CN115785269A - anti-PD-L1 antibodies and uses thereof - Google Patents

Abstract

The invention discloses an anti-PD-L1 antibody and its application, and relates to the field of antibodies. The invention provides an anti-PD-L1 antibody or a functional fragment thereof that can specifically bind to PD-L1, and its CDRs are selected from (1)～ (14) As shown in any one of the items; the antibody or its functional fragments can specifically block the combination of PD-1 and PD-L1, block the inhibitory effect on lymphocytes, and thus block the immune escape of tumors mechanism, effectively inhibiting the growth of local tumors, and providing a way for the prevention and treatment of tumors.

Description

Anti-PD-L1 antibody and its application

technical field

The present invention relates to the field of antibodies, in particular, to anti-PD-L1 antibodies and applications thereof.

Background technique

T cell-mediated cellular immunity plays an important role in identifying and killing tumor cells, and T cells are compatible with major histocompatibility with specific antigens on the surface of tumor cells through T cell receptors (TCR) Complex (major histocompatibility complex, MHC) combined to recognize tumor cells. The interaction of TCR and MHC molecules is controlled by a series of immune checkpoints, including co-stimulatory and co-inhibitory signals, which can enable T cell activation or inhibition. Among them, PD-1 and its ligand PD-L1 pathway are inhibitory immune checkpoints. After they combine, they convey a co-inhibitory signal, which can inhibit the immune activity of T cells and play an important role in immune tolerance. It is also an important reason for tumor cell immune escape.

Programmed death receptor-1 (programmed death-1, PD-1) is a type I transmembrane protein of the CD28 superfamily member, and is a key immune checkpoint receptor expressed by activated T cells and B cells. Among them, PD-1, as a negative regulator of T cell proliferation, plays an important role in maintaining the body's immune tolerance.

Programmed cell death ligand 1 (PD-L1), also known as surface antigen differentiation cluster 274 (cluster of differentiation 274, CD274) or B7 homolog (B7 homolog 1, B7-H1), is a human A protein in the body, encoded by the CD274 gene, is a 40kDa type I transmembrane protein. PD-L1 is the surface glycoprotein ligand of PD-1, a key immune checkpoint receptor expressed by activated T cells and B cells, and mediates immune suppression. PD-L1 has been implicated in the suppression of immune system responses during chronic infection, pregnancy, allogeneic transplantation, autoimmune disease and cancer. In antigen presenting cells and human cancer cells such as head and neck squamous cell carcinoma, melanoma and brain tumors, thyroid, thymus, esophagus, lung, breast, gastrointestinal tract, colorectal, liver, pancreas, kidney, adrenal cortex, PD-L1 has been found in the bladder, urothelium, ovary and skin. PD-L1 interacts with its receptor PD-1 on T cells and plays an important role in the negative regulation of the immune response; many studies have shown that it is related to the immune escape mechanism of tumors. The microenvironment of the tumor part can induce the expression of PD-L1 on tumor cells, and the expressed PD-L1 is conducive to the occurrence and growth of tumors, and induces the apoptosis of anti-tumor T cells. After PD-1 combines with PD-L1, it transmits inhibitory signals, which can inhibit the proliferation and activity of lymphocytes, inhibit the differentiation of CD4+ T cells into Th1 and Th17 cells, and inhibit the release of inflammatory cytokines, all of which play an important role in the immune response. role of regulation. Under normal circumstances, the combination of PDL-1 and PD-1 can maintain the immune tolerance of peripheral lymphocytes to self-antigens through the above-mentioned effects, thereby preventing the occurrence of autoimmune diseases. However, in the occurrence and development of tumors, the combination of PD-L1 expressed by tumor cells and PD-1 can promote the immune escape of tumors through the inhibitory effect on lymphocytes.

Therefore, the development of anti-PD-L1 antibodies with strong specificity and high affinity will provide the possibility for the treatment of various cancers, and has great application potential and market value.

In view of this, the present invention is proposed.

Contents of the invention

The purpose of the present invention is to provide an anti-PD-L1 antibody and its application.

The present invention is achieved like this:

In a first aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region The variable region includes the CDRs shown in any one of (1) to (14). (1) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.1-3, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.4-6; (2) The amino acid sequence is sequentially shown as SEQ ID No. 1, HCDR1-3 shown in 9 and 10, the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.4-6; (3) the amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.1, 13 and 13 3. The amino acid sequence is sequentially as LCDR1-3 shown in SEQ ID No.15, 5 and 16; (4) The amino acid sequence is sequentially as shown in SEQ ID No.19, 20 and 21 HCDR1-3, and the amino acid sequence is sequentially as SEQ ID No.1-3 LCDR1-3 shown in IDNo.22, 5 and 6; (5) the amino acid sequence is shown in sequence as HCDR1-3 shown in SEQ ID No.25-27, and the amino acid sequence is shown in sequence as SEQ ID No.28, 5 and 29 (6) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.32, 26 and 27, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.33-35; (7) The amino acid sequence HCDR1-3 as shown in SEQ ID No.38-40 in turn, amino acid sequence as LCDR1-3 shown in SEQ ID No.41-43 in turn; (8) amino acid sequence as shown in SEQ ID No.46-48 in turn HCDR1-3, the amino acid sequence is sequentially as LCDR1-3 shown in SEQ ID No.41, 49 and 50; (9) the amino acid sequence is sequentially as HCDR1-3 shown in SEQ ID No.53-55, and the amino acid sequence is as follows LCDR1-3 shown in SEQ ID No.56-58; (10) The amino acid sequence is sequentially shown in HCDR1-3 shown in SEQ ID No.61-63, and the amino acid sequence is sequentially shown in LCDR1 shown in SEQ ID No.64-66 ~3; (11) the amino acid sequence is sequentially shown in HCDR1 ~ 3 shown in SEQ ID No.69 ~ 71, and the amino acid sequence is sequentially shown in LCDR1 ~ 3 shown in SEQ ID No.72, 5 and 73; (12) the amino acid sequence is sequentially Such as HCDR1-3 shown in SEQ ID No.76-78, the amino acid sequence is sequentially shown in LCDR1-3 shown in SEQ ID No.79-81; (13) the amino acid sequence is sequentially shown in SEQ ID No.84-86 HCDR1-3, the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.87, 42 and 50; (14) the amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.1, 90 and 91, the amino acid sequence is sequentially LCDR1-3 shown in SEQ ID No.92, 5 and 6.

In a second aspect, an embodiment of the present invention provides an antibody conjugate, which includes: the antibody or antigen-binding fragment thereof described in the foregoing embodiments.

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

In the fourth aspect, the embodiments of the present invention provide the use of the antibody or antigen-binding fragment thereof as described in the foregoing embodiments in the preparation of products for targeting PD-L1 to diagnose, prevent or treat diseases.

In the fifth aspect, the embodiments of the present invention provide a reagent or kit, which includes the antibody or antigen-binding fragment thereof as described in the foregoing embodiments.

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

In the seventh aspect, the embodiments of the present invention provide a vector containing the isolated nucleic acid described in the foregoing embodiments.

In an eighth aspect, the embodiments of the present invention provide a cell containing the vector described in the foregoing embodiments.

In the ninth aspect, the embodiments of the present invention provide a drug or a pharmaceutical composition, the active ingredients of which include the antibody or antigen-binding fragment thereof as described in the foregoing embodiments, the antibody conjugates as described in the foregoing embodiments, and the antibody conjugates as described in the foregoing embodiments. At least one of the reagents or kits shown in the embodiments, the isolated nucleic acid as described in the foregoing embodiments, the vector as described in the foregoing embodiments, and the cells as described in the foregoing embodiments.

The present invention has the following beneficial effects:

The antibody or antigen-binding fragment thereof provided by the invention can bind to activated T cells and DC cells, and can effectively block the binding between PD-L1 and PD-1;

The anti-PD-L1 antibody provided by the invention can effectively inhibit the growth of local tumors; blocking the PD-1/PD-L1 signal can promote the proliferation of tumor antigen-specific T cells and play a role in killing tumor cells; PD-L1 signaling can upregulate the secretion of IFN-γ in infiltrating CD8 ⁺ T cells, suggesting that blockade of the PD-1/PD-L1 signaling pathway plays a role in the tumor immune response for the purpose of inducing an immune response; select anti-PD-L1 Antibodies combined with tumor vaccines for tumor immunotherapy can effectively enhance the immune activation of tumor vaccines. At present, anti-PD-1/PD-L1 therapy is at the forefront of immunotherapy with its good efficacy and safety, and has become a popular target in the field of lung cancer treatment in the past two years.

In the present invention, a single-chain antibody (scFv) and a bispecific antibody (PD-L1-CD3) were constructed, and at the same time, the anti-tumor effect of the PD-L1-CD3 bispecific antibody of the present invention was verified through an in vitro killing experiment. The results showed that the PD-L1-CD3 bispecific antibody had a significant killing effect on Hela-PD-L1 tumor cells. The invention can be applied to the preparation of medicines for preventing, diagnosing and treating human tumors.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.

Figure 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 cells overexpressed PD-L1 molecules;

Figure 2 is a partial representative result of PD-L1 monoclonal antibody blocking the combination of PD-1 and PD-L1;

Figure 3 shows the in vitro tumor killing results of bispecific antibodies 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, 3211A6 respectively in series with CD3scfv;

Figure 4 is the in vivo anti-tumor analysis results of bispecific antibodies 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, 3211A6 respectively in tandem with CD3scfv were evaluated using the NSG mouse model .

