CN115785269A

CN115785269A - anti-PD-L1 antibodies and uses thereof

Info

Publication number: CN115785269A
Application number: CN202211366304.0A
Authority: CN
Inventors: 仝爱平; 王曾; 卢华庆; 周良学; 魏于全
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-11-01
Filing date: 2022-11-01
Publication date: 2023-03-14
Anticipated expiration: 2042-11-01
Also published as: CN115785269B

Abstract

The invention discloses an anti-PD-L1 antibody and application thereof, relates to the field of antibodies, and provides an anti-PD-L1 antibody capable of being specifically combined with PD-L1 or a functional fragment thereof, wherein CDRs of the anti-PD-L1 antibody are selected from any one of (1) to (14); the antibody or the functional fragment thereof can specifically block the combination of PD-1 and PD-L1 and block the inhibitory action on lymphocyte, thereby blocking the immune escape mechanism of tumor, effectively inhibiting the growth of local tumor and providing a way for the prevention and treatment of tumor.

Description

anti-PD-L1 antibodies and uses thereof

Technical Field

The invention relates to the field of antibodies, in particular to an anti-PD-L1 antibody and application thereof.

Background

T cell-mediated cellular immunity plays an important role in recognizing and killing tumor cells, and T cells recognize tumor cells by binding to Major Histocompatibility Complex (MHC) with specific antigens on the surface of the tumor cells through a T Cell Receptor (TCR). The interaction of TCR and MHC molecules is controlled by a series of immune checkpoints, among which co-stimulatory and co-inhibitory signals, can activate or inhibit T cells. The PD-1 and a ligand PD-L1 pathway thereof are inhibitory immune check points, and after being combined, the PD-1 and the ligand PD-L1 communicate a co-inhibitory signal, so that the immune activity of T cells can be inhibited, the T cells play an important role in immune tolerance and are also an important reason for immune escape of tumor cells.

Programmed death receptor-1 (PD-1) is a type I transmembrane protein of a CD28 super family member, is a key immune check point receptor expressed by activated T cells and B cells, and in a normal body, PD-1 is used as a negative regulatory molecule of T cell proliferation and plays an important role in maintaining immune tolerance of the body.

The Programmed cell death ligand 1 (PD-L1), also known as surface antigen cluster of differentiation 274 (CD274) or B7 homolog (B7 homolog 1, B7-H1), is a protein in the human body and is a 40kDa type I transmembrane protein encoded by the CD274 gene. PD-L1 is a surface glycoprotein ligand for PD-1, and PD-1 is a key immune checkpoint receptor expressed by activated T and B cells and mediates immunosuppression. PD-L1 is associated with suppression of immune system responses during chronic infections, pregnancy, allograft transplantation, autoimmune diseases and cancer. PD-L1 is found on antigen presenting cells and human cancer cells, such as squamous cell carcinoma of the head and neck, melanoma and brain tumors, thyroid, thymus, esophagus, lung, breast, gastrointestinal tract, colorectal, liver, pancreas, kidney, adrenal cortex, bladder, urothelium, ovary, and skin. PD-L1 interacts with a receptor PD-1 on a T cell thereof, and plays an important role in the negative regulation of immune response; many studies have shown that it is associated with the immune escape mechanism of tumors. The microenvironment of the tumor part can induce the expression of PD-L1 on the tumor cells, and the expressed PD-L1 is favorable for the generation and the growth of tumors and induces the apoptosis of anti-tumor T cells. After the PD-1 is combined with the PD-L1, an inhibitory signal is transmitted, so that the proliferation and the activity of lymphocytes can be inhibited, the differentiation of CD4+ T cells to Th1 and Th17 cells can be inhibited, the release of inflammatory cytokines can be inhibited, and the effects of immune negative regulation and control can be achieved. Under normal conditions, the combination of PDL-1 and PD-1 can maintain the immune tolerance of peripheral lymphocytes to self-antigens through the above-mentioned actions, thereby preventing the occurrence of autoimmune diseases. However, in the occurrence and development of tumors, PD-L1 expressed by tumor cells can be combined with PD-1 to promote the immune escape of the tumors through the inhibitory action on lymphocytes.

Therefore, the development of the anti-PD-L1 antibody with strong specificity and high affinity provides possibility for the treatment of various cancers, and has huge application potential and market value.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide an anti-PD-L1 antibody and application thereof.

The invention is realized in the following way:

