CN115785269B - Anti-PD-L1 antibodies and their applications - Google Patents

Abstract

The invention discloses anti-PD-L1 antibodies and their applications, and relates to the field of antibodies. The invention provides anti-PD-L1 antibodies or functional fragments thereof that can specifically bind to PD-L1, and their CDRs are selected from (1) to (14) As shown in any one of the above; the antibody or its functional fragment can specifically block the binding of PD-1 and PD-L1, block the inhibitory effect on lymphocytes, thereby blocking the immune evasion 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 antibodies and their applications

Technical Field

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

Background Art

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

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

Programmed cell death 1ligand 1 (PD-L1), also known as surface antigen cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a protein in humans that is encoded by the CD274 gene and is a 40kDa type I transmembrane protein. PD-L1 is a surface glycoprotein ligand for PD-1, a key immune checkpoint receptor expressed by activated T cells and B cells and mediates immunosuppression. PD-L1 has been implicated in the suppression of immune system responses during chronic infection, pregnancy, allogeneic transplantation, autoimmune diseases, and cancer. PD-L1 has been 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 its receptor PD-1 on T cells and plays an important role in the negative regulation of immune responses; many studies have shown that it is associated with the immune escape mechanism of tumors. The microenvironment of the tumor can induce the expression of PD-L1 on tumor cells. The expressed PD-L1 is conducive to the occurrence and growth of tumors and induces apoptosis of anti-tumor T cells. After PD-1 binds to 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 a role in negative immune 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 PD-L1 expressed by tumor cells can promote the immune escape of tumors through the inhibitory effect on lymphocytes after binding to PD-1.

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

In view of this, the present invention is proposed.

Summary of the invention

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

The present invention is achieved in that:

In the first aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof 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 the CDRs shown in any one of (1) to (14). (1) HCDR1-3 whose amino acid sequences are sequentially as those of SEQ ID Nos. 1-3, and whose amino acid sequences are sequentially as those of LCDR1-3 as those of SEQ ID Nos. 4-6; (2) HCDR1-3 whose amino acid sequences are sequentially as those of SEQ ID Nos. 1, 9 and 10, and whose amino acid sequences are sequentially as those of LCDR1-3 as those of SEQ ID Nos. 4-6; (3) HCDR1-3 whose amino acid sequences are sequentially as those of SEQ ID Nos. 1, 13 and 13, and whose amino acid sequences are sequentially as those of LCDR1-3 as those of SEQ ID Nos. 15, 5 and 16; (4) HCDR1-3 whose amino acid sequences are sequentially as those of SEQ ID Nos. 19, 20 and 21, and whose amino acid sequences are sequentially as those of LCDR1-3 as those of SEQ ID Nos. 22, 5 and 6; (5) HCDR1-3 whose amino acid sequences are sequentially as those of SEQ ID Nos. 25-27, and whose amino acid sequences are sequentially as those of LCDR1-3 as those of SEQ ID Nos. 28, 5 and 29; (6) HCDR1-3 as shown in SEQ ID Nos.32, 26 and 27, and the amino acid sequences are LCDR1-3 as shown in SEQ ID Nos.33-35; (7) HCDR1-3 as shown in SEQ ID Nos.38-40, and the amino acid sequences are LCDR1-3 as shown in SEQ ID Nos.41-43; (8) HCDR1-3 as shown in SEQ ID Nos.46-48, and the amino acid sequences are LCDR1-3 as shown in SEQ ID Nos.41, 49 and 50; (9) HCDR1-3 as shown in SEQ ID Nos.53-55, and the amino acid sequences are LCDR1-3 as shown in SEQ ID Nos.56-58; (10) HCDR1-3 as shown in SEQ ID Nos.61-63, and the amino acid sequences are LCDR1-3 as shown in SEQ ID Nos.64-66; (11) HCDR1-3 as shown in SEQ ID Nos.69-71, and the amino acid sequences are LCDR1-3 as shown in SEQ ID Nos. No. 72, 5 and 73; (12) HCDR1 to 3 with amino acid sequences such as SEQ ID No. 76 to 78, and LCDR1 to 3 with amino acid sequences such as SEQ ID No. 79 to 81; (13) HCDR1 to 3 with amino acid sequences such as SEQ ID No. 84 to 86, and LCDR1 to 3 with amino acid sequences such as SEQ ID No. 87, 42 and 50; (14) HCDR1 to 3 with amino acid sequences such as SEQ ID No. 1, 90 and 91, and LCDR1 to 3 with amino acid sequences such as 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 above embodiment.