Detailed ways

In order to make the purpose, 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. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

First, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region The region includes the CDRs shown in any one of (1) to (14).

(1) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.1-3, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.4-6; (2) The amino acid sequence is sequentially shown as SEQ ID No.1 , HCDR1-3 shown in 9 and 10, the amino acid sequence is sequentially as LCDR1-3 shown in SEQ ID No.4-6; (3) the amino acid sequence is sequentially as HCDR1-3 shown in SEQ ID No.1, 13 and 13 3. The amino acid sequence is sequentially as LCDR1-3 shown in SEQ ID No.15, 5 and 16; (4) The amino acid sequence is sequentially as shown in SEQ ID No.19, 20 and 21 HCDR1-3, and the amino acid sequence is sequentially as SEQ ID No.1-3 LCDR1-3 shown in ID No.22, 5 and 6; (5) the amino acid sequence is sequentially as shown in SEQ ID No.25-27 HCDR1-3, and the amino acid sequence is sequentially as shown in SEQ ID No.28, 5 and 29 LCDR1-3 shown; (6) the amino acid sequence is sequentially as shown in SEQ ID No.32, 26 and 27 HCDR1-3, and the amino acid sequence is sequentially as LCDR1-3 shown in SEQ ID No.33-35; (7) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.38-40, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.41-43; (8) The amino acid sequence is sequentially shown as SEQ ID No.46-48 The amino acid sequences of HCDR1-3 shown in sequence are LCDR1-3 shown in SEQ ID No.41, 49 and 50; LCDR1-3 shown in sequence in SEQ ID No.56-58; (10) The amino acid sequence is shown in sequence in HCDR1-3 shown in SEQ ID No.61-63, and the amino acid sequence is shown in sequence in SEQ ID No.64-66 (11) The amino acid sequence is sequentially shown in HCDR1-3 shown in SEQ ID No.69-71, and the amino acid sequence is sequentially shown in LCDR1-3 shown in SEQ ID No.72, 5 and 73; (12) The amino acid sequence HCDR1-3 as shown in SEQ ID No.76-78 in sequence, and the amino acid sequence as LCDR1-3 as shown in SEQ ID No.79-81 in sequence; (13) the amino acid sequence as shown in SEQ ID No.84-86 in sequence HCDR1-3, the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.87, 42 and 50; (14) the amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.1, 90 and 91, the amino acid sequence is sequentially LCDR1-3 shown in SEQ ID No.92, 5 and 6.

In the present invention, the term "antibody" covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc.) , murine antibody, chimeric antibody, full-length antibody, etc., as long as they exhibit the desired antigen-binding activity.

The "chimeric antibody" of the present invention is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as required, and then clone the variable region gene of the mouse The gene is connected with the human constant region gene to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a 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 major contributors to antigen specificity within the variable domains of antibodies. Combined area. In a specific embodiment of the present invention, CDRs refer to more than two hypervariable regions in the heavy chain and light chain of the antibody.

In the present invention, the complementarity determining region of the heavy chain is represented by HCDR, and the three CDR regions contained in the variable region of the heavy chain: HCDR1, HCDR2 and HCDR3; the complementarity determining region of the light chain is represented by LCDR, and the CDR regions contained in the variable region of the light chain are represented by LCDR, 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, "framework region" or "FR" region refers to the region of the antibody heavy chain variable region and light chain variable region except for the CDRs; the heavy chain framework region can be further subdivided into regions separated by CDRs Open contiguous regions (FR1, FR2, FR3 and FR4), where the heavy chain framework region can be further subdivided into contiguous regions separated by CDRs, comprising the HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region It can be further subdivided into contiguous regions separated by LCDRs, containing the LFR1, LFR2, LFR3, and LFR4 framework regions. The heavy chain variable region is obtained by the following numbered CDRs and FRs (arranged from amino-terminal to carboxy-terminal): HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is composed of the following numbered CDRs and FR (arranged from amino-terminus to carboxyl-terminus) permutation connection obtained: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

In some embodiments, the heavy chain variable region and the light chain variable region are as shown 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 in sequence in SEQ ID No.7-8; (b) the amino acid sequences of the heavy chain variable region and the light chain variable region as shown in 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 as shown in SEQ ID No.17-18 in turn; (d) the heavy chain The amino acid sequences of the variable region and the light chain variable region are as shown in SEQ ID No.23 to 24; (e) the amino acid sequences of the heavy chain variable region and the light chain variable region are as in SEQ ID No.30 ~31; (f) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID No.36~37; (g) the heavy chain variable region and the light chain can be The amino acid sequences of the variable regions are shown in sequence in SEQ ID No.44-45; (h) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in sequence in 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 in 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. ID No.67～68; (k) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in sequence in SEQ ID No.74～75; (1) the heavy chain variable region The amino acid sequences of the variable region of the heavy chain and the variable region of the light chain are shown in sequence in SEQ ID No.82～83; (m) the amino acid sequences of the variable region of the heavy chain and the variable region of the light chain are shown in sequence in SEQ ID No.88～89 (n) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID No.93-94 in turn.