in a first aspect, the embodiments provide an antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region comprising CDRs as set forth in any one of (1) to (14). (1) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No. 1-3, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No. 4-6; (2) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No.1, 9 and 10, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No. 4-6; (3) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No.1, 13 and 13, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No.15, 5 and 16; (4) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No.19, 20 and 21, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No.22, 5 and 6; (5) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No. 25-27, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No.28, 5 and 29; (6) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No.32, 26 and 27, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No. 33-35; (7) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No. 38-40, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No. 41-43; (8) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No. 46-48, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No.41, 49 and 50; (9) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No. 53-55, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No. 56-58; (10) The amino acid sequence is shown as HCDR 1-3 shown by SEQ ID No. 61-63 in sequence, and the amino acid sequence is shown as LCDR 1-3 shown by SEQ ID No. 64-66 in sequence; (11) The amino acid sequence is shown as HCDR 1-3 shown in SEQ ID No. 69-71 in sequence, and the amino acid sequence is shown as LCDR 1-3 shown in SEQ ID No.72, 5 and 73 in sequence; (12) The sequence of the amino acid is shown as HCDR 1-3 shown in SEQ ID No. 76-78, and the sequence of the amino acid is shown as LCDR 1-3 shown in SEQ ID No. 79-81; (13) The amino acid sequence is shown as HCDR 1-3 shown by SEQ ID No. 84-86 in sequence, and the amino acid sequence is shown as LCDR 1-3 shown by SEQ ID No.87, 42 and 50 in sequence; (14) The amino acid sequence is shown as HCDR 1-3 shown in SEQ ID No.1, 90 and 91 in sequence, and the amino acid sequence is shown as LCDR 1-3 shown in SEQ ID No.92, 5 and 6 in sequence.

In a second aspect, embodiments of the invention provide an antibody conjugate comprising: the antibody or antigen binding fragment thereof described in the preceding examples.

In a third aspect, the embodiments of the present invention provide the use of the antibody or the antigen-binding fragment thereof as described in the previous embodiments in the preparation of a detection product for PD-L1 antigen.

In a fourth aspect, embodiments of the invention provide the use of an antibody or antigen-binding fragment thereof as described in the preceding embodiments in the manufacture of a product for targeting PD-L1 for diagnosis, prevention or treatment of disease.

In a fifth aspect, embodiments of the invention provide a reagent or kit comprising an antibody or antigen-binding fragment thereof as described in the preceding embodiments.

In a sixth aspect, embodiments of the invention provide an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof as described in previous embodiments.

In a seventh aspect, embodiments of the invention provide a vector comprising an isolated nucleic acid as described in the previous embodiments.

In an eighth aspect, embodiments of the present invention provide a cell comprising the vector of the previous embodiments.

In a ninth aspect, embodiments of the present invention provide a medicament or pharmaceutical composition, the active ingredient of which comprises at least one of an antibody or antigen-binding fragment thereof as described in the preceding embodiments, an antibody conjugate as described in the preceding embodiments, an agent or kit as described in the preceding embodiments, an isolated nucleic acid as described in the preceding embodiments, a vector as described in the preceding embodiments, and a cell as described in the preceding embodiments.

The invention has the following beneficial effects:

the antibody or the antigen binding fragment thereof provided by the invention can be combined with activated T cells and DC cells, and can effectively block the combination between PD-L1 and PD-1;

the anti-PD-L1 antibody provided by the invention can effectively inhibit local tumor growth; blocking PD-1/PD-L1 signals can promote the proliferation of tumor antigen specific T cells and play a role in killing tumor cells; blocking the relevant PD-L1 signal on tumor cells can up-regulate infiltration CD8 ⁺ Secretion of IFN- γ by T cells, indicating that blockade of the PD-1/PD-L1 signaling pathway plays a role in tumor immune responses with the aim of inducing immune responses; the anti-PD-L1 antibody is selected to be matched with a tumor vaccine for tumor immunotherapy, so that the immune activation of the tumor vaccine can be effectively strengthened. At present, the anti-PD-1/PD-L1 treatment is in the front of immunotherapy with good curative effect and safety, and becomes a hot target in the field of lung cancer treatment in two years.

The invention constructs a single-chain antibody (scFv) and a bispecific antibody (PD-L1-CD 3), and simultaneously verifies the anti-tumor effect of the PD-L1-CD3 bispecific antibody through an in vitro killing experiment, and the result shows that the PD-L1-CD3 bispecific antibody has a remarkable killing effect on Hela-PD-L1 tumor cells. The invention can be applied to the preparation of medicaments for preventing, diagnosing and treating human tumors.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the results of immunofluorescence analysis of the binding of recombinant chimeric antibodies 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, 3211A6 to cell-overexpressed PD-L1 molecules;

FIG. 2 is a partial representative result of the PD-L1 monoclonal antibody blocking the binding of PD-1 to PD-L1;

fig. 3 is the in vitro tumor killing results for bispecific antibodies 2420A7, 62D11, 95C12, 142a11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, 3211A6, in tandem with CD3scfv, respectively;

fig. 4 is a graph of in vivo anti-tumor assay results using the NSG murine model to evaluate 2420A7, 62D11, 95C12, 142a11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, 3211A6 bispecific antibody in tandem with CD3scfv, respectively.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

First, the present invention provides an antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise CDRs as set forth in any one of (1) to (14).