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

In a fourth aspect, embodiments of the present invention provide use of the antibody or antigen-binding fragment thereof as described in the preceding embodiments in the preparation of a product for diagnosing, preventing or treating a disease by targeting PD-L1.

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

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

In a seventh aspect, an embodiment of the present invention provides a vector comprising the isolated nucleic acid described in the preceding embodiment.

In an eighth aspect, an embodiment of the present invention provides a cell comprising the vector described in the preceding embodiment.

In a ninth aspect, an embodiment of the present invention provides a drug or a pharmaceutical composition, the active ingredients of which include at least one of the antibody or antigen-binding fragment thereof as described in the preceding embodiments, the antibody conjugate as described in the preceding embodiments, the reagent or kit as shown in the preceding embodiments, the isolated nucleic acid as described in the preceding embodiments, the vector as described in the preceding embodiments, and the cell as described in the preceding embodiments.

The present invention has the following beneficial effects:

The antibody or antigen-binding fragment thereof provided by the present 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 present 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 signals on tumor cells can upregulate the secretion of IFN-γ by infiltrating CD8 ⁺ T cells, indicating that the blocking of the PD-1/PD-L1 signaling pathway plays a role in the tumor immune response for the purpose of inducing immune response; selecting anti-PD-L1 antibodies in combination 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 hot target in the field of lung cancer treatment in the past two years.

The present invention has constructed single-chain antibody (scFv) and bispecific antibody (PD-L1-CD3), and verified the anti-tumor effect of the PD-L1-CD3 bispecific antibody of the present invention through in vitro killing experiments. The results show that the PD-L1-CD3 bispecific antibody has a significant killing effect on Hela-PD-L1 tumor cells. The present invention can be used to prepare drugs for preventing, diagnosing and treating human tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

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

FIG2 shows some representative results of PD-L1 monoclonal antibodies blocking the binding of PD-1 to PD-L1;

FIG3 shows the in vitro tumor killing results of bispecific antibodies 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, and 3211A6 in tandem with CD3scfv, respectively;

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

DETAILED DESCRIPTION

In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

First, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof 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 the CDRs shown in any one of (1) to (14).