It should be noted that the CDRs shown in (1) to (14) are in one-to-one correspondence with (a) to (n) in sequence.

In some embodiments, the antibody or antigen-binding fragment thereof further includes 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 includes the light chain variable region domain VL and the constant region domain CL. The VL is at the amino terminal of the light chain, and the CL domain is at the carboxyl terminal. The light chain includes the κ chain and the λ chain; the heavy chain of the full-length antibody The chain includes a heavy variable domain, VH, and a constant domain, CH, with the VH at the amino-terminus of the heavy chain and the CH domain at the carboxyl-terminus.

In some embodiments, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

In some embodiments, the species source of the constant region is bovine, 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 any one of F(ab') ₂ , Fab', Fab, Fv and scFv (single-chain antibody) of an antibody. An "antigen-binding fragment" is a portion of an intact antibody that specifically binds to the antigen to which the intact antibody binds. Those skilled in the art can easily understand based on the content described in the present invention that the antigen-binding fragments can be prepared by methods known in the art, for example, enzymatic digestion (including pepsin or papain) and/or splitting disulfide by chemical reduction It can also be obtained by recombinant genetic technology or by automatic peptide synthesizer (such as the automatic peptide synthesizer of Applied BioSystems).

Among them, the structural expression of the single-chain antibody is: 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 human The constant region of an IgG1 antibody.

The structural expression of the bispecific antibody (PD-L1-CD3) is: VL1-(G4S)3-VH1-G4S-VH2-(G4S) _3- VL2; wherein, VH1 is the variable heavy chain of PD-L1 VL1 is the light chain variable region of the anti-PD-L1 antibody, VH2 is the heavy chain variable region of the anti-CD3 antibody, VL2 is the light chain variable region of the anti-CD3 antibody, G4S and (G4S) ₃ are peptide linkers, VH2-(G4S)3-VL2 is CD3 scFv, which uses the amino acid sequence of OKT3 (as shown below):

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

On the other hand, an embodiment of the present invention also provides an antibody conjugate, which comprises: the antibody or antigen-binding fragment thereof described in any of the foregoing embodiments.

In some embodiments, the antibody conjugate further includes a solid-phase carrier coupled to the antibody or antigen-binding fragment thereof. Optionally, the solid phase carrier includes, but is not limited to, at least one of magnetic microspheres, plastic microspheres, plastic particles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.

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

During actual use, those skilled in the art can select other appropriate markers according to detection conditions or actual needs, and no matter what marker is used, it falls within the protection scope of the present invention.

In some embodiments, the label is selected from at least one of fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle-based labels.

The embodiment of the present invention also provides the application of the antibody or antigen-binding fragment thereof as described in the foregoing embodiments in the preparation of a detection product for PD-L1 antigen.

In some embodiments, the product includes any one of a test strip, a reagent, and a kit.

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

On the other hand, the embodiments of the present invention also provide the use of the antibody or antigen-binding fragment thereof as described in any of the foregoing embodiments in the preparation of products for targeting PD-L1 to diagnose, prevent or treat diseases.

Preferably, the disease is selected from any one of breast cancer, lung cancer, gastric 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 reagents, kits and medicines.

In another aspect, an embodiment of the present invention also provides a reagent or kit, which includes the antibody or antigen-binding fragment thereof as described in any of the foregoing embodiments.

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

On the other hand, the embodiments of the present invention also provide a vector, which contains the isolated nucleic acid described in any of the foregoing embodiments.

Said vectors include expression vectors. The "expression vector" in the present invention refers to any recombinant polynucleotide construct, which can directly or indirectly (such as packaged into a virus) introduce the target DNA fragment into the host cell by means of transformation, transfection or transduction Inside, express the gene of interest. One type of vector is a plasmid, a circular double-stranded DNA molecule into which a DNA segment of interest is ligated. Another type of vector is a viral vector, which can ligate and package a target DNA segment into a viral genome (eg, adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cells, the expression of the target gene can be carried out.

Those skilled in the art can also use the nucleic acid sequence of the antibody of the present invention as a template to transcribe it into RNA through in vitro transcription, and further transfect, transduce or transform the RNA into host cells, and can also express the antibody of the present invention or its The functional fragment exerts the biological efficacy of the present invention.

On the other hand, the embodiments of the present invention also provide a cell, which contains the carrier as described in any of the foregoing embodiments.