(1) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No. 1-3, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No. 4-6; (2) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No.1, 9 and 10, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No. 4-6; (3) The amino acid sequences are shown as HCDR 1-3 shown in SEQ ID No.1, 13 and 13 in sequence, and the amino acid sequences are shown as LCDR 1-3 shown in SEQ ID No.15, 5 and 16 in sequence; (4) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No.19, 20 and 21, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No.22, 5 and 6; (5) The amino acid sequence is shown as HCDR 1-3 shown by SEQ ID No. 25-27 in sequence, and the amino acid sequence is shown as LCDR 1-3 shown by SEQ ID No.28, 5 and 29 in sequence; (6) The amino acid sequence is shown as HCDR 1-3 shown by SEQ ID No.32, 26 and 27 in sequence, and the amino acid sequence is shown as LCDR 1-3 shown by SEQ ID No. 33-35 in sequence; (7) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No. 38-40, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No. 41-43; (8) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No. 46-48, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No.41, 49 and 50; (9) The amino acid sequence is shown as HCDR 1-3 shown by SEQ ID No. 53-55 in sequence, and the amino acid sequence is shown as LCDR 1-3 shown by SEQ ID No. 56-58 in sequence; (10) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No. 61-63, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No. 64-66; (11) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No. 69-71, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No.72, 5 and 73; (12) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No. 76-78, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No. 79-81; (13) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No. 84-86, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No.87, 42 and 50; (14) The amino acid sequence is shown as HCDR 1-3 shown in SEQ ID No.1, 90 and 91 in sequence, and the amino acid sequence is shown as LCDR 1-3 shown in SEQ ID No.92, 5 and 6 in sequence.

In the present invention, the term "antibody" encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc.), murine antibodies, chimeric antibodies, full-length antibodies, and the like, so long as they exhibit the desired antigen-binding activity.

The chimeric antibody of the present invention is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, and can reduce the immune response reaction induced by the murine antibody. The construction of chimeric antibody includes the first establishing hybridoma secreting mouse-derived specific monoclonal antibody, the subsequent cloning of variable region gene from mouse hybridoma cell, the subsequent cloning of human antibody constant region gene, connecting the mouse variable region gene and human constant region gene into chimeric gene, inserting the chimeric gene into human carrier, and final expressing the chimeric antibody molecule in eukaryotic industrial system or prokaryotic industrial system.

In the present invention, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, which are the regions within the antibody variable domain that contribute primarily to specific binding to antigen. In a particular embodiment of the invention, CDRs refer to more than 2 hypervariable regions in the heavy and light chains of the antibody.

In the present invention, the heavy chain complementarity determining region is represented by HCDR, and the variable region of the heavy chain contains 3 CDR regions: HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is denoted LCDR, and the light chain variable region contains 3 CDR regions: LCDR1, LCDR2 and LCDR3.

In some embodiments, the antigen of the antibody or antigen-binding fragment thereof comprises PD-L1.

In some embodiments, the light chain variable region and the heavy chain variable region further comprise a framework region.

In the present invention, the "framework region" or "FR" region refers to a region other than CDRs in the antibody heavy chain variable region and light chain variable region; the heavy chain framework regions can be further subdivided into contiguous regions (FR 1, FR2, FR3 and FR 4) separated by CDRs, wherein the heavy chain framework regions can be further subdivided into contiguous regions separated by CDRs comprising HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework regions may be further subdivided into contiguous regions separated by LCDRs, comprising LFR1, LFR2, LFR3 and LFR4 framework regions. The heavy chain variable region is derived from the following numbered CDR and FR (amino terminal to carboxy terminal) arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by linking the following numbered CDRs to an FR (amino-terminal to carboxy-terminal) arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

In some embodiments, the heavy chain variable region and the light chain variable region are as set forth in any one of (a) to (n):

(a) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID No. 7-8 in sequence; (b) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID No. 11-12 in sequence; (c) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID No. 17-18 in sequence; (d) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID No. 23-24 in sequence; (e) The amino acid sequences of the heavy chain variable region and the light chain variable region are sequentially shown as SEQ ID No. 30-31; (f) The amino acid sequences of the heavy chain variable region and the light chain variable region are sequentially shown as SEQ ID No. 36-37; (g) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID No. 44-45 in sequence; (h) The amino acid sequences of the heavy chain variable region and the light chain variable region are sequentially shown as SEQ ID No. 51-52; (i) The amino acid sequences of the heavy chain variable region and the light chain variable region are sequentially shown as SEQ ID No. 59-60; (j) The amino acid sequences of the heavy chain variable region and the light chain variable region are sequentially shown in SEQ ID Nos. 67-68; (k) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID No. 74-75 in sequence; (l) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID No. 82-83 in sequence; (m) the amino acid sequences of the heavy chain variable region and the light chain variable region are sequentially shown as SEQ ID Nos. 88-89; (n) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID No. 93-94 in sequence.

The CDRs shown in (1) to (14) correspond to the CDRs (a) to (n) in this order.

In some embodiments, the antibody or antigen-binding fragment thereof further comprises a constant region. Optionally, the constant region comprises a heavy chain constant region and/or a light chain constant region. The light chain of the full-length antibody comprises a light chain variable region structural domain VL and a constant region structural domain CL, wherein the VL is positioned at the amino terminal end of the light chain, the CL structural domain is positioned at the carboxyl terminal end, and the light chain comprises a kappa chain and a lambda chain; the full-length antibody heavy chain includes a heavy variable region domain, VH, at the amino terminus of the heavy chain and a constant region, CH, domain at the carboxy terminus.

In some embodiments, the constant region is selected from the constant regions of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD.

In some embodiments, the species source of the constant region is a cow, horse, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, or human.