(1) the amino acid sequences of HCDR1 to 3 are as shown in SEQ ID Nos. 1 to 3, and the amino acid sequences of LCDR1 to 3 are as shown in SEQ ID Nos. 4 to 6; (2) the amino acid sequences of HCDR1 to 3 are as shown in SEQ ID Nos. 1, 9 and 10, and the amino acid sequences of LCDR1 to 3 are as shown in SEQ ID Nos. 4 to 6; (3) the amino acid sequences of HCDR1 to 3 are as shown in SEQ ID Nos. 1, 13 and 13, and the amino acid sequences of LCDR1 to 3 are as shown in SEQ ID Nos. 15, 5 and 16; (4) the amino acid sequences of HCDR1 to 3 are as shown in SEQ ID Nos. 19, 20 and 21, and the amino acid sequences of LCDR1 to 3 are as shown in SEQ ID Nos. 22, 5 and 6; (5) the amino acid sequences of HCDR1 to 3 are as shown in SEQ ID Nos. 25 to 27, and the amino acid sequences of LCDR1 to 3 are as shown in SEQ ID Nos. 28, 5 and 29; (6) the amino acid sequences are as shown in SEQ ID HCDR1-3 as shown in SEQ ID Nos.32, 26 and 27, and the amino acid sequences are LCDR1-3 as shown in SEQ ID Nos.33-35; (7) HCDR1-3 as shown in SEQ ID Nos.38-40, and the amino acid sequences are LCDR1-3 as shown in SEQ ID Nos.41-43; (8) HCDR1-3 as shown in SEQ ID Nos.46-48, and the amino acid sequences are LCDR1-3 as shown in SEQ ID Nos.41, 49 and 50; (9) HCDR1-3 as shown in SEQ ID Nos.53-55, and the amino acid sequences are LCDR1-3 as shown in SEQ ID Nos.56-58; (10) HCDR1-3 as shown in SEQ ID Nos.61-63, and the amino acid sequences are LCDR1-3 as shown in SEQ ID Nos.64-66; (11) HCDR1-3 as shown in SEQ ID Nos.69-71, and the amino acid sequences are LCDR1-3 as shown in SEQ ID Nos. (12) HCDR1-3 as shown in SEQ ID Nos.76-78, and LCDR1-3 as shown in SEQ ID Nos.79-81; (13) HCDR1-3 as shown in SEQ ID Nos.84-86, and LCDR1-3 as shown in SEQ ID Nos.87, 42 and 50; (14) HCDR1-3 as shown in SEQ ID Nos.1, 90 and 91, and LCDR1-3 as shown in SEQ ID Nos.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 (e.g., bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc.), murine antibodies, chimeric antibodies, full-length antibodies, etc., as long as they exhibit the desired antigen-binding activity.

The "chimeric antibody" described in the present invention is an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, a hybridoma that secretes mouse-specific monoclonal antibodies must first be established, and then the variable region gene must be cloned from the mouse hybridoma cells, and then the constant region gene of the human antibody must be cloned as needed, and the mouse variable region gene and the human constant region gene must be connected into a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule must be expressed in a eukaryotic or prokaryotic industrial system.

In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region of the heavy chain and light chain of an immunoglobulin, which is the region in the antibody variable domain that mainly contributes to specific binding to an antigen. In a specific embodiment of the present invention, CDRs refers to more than two highly variable regions in the heavy chain and light chain of the antibody.

In the present invention, the heavy chain complementary determining region is represented by HCDR, and the heavy chain variable region contains three CDR regions: HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by LCDR, and the light chain variable region contains three 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 other than CDR in the heavy chain variable region and light chain variable region of an antibody; the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3 and FR4), wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by LCDRs, including LFR1, LFR2, LFR3 and LFR4 framework regions. The heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs (arranged from the amino terminal to the carboxyl terminal): HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs (arranged from the amino terminal to the carboxyl terminal): 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 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 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 shown in 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 in 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 in 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 in 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 shown in 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 shown in SEQ ID No.51-52 in sequence; (i) the amino acid sequences of the heavy chain variable region and the light chain variable region are 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 shown in 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 SEQ ID No.74-75; (l) the amino acid sequences of the heavy chain variable region and the light chain variable region are 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 shown 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.

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

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 domain VL and a constant region domain CL, VL is at the amino terminus of the light chain, the CL domain is at the carboxyl terminus, and the light chain comprises a κ chain and a λ chain; the full-length antibody heavy chain comprises a heavy variable region domain VH and a constant region CH, VH is at the amino terminus of the heavy chain, and the CH domain is 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 of origin of the constant region is 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 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 an antigen bound by the intact antibody. A person skilled in the art will readily understand, based on the contents described in the present invention, that the antigen-binding fragment can be prepared by methods known in the art, for example, by enzymatic digestion (including pepsin or papain) and/or by chemical reduction to split disulfide bonds, or by recombinant genetics techniques or by automatic peptide synthesizers (such as Applied BioSystems' automatic peptide synthesizers).

The structural formula 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 a peptide linker, and huIgG1Fc is the constant region of the human IgG1 antibody.

The structural formula of the bispecific antibody (PD-L1-CD3) is: 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 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 adopts the amino acid sequence of OKT3 (as shown below):

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTS EDSAVYYCARYYDDHYCLDYWGQGTTLTVSS(VH2)GGGGSGGGGSGGGGS((G4S) ₃ )DIQLTQSPAIMSASPGEKVTMTCRASSSVSY MNWYQQKS GTSPKRWIYDTSKVASGVPYRFSGGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(VL2).

