Antibodies that specifically bind to PD-L1 and their antigen-binding fragments

Abstract

The present invention relates to antibodies and antigen-binding fragments thereof that specifically bind to PD-L1, which have high specificity and stability, can enhance IFN-γ secretion, have strong regulatory activity on T cell function, and can significantly inhibit tumor growth in vivo.

Description

[Technical Field]

[0001] The present invention relates to the field of antibody and antibody humanization research, and in particular, the present invention relates to an antibody and antigen-binding fragment thereof that can specifically bind to PD-L1. [Background technology]

[0002] Programmed death-1 (PD-1) is an immune checkpoint molecule that is primarily involved in regulating T cell activation and can regulate the strength and duration of immune responses. Under normal circumstances, PD-1 can induce and maintain self-immune tolerance in living tissues, preventing the immune system from becoming overly activated and damaging self-tissues during inflammatory responses, and thus plays a positive role in preventing the onset of autoimmune diseases. In pathological situations, they are involved in the development and progression of tumor immunity and various autoimmune diseases (Anticancer Agents Med Chem. 2015;15(3):307-13. Hematol Oncol Stem Cell Ther. 2014 Mar;7(1):1-17. Trends Mol Med. 2015 Jan;21(1):24-33. Immunity. 2013 Jul 25;39(1):61-73. J Clin Oncol. 2015 Jun 10;33(17):1974-82.).

[0003] PD-1 ligands include PD-L1 (programmed death ligand 1) and PD-L2 (programmed death ligand 2). These ligands belong to the B7 family. PD-L1 expression is induced on the surface of various immune cells, including T cells, B cells, monocytes, macrophages, DCs, endothelial cells, and epidermal cells, whereas PD-L2 expression is induced only on certain immune cells, including macrophages, DCs, and B cells (Autoimmun Rev, 2013, 12(11):1091-1100. Front Immunol, 2013, 4:481. Nat Rev Cancer, 2012, 12(4):252-264. Trends Mol Med. 2015 Jan;21(1):24-33.).

[0004] PD-L1 is highly expressed on the surface of various tumor cells, including melanoma, lung cancer, renal cancer, breast cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, gastric cancer, pancreatic adenocarcinoma, and intestinal cancer, while PD-L2 is highly expressed in B-cell lymphoma. Tumor cells bind to PD-1 on T cells via highly expressed PD-L1 or PD-L2 and transmit immunoinhibitory signals, thereby inducing immune tolerance to tumor cells and favoring tumor cell growth and metastasis. High expression of PD-1 ligands is closely associated with poor prognosis and drug resistance in tumor patients (Hematol Oncol Stem Cell Ther. 2014 Mar;7(1):1-17.). Further research has found that elevated PD-1 expression on the surface of T cells, especially those infiltrating tumor cells, is also closely associated with poor prognosis (Trends Mol Med. 2015 Jan;21(1):24-33.).

[0005] Numerous studies have demonstrated that antibodies blocking the PD-1 / PD-Ls signaling pathway have antitumor effects. Clinically, PD-1 / PD-Ls blocking antibodies have demonstrated strong and sustained antitumor effects across a wide range of tumors, not just specific tumor types. Second, PD-1 / PD-Ls blocking antibodies have a favorable safety profile, avoiding common side effects associated with chemotherapy and targeted drugs, such as fatigue, low white blood cell count, baldness, diarrhea, and rash, while only producing a few immune-related side effects. The PD-1 antibody nivolumab is commercially available for the treatment of advanced melanoma, non-small cell lung cancer, renal cell carcinoma, and lymphoma, while pembrolizumab is commercially available for the treatment of advanced melanoma, non-small cell lung cancer, and lymphoma. The PD-L1 antibodies atezolizumab are commercially available to treat late-stage non-small cell lung cancer and urothelial carcinoma, durvalumab is commercially available to treat late-stage non-small cell lung cancer and urothelial carcinoma, and avelumab is commercially available to treat late-stage Merkel cell carcinoma.

[0006] There remains a need in the field to develop more effective antibodies that specifically bind to PD-L1. Summary of the Invention

[0007] A first aspect of the present invention relates to an isolated anti-human programmed death ligand 1 (PD-L1) antibody, antigen-binding fragment thereof, or variant thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region and / or a heavy chain variable region, wherein: the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region are set forth in SEQ ID Nos. 20-22, respectively; and / or the amino acid sequence of HCDR1 of the heavy chain variable region is set forth in SEQ ID No. 23, the amino acid sequence of HCDR2 of the heavy chain variable region has at least about 94% identity to any of SEQ ID Nos. 24 or 45-47, and the amino acid sequence of HCDR3 of the heavy chain variable region is set forth in SEQ ID No. 25; or the amino acid sequences of the CDRs of the light chain variable region or heavy chain variable region shown are variant sequences that have at least 70% identity, such as at least 75%, 80%, 85%, 90%, 95% or more identity, to the sequences shown in SEQ ID Nos. 20-25 or 45-47, respectively, and that retain the biological activity of the corresponding parent sequences; or The amino acid sequences of the CDRs of the light chain variable region or heavy chain variable region are mutant sequences obtained by deleting, substituting, and / or adding one or more, for example, one, two, three, or more, amino acid residues from the sequences shown in SEQ ID NOs: 20-25 or 45-47, respectively, while retaining the biological activity of the corresponding parent sequences.