In some embodiments, the antigen-binding fragment is selected from the group consisting of F (ab') ₂ Any one of Fab', fab, fv and scFv (single chain antibody). An "antigen-binding fragment" is a portion of an intact antibody that specifically binds to the antigen to which the intact antibody binds. As will be readily understood by those skilled in the art based on the teachings herein, antigen-binding fragments can be prepared by methods known in the art, for example, enzymatic digestion (including pepsin or papain) and/or by chemical reduction to cleave disulfide bonds, and can be synthesized by recombinant genetic techniques or by automated peptide synthesizers (e.g., those of Applied BioSystems).

Wherein the structure of the single-chain antibody is represented by the formula: VH- (G4S) 3-VL-huIgG1Fc, wherein VH is the heavy chain variable region, VL is the light chain variable region, (G4S) 3 is the peptide linker, and huIgG1Fc is the constant region of human IgG1 antibody.

The structure of the bispecific antibody (PD-L1-CD 3) is represented by the formula: VL1- (G4S) 3-VH1-G4S-VH2- (G4S) ₃ -VL2; wherein VH1 is the heavy chain variable region of PD-L1, VL1 is the light chain variable region of anti-PD-L1 antibody, VH2 is the heavy chain variable region of anti-CD 3 antibody, VL2 is the light chain variable region of anti-CD 3 antibody, G4S and (G4S) ₃ Is a peptide linker, VH2- (G4S) 3-VL2 is CD3scFv using the amino acid sequence of OKT3 (shown below):

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTS EDSAVYYCARYYDDHYCLDYWGQGTTLTVSS(VH2)GGGGSGGGGSGGGGS((G4S) ₃ )DIQLTQSPAIMSASPGEKVTMTCRASSSVSY MNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(VL2)。

in another aspect, embodiments of the present invention also provide an antibody conjugate, comprising: an antibody or antigen-binding fragment thereof as described in any of the preceding examples.

In some embodiments, the antibody conjugate further comprises a solid support conjugated to the antibody or antigen-binding fragment thereof. Optionally, the solid support includes, but is not limited to, at least one of magnetic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon, and nitrocellulose membranes.

In some embodiments, the antibody conjugate further comprises a detectable label conjugated to the antibody or antigen-binding fragment thereof.

In the actual use process, one skilled in the art can select other suitable markers according to the detection conditions or actual needs, and whatever marker is used falls within the protection scope of the present invention.

In some embodiments, the label is selected from at least one of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent agent, and a nanoparticle-based label.

The embodiment of the invention also provides application of the antibody or the antigen binding fragment thereof in preparing a detection product of the PD-L1 antigen.

In some embodiments, the product comprises any of a dipstick, a reagent and a kit.

In some embodiments, the method of detection is selected from: ELISA, immunofluorescence, chemiluminescence immunoassay, western blot, immunochromatography, electrochemical immunoassay and magnetic bead method.

In another aspect, embodiments of the invention also provide a use of the antibody or antigen binding fragment thereof according to any of the preceding embodiments in the manufacture of a product for targeting PD-L1 for diagnosis, prevention or treatment of a disease.

Preferably, the disease is selected from any one of breast cancer, lung cancer, stomach cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma or melanoma;

preferably, the product is selected from any one of a reagent, a kit and a medicament.

In another aspect, the embodiments of the present invention also provide a reagent or a kit, which includes the antibody or the antigen-binding fragment thereof according to any of the preceding embodiments.

In another aspect, embodiments of the invention also provide an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof as described in any of the preceding embodiments.

In another aspect, the embodiments of the present invention also provide a vector comprising the isolated nucleic acid of any of the preceding embodiments.

The vector includes an expression vector. The term "expression vector" as used herein refers to any recombinant polynucleotide construct which can be transformed, transfected or transduced to introduce a DNA fragment of interest directly or indirectly (e.g., into a virus) into a host cell for expression of a gene of interest. One type of vector is a plasmid, i.e., a circular double-stranded DNA molecule, which can link a DNA fragment of interest into a plasmid loop. Another type of vector is a viral vector, which ligates the DNA segment of interest into a viral genome (e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors have entered the host cell, expression of the desired gene can be carried out.

The skilled person can also express the antibody or functional fragment thereof of the present invention by in vitro transcription using the nucleic acid sequence of the antibody of the present invention as a template, transcribing the RNA, further transfecting, transducing or transforming the RNA into a host cell, and performing the biological effect of the present invention.

In another aspect, embodiments of the present invention also provide a cell containing a vector as described in any of the previous embodiments.

In some embodiments, the cell is a host cell, including prokaryotic host cells, eukaryotic host cells, and phage. The prokaryotic host cell can be escherichia coli, streptomycete or bacillus subtilis and the like. The eukaryotic host cell can be 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, per6, saccharomyces cerevisiae, pichia pastoris, hansenula, candida, part of insect cells and plant cells. 293 series cells, per6 cells and CHO cells are commonly used mammalian cells for the production of antibodies or recombinant proteins and are well known to those of ordinary skill in the art.

In another aspect, embodiments of the invention provide a method of producing an antibody or antigen-binding fragment thereof as described in any of the preceding embodiments, comprising culturing a cell that expresses the antibody or antigen-binding fragment thereof.

Based on the disclosure of the amino acid sequence of the antibody, it is easy for those skilled in the art to prepare the antibody by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by separating and purifying the antibody from the culture product of recombinant cells capable of recombinantly expressing the antibody as described above, and therefore, it is within the scope of the present invention to prepare the antibody of the present invention by any technique.