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

In some embodiments, the antibody conjugate further comprises a solid phase carrier coupled to the antibody or its antigen binding fragment. Optionally, the solid phase carrier includes but is not limited to at least one of magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

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

In actual use, those skilled in the art can select other suitable markers according to the detection conditions or actual needs. No matter which marker is used, it belongs to 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 label.

The embodiments of the present invention also provide the use of the antibody or antigen-binding fragment thereof as described in the above embodiments in preparing 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, chemiluminescent immunoassay, Western blot, immunochromatography, electrochemical immunoassay and magnetic bead method.

On the other hand, an embodiment of the present invention further provides the use of the antibody or antigen-binding fragment thereof as described in any of the foregoing embodiments in the preparation of a product for diagnosing, preventing or treating a disease by targeting PD-L1.

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 a reagent, a kit and a medicine.

On the other hand, an embodiment of the present invention further provides a reagent or a kit, which includes the antibody or antigen-binding fragment thereof as described in any of the above embodiments.

On the other hand, an embodiment of the present invention further provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof as described in any of the above embodiments.

On the other hand, an embodiment of the present invention further provides a vector comprising the isolated nucleic acid described in any of the above embodiments.

The vector includes an expression vector. The "expression vector" described 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 a host cell by transformation, transfection or transduction to express the target gene. One type of vector is a plasmid, i.e., a circular double-stranded DNA molecule, which can connect the target DNA fragment to the plasmid ring. Another type of vector is a viral vector, which can connect and package the target DNA fragment into a viral genome (such as adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cell, the target gene can be expressed.

A person skilled in the art can also use in vitro transcription to transcribe the nucleic acid sequence of the antibody of the present invention as a template into RNA, and further transfect, transduce or transform the RNA into a host cell to express the antibody of the present invention or a functional fragment thereof to exert the biological efficacy of the present invention.

On the other hand, an embodiment of the present invention further provides a cell, which contains the vector as described in any of the above 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 bacteriophage. The prokaryotic host cell may be Escherichia coli, Streptomyces, or Bacillus subtilis, etc. The eukaryotic host cell may be 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, Per6, Saccharomyces cerevisiae, Pichia pastoris, Hansen yeast, Candida, some insect cells, and plant cells. 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins, and are well known to those of ordinary skill in the art.

On the other hand, an embodiment of the present invention provides a method for producing the antibody or antigen-binding fragment thereof as described in any of the above embodiments, which comprises culturing cells capable of expressing the antibody or antigen-binding fragment thereof.

Based on the amino acid sequence of the antibody disclosed in the present invention, those skilled in the art can prepare the antibody by genetic engineering technology or other technologies (chemical synthesis, recombinant expression), for example, separating and purifying the antibody from the culture product of a recombinant cell capable of recombinantly expressing the antibody as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody of the present invention, it belongs to the protection scope of the present invention.

On the other hand, an embodiment of the present invention further provides a drug or pharmaceutical composition, the active ingredient of which includes at least one of the antibody or antigen-binding fragment thereof as described in any of the preceding embodiments, the antibody conjugate as described in any of the preceding embodiments, the reagent or kit as shown in any of the preceding embodiments, the isolated nucleic acid as described in any of the preceding embodiments, the vector as described in any of the preceding embodiments, and the cell as described in any of the preceding embodiments.

The "pharmaceutical composition" of the present invention refers to a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient that are combined together to achieve a specific purpose. In certain embodiments, the pharmaceutical composition includes a combination separated in time and/or space, as long as they can work together to achieve the purpose of the present invention. Some pharmaceutical compositions are achieved by co-administering some pharmaceutically acceptable ingredients or compounds to enhance the biological efficacy of the present invention or reduce the side effects of the drug (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 administration to an organism, facilitate the absorption of active ingredients, enhance stability or targeting, prolong the half-life, and thus better exert the biological efficacy of the present invention.