[0008] Here, the variant is one selected from a chimeric antibody, a humanized antibody, or a fully human antibody.

[0009] In some embodiments, the antibody heavy chain constant region sequence is selected from the constant region sequence of any of human IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, and / or the antibody light chain constant region sequence is selected from the κ chain or λ chain. Preferably, the heavy chain constant region sequence is selected from the constant region sequence of IgG1 or IgG4, and / or the light chain constant region sequence is selected from the constant region sequence of the κ light chain.

[0010] In some embodiments, the amino acid sequence of the light chain variable region of the PD-L1 chimeric antibodies and functional fragments thereof is set forth in SEQ ID NO. 18, or a sequence that is at least 70% identical, for example at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, to the sequence set forth in SEQ ID NO. 18, and retains the biological activity of the corresponding parent sequence, or a variant sequence obtained by deleting, substituting, and / or adding one or more amino acid residues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more, to the sequence set forth in SEQ ID NO. 18, which retains the biological activity of the corresponding parent sequence; and / or the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO. 19, or a variant sequence that is at least 70% identical, for example at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, to the sequence set forth in SEQ ID NO. 18, and retains the biological activity of the corresponding parent sequence. or a variant sequence obtained by deleting, substituting and / or adding one or more amino acid residues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more, to the sequence set forth in SEQ ID NO. 19, and retaining the biological activity of the corresponding parent sequence.

[0011] The amino acid sequences of the light chain constant region and heavy chain constant region of the PD-L1 chimeric antibody and functional fragment thereof are set forth in SEQ ID NO. 26 and SEQ ID NO. 27, respectively, or have at least 70% identity, for example at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.4%, 99.7% or more identity, to the sequences set forth in SEQ ID NO. 26 and SEQ ID NO. 27, respectively, and retain the biological activity of the corresponding parent sequences; or are variant sequences obtained by deleting, substituting, and / or adding one or more amino acid residues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more, to the sequences set forth in SEQ ID NO. 26 and SEQ ID NO. 27, which retain the biological activity of the corresponding parent sequences.

[0012] In some embodiments, the light chain variable region framework regions of the anti-human PD-L1 antibody, antigen-binding fragment thereof, or variant thereof comprise FR-L1, FR-L2, FR-L3, and FR-L4, and the heavy chain variable region framework regions comprise FR-H1, FR-H2, FR-H3, and FR-H4, wherein:

[0013] The amino acid sequence of the FR-L1 is shown in SEQ ID NO. 54, The amino acid sequence of the FR-L2 is as set forth in SEQ ID NO. 55, or the amino acid sequence obtained by further substituting any one of the following substitutions or any combination thereof: The second amino acid Y is replaced by I, The third amino acid Q is replaced by H, The ninth amino acid, A, is replaced by S. The amino acid sequence of the FR-L3 is shown in SEQ ID NO. 56, The amino acid sequence of the FR-L4 is shown in SEQ ID NO. 57, The amino acid sequence of the FR-H1 is set forth in SEQ ID NO. 58, or an amino acid sequence obtained by further substituting any one of the following substitutions or any combination thereof: The first amino acid Q is replaced by E, The 23rd amino acid, K, is replaced by T. The amino acid sequence of FR-H2 is shown in SEQ ID NO. 59, or an amino acid sequence obtained by the following substitutions: The 13th amino acid, M, is replaced by I, The amino acid sequence of the FR-H3 is set forth in SEQ ID NO. 60, or an amino acid sequence obtained by any one of the following substitutions or any combination thereof: The second amino acid V is replaced by A, The eighth amino acid, E, is replaced by T, The 11th amino acid, S, is replaced by N, the 31st amino acid A is substituted with G, and / or The amino acid sequence of FR-H4 is shown in SEQ ID NO.61.