In another aspect, the present invention also provides a medicament or pharmaceutical composition, wherein the effective component comprises at least one of the antibody or antigen binding fragment thereof according to any of the preceding embodiments, an antibody conjugate according to any of the preceding embodiments, a reagent or kit according to any of the preceding embodiments, an isolated nucleic acid according to any of the preceding embodiments, a vector according to any of the preceding embodiments, and a cell according to any of the preceding embodiments.

The term "pharmaceutical composition" as used herein means a combination of at least one drug and optionally a pharmaceutically acceptable carrier or adjuvant, which are combined together to achieve a particular purpose. In certain embodiments, the pharmaceutical compositions include temporally and/or spatially separated combinations, so long as they are capable of acting together to achieve the objectives of the present invention. Some pharmaceutical compositions are prepared by co-administering pharmaceutically acceptable ingredients or compounds to achieve the biological efficacy of the invention or to reduce side effects of the drug (e.g., may be used in combination with other anti-tumor drugs to enhance anti-tumor efficacy). Other pharmaceutical compositions are intended to facilitate administration to the organism, facilitate absorption of the active ingredient, enhance stability or targeting, prolong half-life, and further exert the biological efficacy of the present invention better.

In another aspect, the invention also provides a method of targeting PD-L1 for diagnosis, prevention, or treatment of a disease, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acids, vectors, cells, and pharmaceutical compositions of the invention.

The disease is selected from breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, renal cancer, bladder cancer, pancreatic cancer, glioma or melanoma, etc.

An "effective amount" as used herein refers to a dosage sufficient to show its benefits to the subject to which it is administered. The actual amount administered, as well as the rate and time course of administration, will depend on the subject's own condition and severity. The prescription for treatment is ultimately determined by a physician, usually taking into account the individual condition of the patient, the site of delivery, the method of administration, the severity of the disease and other factors conventional to physicians.

The term "subject" as used herein refers to a mammal, such as a human, but may also be other animals, such as wild animals, domestic animals or laboratory animals.

In another aspect, the invention also provides a method of detecting a PD-L1 antigen, comprising detecting the antigen using an antibody or antigen-binding fragment thereof provided in any of the preceding examples.

The detection method includes but is not limited to ELISA, immunochromatography, immunohistochemistry, immunofluorescence and flow detection, and can also be used for antigen tracing of cells, tissues or living bodies, for example, the antibody or functional fragment thereof of the present invention can be fluorescently or isotopically labeled for antigen tracing.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

(1) Preparation of PD-L1 recombinant protein: the nucleotide sequence encoding human PD-L1 was synthesized by the general biology of Anhui. PCR amplified and subcloned into pcDNA3.1 expression vector. Then, the extracellular domain of PD-L1 was subcloned into pcdna3.1 expression vector carrying Fc or His tag at the C-terminal, respectively. Wherein the Fc tag comprises human-derived Fc (hFc) and murine-derived Fc (mFc). By transient transfection of 293FT, using FreeStyle ^TM Serum-free medium (Life Technologies) is cultured for 5-7 days in a shaking flask, supernatant is collected, centrifugal ultrafiltration is carried out, and then recombinant PD-L1 Protein carrying Fc or His labels is purified through Protein A/G or NTA-Ni affinity chromatography and a molecular sieve chromatographic column.

(2) Preparation of stable cell lines expressing human PD-L1 antigen: and (3) constructing a full-length sequence for encoding the human PD-L1 into a lentiviral vector carrying plenti-eGFP, and packaging into lentiviral particles. Hela cells were cultured in DMEM containing 10% fetal bovine serum. Infection of hela cells was performed with concentrated virus solution. After 48 hours, flow sorting is carried out, the cells are cultured to a 96-well plate, monoclonal stable cell strains are screened and identified, and Hela-PD-L1-EGFP cells which stably express PL-L1 are protected.

Example 2: preparation of anti-PD-L1 monoclonal antibody

Animal immunization: balb/c female mice 5-6 weeks old were used as immunized animals at an immunizing dose of 100. Mu.g/mouse. For the first immunization, 100. Mu.l Freund's complete adjuvant (Sigma) was mixed with an equal volume of recombinant PD-L1 protein, emulsified well and injected subcutaneously in several spots. Every 2 weeks, recombinant protein was mixed with an equal volume of incomplete Freund's adjuvant (Sigma), emulsified thoroughly and injected subcutaneously in multiple spots. 4 times of boosting immunization, and 10 days after the last boosting immunization, cutting the tail and collecting blood to detect the titer of the antibody of the mouse. 3 days before cell fusion, 100. Mu.g of recombinant protein was subjected to intraperitoneal impact once.