On the other hand, the present invention also provides a method for targeting PD-L1 to diagnose, prevent or treat a disease, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell and pharmaceutical composition of the present invention to a subject in need thereof.

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, etc.

The "effective amount" of the present invention refers to a dose sufficient to show its benefit to the object to which it is administered. The actual amount administered, as well as the rate and time course of administration will depend on the individual condition and severity of the person being treated. The prescription of treatment is ultimately determined by the doctor, who usually takes into account the individual condition of the patient, the delivery site, the method of administration, the severity of the disease, and other conventional factors for the doctor.

The "subject" described in the present invention refers to mammals, such as humans, but may also be other animals, such as wild animals, livestock or experimental animals.

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

Detection methods include, but are not limited to, ELISA, immunochromatography, immunohistochemistry, immunofluorescence and flow cytometry, and can also be used for antigen tracing in cells, tissues or living organisms. For example, the antibodies of the present invention or their functional fragments can be fluorescently or isotopically labeled for antigen tracing.

The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

Example 1

(1) Preparation of PD-L1 recombinant protein: The nucleotide sequence encoding human PD-L1 was synthesized by Anhui General Biotechnology Co., Ltd. PCR amplification and subcloning into the pcDNA3.1 expression vector. Then, the extracellular domain of PD-L1 was subcloned into the pcDNA3.1 expression vector carrying an Fc or His tag at the C-terminus. The Fc tag includes human Fc (hFc) and mouse Fc (mFc). After transient transfection of 293FT, FreeStyle ^TM serum-free medium (Life Technologies) was used for shake flask culture for 5-7 days, the supernatant was collected, subjected to centrifugal ultrafiltration, and then purified by Protein A/G or NTA-Ni affinity chromatography and molecular sieve chromatography columns.

(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 and cultured to 96-well plates for monoclonal stable cell line screening and identification, 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 Freund's complete adjuvant (Sigma) was mixed with an equal volume of recombinant PD-L1 protein, and multiple subcutaneous injections were performed after sufficient emulsification. Every 2 weeks, an equal volume of Freund's incomplete adjuvant (Sigma) was mixed with the recombinant protein, and multiple subcutaneous injections were performed after sufficient emulsification. The booster immunization was performed 4 times in total. On the 10th day after the last booster immunization, the mouse antibody titer was detected by cutting the tail and collecting blood. 3 days before cell fusion, 100 μg of recombinant protein was intraperitoneally impacted once.

Cell fusion and hybridoma screening: Under sterile conditions, lymph nodes at the base of the mouse hind legs were taken to prepare a suspension rich in B cells, and cells were fused with SP2/0 cells according to the classic PEG (Sigma) method. The fused cells were resuspended in HAT medium for culture. Fresh HAT medium was used for culture on the 5th and 10th days after fusion. ELISA, immunofluorescence and flow cytometry were performed on days 11-15 after fusion to screen positive clones. ELISA screening was performed using a 96-well plate, and the PD-L1 recombinant protein was coated on the bottom of the well plate at 100 ng/well overnight at 4 degrees Celsius. 50 μl of hybridoma culture supernatant was used as the primary antibody, and HRP-coupled anti-mouse IgG antibody and chemiluminescent reagent (Biyuntian Biotechnology Co., Ltd.) were used for color development, and the value was read on an enzyme reader at a wavelength of 450 nm. Immunofluorescence staining used Hela cell lines that stably expressed PD-L1. Briefly, the cell lines were cultured in 96-well plates, 50 μl of hybridoma supernatant was added as the primary antibody, incubated at 4 degrees for 2 hours, washed three times with PBS, and Cy3-labeled Goat Anti-Mouse IgG (Proteintech) was used as the secondary antibody. The cells were incubated at room temperature for 1 hour, washed three times with PBS, and images were collected using a fluorescence microscope.

