturing a recombinant cell of any one of claims 40 to 43.

46. 46. ​​A medicament comprising the antibody or antigen-binding fragment of any one of claims 1 to 15, the bispecific binding molecule of any one of claims 16 to 37, the polynucleotide of claim 38, the expression vector of claim 39, the recombinant cell of any one of claims 40 to 43, or the composition of claim 44.

47. 42. A kit comprising the antibody or antigen-binding fragment of any one of claims 1 to 15, the bispecific binding molecule of any one of claims 16 to 37, the polynucleotide of claim 38, the expression vector of claim 39, and the recombinant cell of any one of claims 40 to 43.

48. Use of the antibody or antigen-binding fragment of any one of claims 1 to 15, the bispecific binding molecule of any one of claims 16 to 37, the polynucleotide of claim 38, the expression vector of claim 39, the recombinant cell of any one of claims 40 to 43, or the composition of claim 47 in the preparation of a medicament for preventing and / or treating a PDL1-mediated associated disease.

49. 49. The use according to claim 48, wherein the PDL1-mediated associated disease comprises cancer.

50. The cancer is The use according to claim 49, comprising at least one selected from lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

51. Use of the bispecific binding molecule of any one of claims 16 to 37, the polynucleotide of claim 38, the expression vector of claim 39, the recombinant cell of any one of claims 40 to 43 or the composition of claim 47 in the preparation of a medicament for preventing and / or treating a disease mediated by PDL1 and CD3.

52. The use according to claim 51, wherein the PDL1 and CD3 mediated associated disease comprises cancer.

53. The cancer is 53. The use of claim 52, comprising at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

54. Use of the antibody or antigen-binding fragment of any one of claims 1 to 15, the bispecific antibody of any one of claims 16 to 37, the polynucleotide of claim 38, the expression vector of claim 39, or the recombinant cell of any one of claims 40 to 43 in the preparation of a kit for detecting PDL1.

55. Use of the bispecific binding molecule of any one of claims 16 to 37, the polynucleotide of claim 38, the expression vector of claim 39, or the recombinant cell of any one of claims 40 to 43 in the preparation of a kit for detecting PDL1 and / or CD3.

56. Use of the antibody or antigen-binding fragment of any one of claims 1 to 15, the bispecific binding molecule of any one of claims 16 to 37, the polynucleotide of claim 38, the expression vector of claim 39, the recombinant cell of any one of claims 40 to 43, the composition of claim 44, or the drug of claim 46 in the prevention or treatment of a disease including cancer whose cancer cells are positive for PDL1 on their surface.

57. 57. The use of claim 56, wherein the cancer is selected from at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

58. 1. A method for preventing or treating a tumor, comprising administering to a subject: The antibody or antigen-binding fragment of any one of claims 1 to 15. A bispecific binding molecule according to any one of claims 16 to 37.

39. The polynucleotide of claim 38.

40. The expression vector of claim 39. The recombinant cell according to any one of claims 40 to 43.

45. The composition of claim 44.

47. A method for preventing or treating a tumor, comprising administering at least one of the drugs described in claim 46.

59. 59. The method of claim 58, wherein the tumor cell surface PDL1 is positive, and the tumor is selected from at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

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