Cell fusion and hybridoma screening: under aseptic conditions, the hind leg root lymph node from the mouse was used to prepare a B cell-rich suspension, which was subjected to cell fusion with SP2/0 cells according to the classical PEG (Sigma) method. The fused cells were resuspended in HAT medium for culture. Culture was performed using fresh HAT medium on days 5 and 10 after fusion. ELISA, immunofluorescence and flow analysis are carried out 11-15 days after fusion, and positive clones are screened. ELISA screening was performed using 96-well plates, PD-L1 recombinant protein was coated onto the bottom of the plate at 100 ng/well overnight at 4 deg.C, 50. Mu.l hybridoma culture supernatant was used as the primary antibody, developed using HRP-conjugated anti-mouse IgG antibody and chemiluminescent reagent (Biyuntian Biotech Co., ltd.), and read in a microplate reader at 450nm wavelength. Immunofluorescence staining using stably expressing PD-L1 Hela cell strain, briefly, cell strain in 96 well plate adherent culture, adding 50 u L hybridoma supernatant as primary antibody, 4 degrees incubation for 2 hours, PBS washing 3 times, cy3 labeled Goat Anti-Mouse IgG (Proteintech) as secondary antibody, room temperature incubation for 1 hours, PBS washing 3 times, using fluorescence microscope to collect images.

Based on the above results of ELISA analysis and immunofluorescence analysis, 14 optimal hybridoma clones (designated 2420A7, 62D11, 95C12, 142a11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, 3211A6, respectively) were finally determined for subsequent experiments such as sequence cloning and affinity analysis.

Example 3

Hybridoma antibody variable region sequence cloning: optimal hybridoma clone cells in the logarithmic growth phase were collected, RNA extracted with Trizol (Invitrogen) and reverse transcribed (PrimeScript) ^TM Reverse Transcriptase, takara). And (3) carrying out PCR amplification on cDNA obtained by reverse transcription by using mouse Ig-Primer Set (Novagen) and then sequencing to finally obtain the sequences of the heavy chain and light chain variable regions. Wherein the variable region CDR sequences of the heavy and light chains are shown in Table 1.

TABLE 1 CDR sequences contained in the variable regions of the murine mAb heavy and light chains

Example 4: binding analysis of recombinant chimeric antibody to hela cervical cancer cell overexpression PD-L1

Cloning human (IgG 1) heavy chain constant region and human light chain constant region into pcDNA3.1 (Invitrogen) plasmid vector, then constructing VH and VL gene segments of hybridoma clone 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, 3211A6 on genetic recombination vector with human IgG1 heavy chain constant region and human IgG kappa light chain constant region respectively to obtain recombinant chimeric antibody heavy chain expression vector and light chain expression vector, transiently transfecting 293FT using FreeStyle ^TM Culturing in serum-free medium (Life Technologies) in shake flask for 5-7 days, collecting supernatant, centrifuging, ultrafiltering, and performing ultrafiltrationAnd purifying by Protein A/G affinity chromatography and a molecular sieve chromatographic column to obtain the corresponding anti-PD-L1 recombinant monoclonal antibody.

Human IgG1 heavy chain constant region sequence (SEQ ID No. 95):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

human IgG kappa light chain constant region sequence (SEQ ID No. 96):

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

Hela-PD-L1-EGFP cells were plated in 24-well cell culture dishes, and the next day recombinant chimeric antibodies 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, 3211A6 were used as primary antibodies, CY 3-labeled Goat Anti-human IgG (H + L) (Biyuntan Biotech) was used as secondary antibodies, and they were observed with a fluorescent confocal lens and photographed. As shown in fig. 1, it is shown by the results of fig. 1 that: the recombinant chimeric antibodies 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6 and 3211A6 can specifically bind to cell Hela-PD-L1-EGFP.

Example 5: binding assay of hybridoma antibodies to murine PD-L1

A cDNA clone of murine PD-L1 was purchased from Okayama, and then a stably expressing cell line CHO-mPD-L1 of murine PD-L1 was constructed as in example 1. Supernatants from hybridomas 2420A7, 62D11, 95C12, 142a11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, and 3211A6 were then immunofluorescent-stained as in example 2. The results indicate that the monoclonal antibody is not capable of binding to murine PD-L1.

Example 6: in vitro binding affinity and kinetics experiments

This example was measured by Surface Plasmon Resonance (SPR) and analyzed using a Biacore 8K instrument from GE. The PD-L1-His recombinant protein is covalently connected to a CM5 (GE) chip by using a kit provided by Biacore and adopting a standard amino coupling method, then the single-chain antibody to be detected is diluted in the same buffer solution according to different concentration gradients for sample injection, and the regeneration is carried out by matching a regeneration reagent in the kit after the sample injection. Analysis and collection of data was performed using Biacore 8K suite analysis software. The results obtained are shown in Table 2 below.

TABLE 2 affinity

Example 7: PD-L1 monoclonal antibody blocks binding of PD-1 and PD-L1

The corresponding types of anti-PD-L1 recombinant monoclonal antibodies 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6 and 3211A6 are obtained by the Protein A/G affinity chromatography and the molecular sieve chromatographic column purification of the implementation case 4. And (3) paving the Hela-PD-L1-EGFP cells in a 96-well plate, and performing immunofluorescence verification of PD-L1 antibody blocking PD-1 combined PD-L1 the next day. After cell fixation, washing twice with PBS (phosphate buffer solution), taking protein of PD-1-mFc as a primary antibody, incubating for 1h at 37 ℃, washing twice with PBS, then respectively adding 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6 and 3211A6 monoclonal antibodies for blocking, and taking a hole without anti-PD-L1 antibody as a control. After incubation at 37 ℃ for 1h, PBS was washed twice, CY 3-labeled Goat Anti-Mouse IgG (H + L) (Byuntian Biotech Co.) was added as a secondary antibody, and observed and photographed with a fluorescence confocal microscope. Representative results are shown in FIG. 2, and the results show that PD-1-mFc protein can bind to PD-L1 on tumor cells, but the binding amount of PD-1 protein is obviously reduced after 10ug/ml of anti-PD-L1 monoclonal antibody is added, which indicates that the PD-L1 monoclonal antibodies can obviously block the binding of PD-1 to PD-L1.