In some embodiments, the cell is a host cell, and the host cell includes a prokaryotic host cell, a eukaryotic host cell, and a phage. The prokaryotic host cell may be Escherichia coli, Streptomyces or Bacillus subtilis, etc. The eukaryotic host cells may be 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, Per6, Saccharomyces cerevisiae, Pichia pastoris, Hansenula, Candida, some 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, an embodiment of the present invention provides a method for producing the antibody or antigen-binding fragment thereof as described in any of the preceding embodiments, comprising culturing cells capable of expressing the antibody or antigen-binding fragment thereof.

On the basis of the amino acid sequence of the antibody disclosed in the present invention, those skilled in the art can use genetic engineering technology or other techniques (chemical synthesis, recombinant expression) to prepare the antibody, for example, from the antibody capable of recombinant expression as described in any one of the above It is easy for those skilled in the art to obtain the antibody by separating and purifying it from the cultured product of recombinant cells. Based on this, no matter what technique is used to prepare the antibody of the present invention, it falls within the protection scope of the present invention.

On the other hand, the embodiments of the present invention also provide a drug or a pharmaceutical composition, the active ingredient of which includes the antibody or antigen-binding fragment thereof as described in any of the foregoing embodiments, the antibody conjugate as described in any of the foregoing embodiments , at least one of the reagent or kit as described in any of the foregoing embodiments, the isolated nucleic acid as described in any of the foregoing embodiments, the vector as described in any of the foregoing embodiments, and at least one of the cells as described in any of the foregoing embodiments .

The "pharmaceutical composition" in the present invention refers to a combination of at least one drug and optionally a pharmaceutically acceptable carrier or adjuvant combined together to achieve a specific purpose. In certain embodiments, the pharmaceutical composition includes temporally and/or spatially separated combinations as long as they can act together to achieve the purpose of the present invention. Some pharmaceutical compositions are used in combination with some pharmaceutically acceptable ingredients or compounds to enhance the biological efficacy of the present invention or reduce drug side effects (for example, it can be used in combination with other anti-tumor drugs to enhance the anti-tumor effect). The purpose of other pharmaceutical compositions is to promote the administration to organisms, facilitate the absorption of active ingredients, enhance stability or targeting, prolong half-life, and then better exert the biological efficacy of the present invention.

On the other hand, the present invention also provides a method for diagnosing, preventing or treating diseases by targeting PD-L1, which comprises administering a therapeutically effective amount of the antibody of the present invention or its antigen-binding fragment, nucleic acid, Vectors, cells and pharmaceutical compositions.

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

The "effective amount" mentioned in the present invention refers to the dose sufficient to show the benefit to the administered object. The actual amount administered, as well as the rate and time course of administration, will depend upon the individual condition and severity of the individual being treated. Prescribing a treatment is ultimately a decision made by the physician, usually taking into account the individual patient's condition, site of delivery, method of administration, severity of disease, and other factors routine to the physician.

The "subject" in the present invention refers to mammals, such as human beings, but it can also be other animals, such as wild animals, domestic animals or experimental animals.

On the other hand, the present invention also provides a method for detecting PD-L1 antigen, which comprises using the antibody or antigen-binding fragment thereof provided in any of the foregoing embodiments to detect the antigen.

Detection methods include but are 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, antibodies of the present invention or functional fragments thereof can be used for Fluorescent or isotopic labeling for antigen tracking.

The characteristics and performance of the present invention will be described in further detail below in conjunction with the examples.

Example 1

(1) Preparation of PD-L1 recombinant protein: The nucleotide sequence encoding human PD-L1 was synthesized by Anhui General Biology Company. PCR amplified and subcloned into pcDNA3.1 expression vector. Then, the extracellular domain of PD-L1 was subcloned into the pcDNA3.1 expression vector carrying Fc or His tag at the C-terminus, respectively. The Fc tags include human Fc (hFc) and mouse Fc (mFc). 293FT was transiently transfected, cultured in shake flasks with FreeStyle ^TM serum-free medium (Life Technologies) for 5-7 days, the supernatant was collected, subjected to centrifugal ultrafiltration, and then passed through Protein A/G or NTA-Ni affinity chromatography and molecular sieve Chromatographic column purification of recombinant PD-L1 protein carrying Fc or His tag.

(2) Preparation of stable cell lines expressing human PD-L1 antigen: the full-length sequence encoding human PD-L1 was constructed into a lentiviral vector carrying plenti-eGFP, and packaged into lentiviral particles. Hela cells were cultured in DMEM containing 10% fetal bovine serum. HeLa cells were infected with concentrated virus solution. After 48 hours, flow sorting was performed, cultured into a 96-well plate, and monoclonal stable cell lines were screened and identified, and Hela-PD-L1-EGFP cells stably expressing PL-L1 were preserved.