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

Example 3

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

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

Example 4: Binding analysis of recombinant chimeric antibodies to HeLa cervical cancer cells overexpressing PD-L1

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 VH and VL gene fragments of the hybridoma clones 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, and 3211A6 were constructed into the gene recombination vectors containing the human IgG1 heavy chain constant region and the human IgGκ light chain constant region, respectively, to obtain the recombinant chimeric antibody heavy chain expression vector and light chain expression vector, which were transiently transfected into 293FT and cultured in shake flasks using FreeStyle ^™ serum-free medium (Life Technologies) for 5-7 days. The supernatant was collected, ultrafiltered by centrifugation, and then expressed by Protein The corresponding type of anti-PD-L1 recombinant monoclonal antibody was obtained by A/G affinity chromatography and molecular sieve chromatography column purification.

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

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Human IgGκ light chain constant region sequence (SEQ ID No.96):

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Hela-PD-L1-EGFP cells were plated in a 24-well cell culture dish. 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) (Biyuntian Biotech) was used as secondary antibody, and the cells were observed and photographed using fluorescence confocal microscopy. As shown in Figure 1, the results in Figure 1 show that the recombinant chimeric antibodies 2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, and 3211A6 can specifically bind to Hela-PD-L1-EGFP cells.

Example 5: Binding analysis of hybridoma antibodies to murine PD-L1

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

Example 6: In vitro binding affinity and kinetics experiments

This example uses the surface plasmon resonance (SPR) method for determination, and uses the GE Biacore 8K instrument 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 according to different concentration gradients and injected. After injection, it was regenerated with the regeneration reagent in the kit. The data was analyzed and collected using the Biacore 8K supporting analysis software. The results are shown in Table 2 below.

Table 2 Affinity

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

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

The structural representation of the PD-L1-CD3 bispecific antibody is: 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 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 MNWYQQKS GTSPKRWIYDTSKVASGVPYRFSGGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(VL2).

Preparation of effector cells and target cells: Peripheral blood was drawn from healthy donors, PBMC was separated, and T cells were separated using the Miltenyi T Cell Isolation Kit. The separated T cells were cultured in X-VIVO (LONZA) medium containing 5% AB serum. The 6-well plate coated with TC was pre-incubated with 1 ml of coating solution containing 50 ng/ml anti-human CD3 antibody (PeproTech) and 50 ng/ml CD28 antibody (PeproTech) at 37°C for 2 h, and the coating solution was removed before use. The cells were inoculated into the 6-well plate coated with antibodies at 1 ml/well. After stimulation and culture for 48 hours, IL-2 (100 U/mL) and IL-15 (10 ng/mL) were added as activation factors to continue the culture. The target cells selected were Hela-PD-L1-EGFP overexpressing PD-L1 and cultured in DMEM high-glucose culture 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. After overnight culture, 0 ng/ml (Control) or 1 ng/ml of bispecific antibody was added. At the same time, T cells were added at a ratio of 1:1, 2:1, 4:1 and 8:1 between the effector and target of T cells and tumor cells, respectively. After 24 hours, the killing effect of T cells on tumor cells was observed. The representative results are shown in Figure 3. The experimental results show that compared with the group without adding bispecific antibody (Control), Fourteen PD-L1-CD3 bispecific antibodies (2420A7, 62D11, 95C12, 142A11, 151D1, 3518G5, 2217B3, 3718B12, 521H2, 2111C9, 2616B4, 3920F1, 3821G6, 3211A6) had significant killing effects on Hela-PD-L1-EGFP tumor cells (*P<0.05, **P<0.01, ***P<0.001), and there was no significant difference among the antibodies.