Example 8: in vitro killing experiment of PD-L1-CD3 bispecific antibody

The structure of the PD-L1-CD3 bispecific antibody is represented by the formula: VL1- (G4S) 3-VH1-G4S-VH2- (G4S) ₃ -VL2; wherein VH1 is a heavy chain variable region of PD-L1, VL1 is a light chain variable region of the anti-PD-L1 antibody provided by the invention, VH2 is a heavy chain variable region of the anti-CD 3 antibody, VL2 is a light chain variable region of the anti-CD 3 antibody, G4S and (G4S) ₃ For peptide linker, VH2- (G4S) 3-VL2 is CD3scFv using the amino acid sequence of OKT3 (shown below):

preparation of effector and target cells: peripheral blood was collected from healthy donors, PBMC was isolated, T cells were isolated using a T Cell Isolation Kit (Miltenyi T Cell Isolation Kit), and the isolated T cells were cultured in X-VIVO (LONZA) medium containing 5% AB serum, and TC-coated 6-well plates were incubated with 1ml of coating solution containing 50ng/ml of anti-human CD3 antibody (PeproTech) and 50ng/ml of CD28 antibody (PeproTech) at 37 ℃ for 2 hours before use, and the coating solution was removed before use. Cells were seeded at 1 mL/well in antibody-coated 6-well plates and cultured for 48 hours under stimulation, and then continued to be cultured supplemented with IL-2 (100U/mL) and IL-15 (10 ng/mL) as activating factors. Target cells are selected from Hela-PD-L1-EGFP overexpressed by PD-L1 and cultured in DMEM high-glucose culture solution added with 10% fetal bovine serum (Gibco).

Cell co-culture experiment: adding Hela-PD-L1-EGFP tumor cells in logarithmic growth phase into a 96-well plate, after overnight culture, adding 0ng/ml (Control) or 1ng/ml bispecific antibody, and simultaneously adding 1: 1. 2: 1. 4:1 and 8:1, and after 24 hours, the killing effect of T cells on tumor cells was observed, and representative results are shown in fig. 3, and the experimental results showed that fourteen PD-L1-CD3 bispecific antibodies (2420 A7, 62D11, 95C12, 142a11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, 3211 A6) had a significant killing effect on Hela-PD-L1-EGFP tumor cells (P <0.05, # P <0.01, # P < 0.001) compared to the group without bispecific antibody (Control), and there was no significant difference between the antibodies.

Example 9: xenograft mouse model antitumor experiment

This example used a xenograft mouse model to evaluate the in vivo anti-tumor activity of PD-L1-targeted bispecific antibodies 2420A7, 62D11, 95C12, 142a11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, 3211A6. An immunodeficient mouse model was used for the evaluation.

NCG severe immunodeficiency mouse model: NCG severe immunodeficiency mice were purchased from the university of Nanjing model animal institute, and 2 × 106 logarithmic growth phase a375 cells were inoculated subcutaneously in the right hind back of NCG mice. The tumor grows to 200mm after about 6 days ³ Thereafter, mice with uniform tumor volumes were randomly grouped into 5 mice each. An equal volume of saline to the administration was set as a control group. The bispecific antibody was administered intraperitoneally at 30 μ g/mouse 1 time every 3 days for a total of 4 times. Mice were weighed every 3 days and tumor size was measured. The mean volume of the transplanted tumor was calculated according to the formula V =1/2 (L × W2), where L represents the length of the tumor volume and W represents the width of the tumor volume. When the tumor volume of the mice reaches 2000mm ³ Or the tumor surface is obviously broken, the mice are killed, and the animal experiment is finished. All data were mean ± s.d.p values obtained using unpaired two-tailed student t-test. As shown in fig. 4, the results of fig. 4 revealed that the fourteen PD-L1-CD3 bispecific antibodies (2420 A7, 62D11, 95C12, 142a11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, 3211 A6) all had significant inhibitory effects on the growth of a375 tumor cells (P) compared to the non-administered control group (see fig. 4)<0.05、**P<0.01、***P<0.001 2217B3/2616B4/3718B12-CD3 group has the best inhibition effect on P<0.001。

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise CDRs as set forth in any one of (1) to (14);

(1) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No. 1-3, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No. 4-6;

(2) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No.1, 9 and 10, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No. 4-6;

(3) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No.1, 13 and 13, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No.15, 5 and 16;

(4) The amino acid sequences are shown as HCDR 1-3 shown in SEQ ID No.19, 20 and 21 in sequence, and the amino acid sequences are shown as LCDR 1-3 shown in SEQ ID No.22, 5 and 6 in sequence;

(5) The amino acid sequence is shown as HCDR 1-3 shown by SEQ ID No. 25-27 in sequence, and the amino acid sequence is shown as LCDR 1-3 shown by SEQ ID No.28, 5 and 29 in sequence;