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

Animal immunization: 5-6 week-old Balb/c female mice were used as immunized animals, and the immunization dose was 100 μg/mouse. For the first immunization, 100 μl of complete Freund's adjuvant (Sigma) was mixed with an equal volume of recombinant PD-L1 protein, fully emulsified, and injected subcutaneously at multiple points. Every two weeks, the recombinant protein was mixed with an equal volume of Freund's incomplete adjuvant (Sigma), fully emulsified, and injected subcutaneously at multiple points. A total of 4 booster immunizations were performed, and on the 10th day after the last booster immunization, blood was collected from the tail to detect the antibody titer of the mice. Three days before cell fusion, 100 μg of recombinant protein was injected into the abdominal cavity once.

Cell fusion and hybridoma screening: Under aseptic conditions, the lymph nodes of the hind legs of the mice were collected to prepare a suspension rich in B cells, and the cells were fused with SP2/0 cells according to the classic PEG (Sigma) method. The fused cells were resuspended in HAT medium for culture. On the 5th and 10th day after fusion, fresh HAT medium was used for culture. ELISA, immunofluorescence and flow cytometry analysis were performed 11-15 days after fusion to screen positive clones. ELISA screening was carried out on a 96-well plate. PD-L1 recombinant protein was coated on the bottom of the well plate overnight at 4 degrees Celsius at an amount of 100 ng/well. 50 μl of hybridoma culture supernatant was used as the primary antibody, and an HRP-coupled anti-mouse IgG antibody was used. Develop color with chemiluminescence reagent (Beiyuntian Biotechnology Co., Ltd.), and read the value at a wavelength of 450nm on a microplate reader. The Hela cell line stably expressing PD-L1 was used for immunofluorescence staining. In short, the cell line was adhered to a 96-well plate, and 50 μl of hybridoma supernatant was added as the primary antibody, incubated at 4 degrees for 2 hours, washed with PBS three times, Cy3-labeled Goat Anti-Mouse IgG (Proteintech) was used as the secondary antibody, incubated at room temperature for 1 hour, washed 3 times with PBS, and images were collected using a fluorescence microscope.

According to the above ELISA analysis and immunofluorescence analysis results, 14 optimal hybridoma clones (respectively named 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6 , 3211A6), for subsequent experiments such as sequence cloning and affinity analysis.

Example 3

Hybridoma antibody variable region sequence cloning: the optimal hybridoma clone cells in the logarithmic growth phase were collected, RNA was extracted with Trizol (Invitrogen) and reverse transcribed (PrimeScript ^™ Reverse Transcriptase, Takara). The cDNA obtained by reverse transcription was amplified by mouse Ig-Primer Set (Novagen) and then sequenced to obtain the variable region sequences of the heavy chain and light chain. The variable region CDR sequences of the heavy chain and the light chain are shown in Table 1.

Table 1 The CDR sequences contained in the heavy chain and light chain variable regions of mouse monoclonal antibodies

Example 4: Binding analysis of recombinant chimeric antibody to PD-L1 overexpressed in HeLa cervical cancer cells

The human (IgG1) heavy chain constant region, and the human light chain constant region were cloned into the pcDNA3.1 (Invitrogen) plasmid vector, and then the hybridoma clones 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2 , 2111C9, 2616B4, 3920F1, 3821G6, and 3211A6 VH and VL gene fragments were respectively constructed on the gene recombination vectors of human IgG1 heavy chain constant region and human IgGκ light chain constant region to obtain recombinant chimeric antibody heavy chain expression vector and light chain Expression vector, through transient transfection of 293FT, using FreeStyle ^TM serum-free medium (Life Technologies) shake flask culture for 5-7 days, collecting supernatant, centrifugal ultrafiltration, and then through Protein A/G affinity chromatography and molecular sieve chromatography Column purification to obtain the corresponding type of anti-PD-L1 recombinant monoclonal antibody.

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

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

Human IgGκ 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 and 3211A6 were used as primary antibodies, and CY3-labeled Goat Anti-human IgG (H+L) (Beyontian Biotechnology Co., Ltd.) was used as secondary antibodies, which were observed and photographed with fluorescent confocal. As shown in Figure 1, the results in Figure 1 show that: recombinant chimeric antibodies 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, 3211A6 can interact with cells Hela-PD- L1-EGFP specifically binds.

Example 5: Binding analysis of hybridoma antibody to mouse PD-L1

The cDNA clone of murine PD-L1 was purchased from Sino Biological, and then the stable expression cell line CHO-mPD-L1 of murine PD-L1 was constructed according to Example 1. Then the supernatants of hybridomas 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, and 3211A6 were taken for immunofluorescence staining according to Example 2. The results showed that the monoclonal antibody could not bind to murine PD-L1.