Example 9: Anti-tumor experiment in xenograft mouse model

This example uses 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, and 3211A6. 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 the logarithmic growth phase were inoculated subcutaneously on the right back of the NCG mice. After about 6 days when the tumor grew to ^200mm3 , the mice with uniform tumor volume were randomly divided into groups, with 5 mice in each group. A control group was set up with a saline solution of the same volume as the drug administration. The bispecific antibody was administered intraperitoneally, 30μg/mouse, once every 3 days, for a total of 4 times. The mice were weighed and the tumor size was measured every 3 days. The average volume of the transplanted tumor was calculated according to the formula V=1/2(L×W2), where L represents the length of the tumor and W represents the width of the tumor. When the mouse tumor volume reached ^2000mm3 or the tumor surface showed obvious ulceration, the mouse was killed and the animal experiment was terminated. All data are mean ± sdP values were obtained using an unpaired two-tailed Student's t-test. The experimental results are shown in Figure 4. It can be seen from the results in Figure 4 that compared with the control group without drug administration, the fourteen 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), among which the 2217B3/2616B4/3718B12-CD3 group had the best inhibitory effect***P<0.001.

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

Claims (21)

1. An anti-PD-L1 antibody or an antigen-binding fragment thereof, characterized in that the antibody or an antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region The chain variable region includes CDRs shown in any of the following; The amino acid sequence is HCDR1~3 shown in SEQ ID No. 38~40, and the amino acid sequence is LCDR1~3 shown in SEQ ID No. 41~43; The amino acid sequence is HCDR1~3 shown in SEQ ID No. 53~55, and the amino acid sequence is LCDR1~3 shown in SEQ ID No. 56~58; The amino acid sequence is HCDR1~3 shown in SEQ ID No. 69~71, and the amino acid sequence is LCDR1~3 shown in SEQ ID No. 72, 5 and 73. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region and the heavy chain variable region further comprise a framework region. 3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain variable region and the light chain variable region are as shown in any one: The amino acid sequences of the heavy chain variable region and light chain variable region are shown in SEQ ID No. 44~45; The amino acid sequences of the heavy chain variable region and light chain variable region are shown in SEQ ID No. 59~60; The amino acid sequences of the heavy chain variable region and light chain variable region are shown in SEQ ID No. 74~75 in sequence. 4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof further includes a constant region. 5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD. 6. The antibody or antigen-binding fragment thereof according to claim 4, wherein the species source of the constant region is cattle, horse, pig, sheep, rat, mouse, dog, cat, rabbit, donkey , deer, mink, chicken, duck, goose or human. 7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that the antibody is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, multispecific antibodies, murine antibodies, and chimeric antibodies. and full-length antibodies. 8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that the antigen-binding fragment is selected from the group consisting of F(ab') ₂ , Fab', Fab, Fv and scFv of antibodies. any kind. 9. An antibody conjugate, characterized in that it includes: the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8. 10. The antibody conjugate according to claim 9, wherein the antibody conjugate further comprises a solid-phase carrier coupled to an antibody or an antigen-binding fragment thereof. 11. The antibody conjugate according to claim 9, wherein the antibody conjugate further comprises a detectable label coupled to the antibody or its antigen-binding fragment. 12. Application of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in the preparation of a detection product for PD-L1 antigen. 13. The application according to claim 12, characterized in that the product includes any one of test strips, reagents and test kits. 14. The application according to claim 12, characterized in that the detection method is selected from: ELISA, immunofluorescence method, chemiluminescence immunoassay, Western blot, immunochromatography, electrochemical immunoassay and magnetic bead method. any of them. 15. Application of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in the preparation of products for diagnosing or treating diseases by targeting PD-L1, and the diseases are selected from breast cancer, lung cancer, Any of stomach, bowel, esophageal, ovarian, cervical, kidney, bladder, pancreatic, glioma or melanoma. 16. The application according to claim 15, characterized in that the product is selected from any one of reagents, kits and medicines. 17. A reagent or kit, characterized in that it includes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8. 18. An isolated nucleic acid, characterized in that it encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8. 19. A vector, characterized in that it contains the isolated nucleic acid according to claim 18. 20. A cell, characterized in that it contains the vector of claim 19. 21. A medicine or pharmaceutical composition, characterized in that its active ingredients include the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the antibody according to any one of claims 9 to 11 At least one of the conjugate, the reagent or kit as claimed in claim 17, the isolated nucleic acid as claimed in claim 18, the vector as claimed in claim 19 or the cell as claimed in claim 20.