(6) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No.32, 26 and 27, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No. 33-35;

(7) The amino acid sequence is shown as HCDR 1-3 shown by SEQ ID No. 38-40 in sequence, and the amino acid sequence is shown as LCDR 1-3 shown by SEQ ID No. 41-43 in sequence;

(8) The amino acid sequence is shown as HCDR 1-3 shown by SEQ ID No. 46-48 in sequence, and the amino acid sequence is shown as LCDR 1-3 shown by SEQ ID No.41, 49 and 50 in sequence;

(9) The amino acid sequence is shown as HCDR 1-3 shown by SEQ ID No. 53-55 in sequence, and the amino acid sequence is shown as LCDR 1-3 shown by SEQ ID No. 56-58 in sequence;

(10) The amino acid sequence is shown as HCDR 1-3 shown by SEQ ID No. 61-63 in sequence, and the amino acid sequence is shown as LCDR 1-3 shown by SEQ ID No. 64-66 in sequence;

(11) The amino acid sequence is shown as HCDR 1-3 shown in SEQ ID No. 69-71 in sequence, and the amino acid sequence is shown as LCDR 1-3 shown in SEQ ID No.72, 5 and 73 in sequence;

(12) The sequence of the amino acid is shown as HCDR 1-3 shown in SEQ ID No. 76-78, and the sequence of the amino acid is shown as LCDR 1-3 shown in SEQ ID No. 79-81;

(13) The amino acid sequence is sequentially shown as HCDR 1-3 shown as SEQ ID No. 84-86, and the amino acid sequence is sequentially shown as LCDR 1-3 shown as SEQ ID No.87, 42 and 50;

(14) The amino acid sequence is shown as HCDR 1-3 shown in SEQ ID No.1, 90 and 91 in sequence, and the amino acid sequence is shown as LCDR 1-3 shown in SEQ ID No.92, 5 and 6 in sequence.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antigen of the antibody or antigen-binding fragment thereof comprises PD-L1;

preferably, the light chain variable region and the heavy chain variable region further comprise a framework region;

preferably, the heavy chain variable region and the light chain variable region are as defined in any one of (a) to (n);

(a) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID No. 7-8 in sequence;

(b) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID No. 11-12 in sequence;

(c) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID No. 17-18 in sequence;

(d) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID No. 23-24 in sequence;

(e) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID No. 30-31 in sequence;

(f) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID No. 36-37 in sequence;

(g) The amino acid sequences of the heavy chain variable region and the light chain variable region are sequentially shown in SEQ ID No. 44-45;

(h) The amino acid sequences of the heavy chain variable region and the light chain variable region are sequentially shown as SEQ ID No. 51-52;

(i) The amino acid sequences of the heavy chain variable region and the light chain variable region are sequentially shown as SEQ ID No. 59-60;

(j) The amino acid sequences of the heavy chain variable region and the light chain variable region are sequentially shown in SEQ ID Nos. 67-68;

(k) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID No. 74-75 in sequence;

(l) The amino acid sequences of the heavy chain variable region and the light chain variable region are sequentially shown in SEQ ID No. 82-83;

(m) the amino acid sequences of the heavy chain variable region and the light chain variable region are sequentially shown as SEQ ID Nos. 88-89;

(n) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID No. 93-94 in sequence;

preferably, the antibody or antigen-binding fragment thereof further comprises a constant region;

preferably, the constant region is selected from the constant regions of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD;

preferably, the species source of the constant region is cattle, horses, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese or humans;

preferably, the antibody is selected from any one of a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a murine antibody, a chimeric antibody and a full-length antibody;

preferably, the antigen-binding fragment is selected from the group consisting of F (ab') ₂ Any one of Fab', fab, fv and scFv.

3. An antibody conjugate, comprising: the antibody or antigen-binding fragment thereof of claim 1 or 2;

preferably, the antibody conjugate further comprises a solid support conjugated to the antibody or antigen-binding fragment thereof;

preferably, the antibody conjugate further comprises a detectable label conjugated to the antibody or antigen-binding fragment thereof.

4. Use of the antibody or antigen-binding fragment thereof of claim 1 or 2 for the preparation of a detection product for PD-L1 antigen;

preferably, the product comprises any one of a test paper, a reagent and a kit;

preferably, the method of detection is selected from: ELISA, immunofluorescence, chemiluminescence immunoassay, western blot, immunochromatography, electrochemical immunoassay and magnetic bead method.

5. Use of the antibody or antigen-binding fragment thereof of claim 1 or 2 in the manufacture of a product for targeting PD-L1 for diagnosis, prevention or treatment of a disease;

6. A reagent or kit comprising the antibody or antigen-binding fragment thereof of claim 1 or 2.

7. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of claim 1 or 2.

8. A vector comprising the isolated nucleic acid of claim 7.

9. A cell comprising the vector of claim 8.

10. A medicament or pharmaceutical composition comprising as active ingredient at least one of an antibody or antigen-binding fragment thereof according to claim 1 or 2, an antibody conjugate according to claim 3, an agent or kit according to claim 6, an isolated nucleic acid according to claim 7 and a vector according to claim 8 or a cell according to claim 9.