Embodiment 6: in vitro binding affinity and kinetic experiments

In this embodiment, the surface plasmon resonance (SPR) method is used for measurement, and the Biacore 8K instrument of GE Company is used for analysis. Using the kit provided by Biacore, the PD-L1-His recombinant protein was covalently linked to the CM5(GE) chip using the standard amino coupling method, and then the single-chain antibody to be tested was diluted in the same buffer at different concentrations After sample injection, the sample is regenerated with the regeneration reagent in the kit. Data analysis and collection were carried out using Biacore 8K supporting analysis software. The results obtained are shown in Table 2 below.

Table 2 Affinity

Example 7: PD-L1 monoclonal antibody blocks the 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 were obtained by Purifying Example 4 through Protein A/G affinity chromatography and molecular sieve chromatography column , 3920F1, 3821G6, 3211A6. The Hela-PD-L1-EGFP cells were plated in a 96-well plate, and the PD-L1 antibody was used to block PD-1 binding to PD-L1 immunofluorescence verification the next day. After the cells were fixed, they were washed twice with PBS, and PD-1-mFc protein was used as the primary antibody, incubated at 37°C for 1 h, washed twice with PBS, and then 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, and 3211A6 monoclonal antibodies were used for blocking, and the wells without anti-PD-L1 antibody were used as controls. Incubate at 37°C for 1 h, wash with PBS twice, then add CY3-labeled Goat Anti-Mouse IgG (H+L) (Beyontian Biotechnology Co., Ltd.) as the secondary antibody, and observe and take pictures with fluorescent confocal. Representative results are shown in Figure 2. The results show that PD-1-mFc protein can bind to PD-L1 on tumor cells, but after adding 10ug/ml anti-PD-L1 monoclonal antibody, the PD-1 protein The binding amount was significantly reduced, indicating that these PD-L1 monoclonal antibodies can significantly block the binding of PD-1 and PD-L1.

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

The structural expression of the PD-L1-CD3 bispecific antibody is: VL1-(G4S)3-VH1-G4S-VH2-(G4S) ₃ -VL2; wherein, VH1 is the variable region of the heavy chain of PD-L1, and VL1 is The light chain variable region of the anti-PD-L1 antibody provided by the present invention, VH2 is the heavy chain variable region of the anti-CD3 antibody, VL2 is the light chain variable region of the anti-CD3 antibody, G4S and (G4S) ₃ are peptide linkers, VH2-(G4S)3-VL2 is CD3 scFv, which uses the amino acid sequence of OKT3 (as shown below):

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

Preparation of effector cells and target cells: extract peripheral blood from healthy donors, isolate PBMCs, use T cell isolation kit (Miltenyi T Cell Isolation Kit) to isolate T cells, and culture the isolated T cells with X- VIVO (LONZA) medium, pre-incubate the TC-treated 6-well plate with 1ml of coating solution containing 50ng/ml anti-human CD3 antibody (PeproTech) and 50ng/ml CD28 antibody (PeproTech) at 37°C for 2h, remove before use Coating solution. Cells were inoculated into antibody-coated 6-well plates at 1 ml/well, and after stimulation for 48 hours, IL-2 (100 U/mL) and IL-15 (10 ng/mL) were supplemented as activating factors to continue the culture. Target cells were selected from Hela-PD-L1-EGFP overexpressing PD-L1 and cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum (Gibco).

Cell co-culture experiment: Hela-PD-L1-EGFP tumor cells in the logarithmic growth phase were added to a 96-well plate, and after overnight culture, 0ng/ml (Control) or 1ng/ml bispecific antibody was added, and at the same time according to T The effect-to-target ratios of cells and tumor cells were 1:1, 2:1, 4:1, and 8:1. T cells were added into the ratio. After 24 hours, the killing effect of T cells on tumor cells was observed. The representative results are shown in the figure As shown in 3, the experimental results showed that compared with the no bispecific antibody group (Control), fourteen PD-L1-CD3 bispecific antibodies (2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12 , 521H2, 2111C9, 2616B4, 3920F1, 3821G6, 3211A6) had significant killing effect on Hela-PD-L1-EGFP tumor cells (*P<0.05, **P<0.01, ***P<0.001), each antibody There is no significant difference between.

Example 9: Anti-tumor experiment of xenograft mouse model

In this example, a xenograft mouse model was used to evaluate PD-L1-targeted bispecific antibodies 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, 3211A6 In vivo antitumor activity. An immunodeficient mouse model was used for evaluation.

NCG severe immunodeficiency mouse model: NCG severe immunodeficiency mice were purchased from the Model Animal Institute of Nanjing University, and 2×106 A375 cells in logarithmic growth phase were inoculated subcutaneously on the right back of NCG mice. After the tumors grew to 200mm ³ about 6 days later, the mice with uniform tumor volumes were divided into random groups, with 5 mice in each group. Set the same volume of normal saline as the control group. The administration method of the bispecific antibody was intraperitoneal administration, 30 μg/monkey, administered once every 3 days, and administered 4 times in total. Mice were weighed and tumor size measured every 3 days. The average volume of the transplanted tumor was calculated according to the formula V=1/2(L×W2), wherein L represents the length of the tumor body, and W represents the width of the tumor body. When the tumor volume of the mice reached 2000 mm ³ or the surface of the tumor was obviously ruptured, the mice were sacrificed to end the animal experiment. All data are means ± sdP values obtained using unpaired two-tailed Student's t-test. The experimental results are shown in Figure 4. From the results in Figure 4, it can be seen that compared with the control group without administration, fourteen kinds of PD-L1-CD3 bispecific antibodies (2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3 , 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, 3211A6) all had significant inhibitory effects on the growth of A375 tumor cells (*P<0.05, **P<0.01, ***P<0.001), in which 2217B3/ The 2616B4/3718B12-CD3 group had the best inhibitory effect ***P<0.001.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. An antibody or an antigen-binding fragment thereof, wherein the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region comprise The CDRs shown in any one of (1) to (14); (1) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.1-3, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.4-6; (2) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.1, 9 and 10, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.4-6; (3) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.1, 13 and 13, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.15, 5 and 16; (4) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.19, 20 and 21, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.22, 5 and 6; (5) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.25-27, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.28, 5 and 29; (6) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.32, 26 and 27, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.33-35; (7) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.38-40, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.41-43; (8) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.46-48, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.41, 49 and 50; (9) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.53-55, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.56-58; (10) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.61-63, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.64-66; (11) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.69-71, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.72, 5 and 73; (12) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.76-78, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.79-81; (13) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.84-86, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.87, 42 and 50; (14) The amino acid sequence is sequentially shown as HCDR1-3 shown in SEQ ID No.1, 90 and 91, and the amino acid sequence is sequentially shown as LCDR1-3 shown in SEQ ID No.92, 5 and 6. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen of the antibody or antigen-binding fragment thereof comprises PD-L1; Preferably, said light chain variable region and heavy chain variable region further comprise a framework region; Preferably, the heavy chain variable region and the light chain variable region are shown 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 in sequence as SEQ ID No.7-8; (b) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in sequence as SEQ ID No.11-12; (c) the amino acid sequences of the heavy chain variable region and light chain variable region are shown in sequence as SEQ ID No.17-18; (d) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in sequence as SEQ ID No.23-24; (e) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in sequence as SEQ ID No.30-31; (f) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in sequence 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 in sequence as SEQ ID No.44-45; (h) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in sequence as SEQ ID No.51-52; (i) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in sequence as SEQ ID No.59-60; (j) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in sequence as SEQ ID No.67-68; (k) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in sequence as SEQ ID No.74-75; (1) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in sequence as SEQ ID No.82-83; (m) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in sequence as SEQ ID No.88-89; (n) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in sequence as SEQ ID No.93-94; Preferably, the antibody or antigen-binding fragment thereof further comprises a constant region; Preferably, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; Preferably, the species source of the constant region is bovine, horse, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Preferably, the antibody is selected from any one of monoclonal antibodies, polyclonal antibodies, multispecific antibodies, murine antibodies, chimeric antibodies and full-length antibodies; Preferably, the antigen-binding fragment is selected from any one of F(ab') ₂ , Fab', Fab, Fv and scFv of antibodies. 3. An antibody conjugate, characterized in that it comprises: the antibody or antigen-binding fragment thereof according to claim 1 or 2; Preferably, the antibody conjugate further includes a solid-phase carrier coupled to the antibody or its antigen-binding fragment; Preferably, the antibody conjugate further comprises a detectable label conjugated to the antibody or antigen-binding fragment thereof. 4. Application of the antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of PD-L1 antigen detection products; Preferably, the product includes any one of test paper, reagent and kit; Preferably, the detection method is selected from any one of ELISA, immunofluorescence, chemiluminescence immunoassay, Western blot, immunochromatography, electrochemical immunoassay and magnetic bead method. 5. Use of the antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of products for targeting PD-L1 to diagnose, prevent or treat diseases; Preferably, the disease is selected from any one of breast cancer, lung cancer, gastric 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 reagents, kits and medicines. 6. A reagent or kit, characterized in that it comprises the antibody or antigen-binding fragment thereof according to claim 1 or 2. 7. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to claim 1 or 2. 8. A vector comprising the isolated nucleic acid of claim 7. 9. A cell comprising the vector according to claim 8. 10. A medicine or pharmaceutical composition, characterized in that its active ingredient comprises the antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, the antibody conjugate as claimed in claim 3, the antibody conjugate as claimed in claim 6 At least one of the indicated reagent or kit, the isolated nucleic acid as claimed in claim 7 and the vector as claimed in claim 8 or the cell as claimed in claim 9.

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