[0014] In some embodiments, the amino acid sequence of the light chain variable region is set forth in any of SEQ ID Nos. 38, 39, or 44, and / or the amino acid sequence of the heavy chain variable region is set forth in any of SEQ ID Nos. 30-37, 40-43, 48-53, or a variant sequence having at least 70% identity, e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, to the amino acid sequence set forth in SEQ ID Nos. 30-44 or 48-53, and retaining the biological activity of the corresponding parent sequence, or a variant sequence obtained by deleting, substituting, and / or adding one or more amino acid residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more, to the sequence set forth in SEQ ID Nos. 30-44 or 48-53, and retaining the biological activity of the corresponding parent sequence.

[0015] Preferably, the light chain variable region sequence has the amino acid sequence shown in SEQ ID NO. 38, and / or the heavy chain variable region sequence has an amino acid sequence selected from SEQ ID NOs. 30-37; or the light chain variable region sequence has the amino acid sequence set forth in SEQ ID NO. 39, and / or the heavy chain variable region sequence has an amino acid sequence selected from SEQ ID NOs. 30-37 or 48-50; or The light chain variable region sequence has the amino acid sequence shown in SEQ ID NO. 44, and / or the heavy chain variable region sequence has an amino acid sequence selected from SEQ ID NOs. 40-43 or 51-53.

[0016] More preferably, the light chain variable region sequence has the amino acid sequence shown in SEQ ID NO. 39, and / or the heavy chain variable region sequence has an amino acid sequence selected from SEQ ID NO. 36 or 48-50; or The light chain variable region sequence has the amino acid sequence shown in SEQ ID NO. 44, and / or the heavy chain variable region sequence has an amino acid sequence selected from SEQ ID NO. 41 or 51-53.

[0017] Preferably, the amino acid sequences of the light chain constant region and the heavy chain constant region are shown in SEQ ID NO. 26 and SEQ ID NO. 27, respectively.

[0018] In this field, when identity is referred to, the length of the amino acid sequence must be a natural number, so the actual calculated identity value may not be a finite percentage such as 95%, but may be a number close to a percentage such as 95%. For example, in the variable region sequence of amino acid residue 117, if only one amino acid residue is changed, the corresponding identity percentage is actually close to 99.15%, but for convenience, such a number is expressed as 99% in this specification.

[0019] In some embodiments, the antigen-binding fragment is one or more selected from F(ab')2, Fab', Fab, Fd, Fv, scFv, bispecific antibody, camel antibody, CDR, and minimal antibody recognition unit (dAb), and preferably the antigen-binding fragment is Fab, F(ab')2, or scFv.

[0020] A second aspect of the present invention relates to an isolated nucleic acid molecule selected from: (1) DNA or RNA encoding the anti-human PD-L1 antibody, antigen-binding fragment thereof, or variant thereof according to the first aspect; (2) A nucleic acid that is completely complementary to the DNA or RNA defined in (1).

[0021] A third aspect of the present invention relates to a vector comprising operatively ligated nucleic acid molecules according to the second aspect, preferably said vector being an expression vector.

[0022] A fourth aspect of the present invention relates to a host cell comprising a nucleic acid molecule according to the second aspect or a vector according to the third aspect.

[0023] A fifth aspect of the present invention relates to a composition comprising an anti-human PD-L1 antibody, antigen-binding fragment or variant thereof according to the first aspect, a nucleic acid molecule according to the second aspect, a vector according to the third aspect or a host cell according to the fourth aspect, and a pharmaceutically acceptable carrier, diluent or excipient.

[0024] A sixth aspect of the present invention relates to a method for producing an anti-human PD-L1 antibody, antigen-binding fragment thereof, or variant thereof according to the first aspect, said method comprising:

[0025] allowing the host cell of the fourth aspect to express the anti-human PD-L1 antibody, or antigen-binding fragment thereof, or variant thereof, under culture conditions suitable for expression, and optionally isolating and purifying the resulting product.

[0026] A seventh aspect of the present invention relates to the use of an anti-human PD-L1 antibody according to the first aspect, an antigen-binding fragment thereof or a variant thereof, a nucleic acid molecule according to the second aspect, a vector according to the third aspect, or a host cell according to the fourth aspect in the manufacture of a medicament for the prevention and / or treatment of a PD-L1-mediated disease or condition, such as an autoimmune disease, an immune response to a transplant, an allergic reaction, an infectious disease, a neurodegenerative disease, or a tumor.

[0027] An eighth aspect of the present invention relates to an anti-human PD-L1 antibody according to the first aspect, an antigen-binding fragment thereof or a variant thereof, a nucleic acid molecule according to the second aspect, a vector according to the third aspect or a host cell according to the fourth aspect for the prevention and / or treatment of a PD-L1-mediated disease or condition, such as an autoimmune disease, an immune response to a transplant, an allergic reaction, an infectious disease, a neurodegenerative disease or a tumor.

[0028] A ninth aspect of the invention relates to a method for preventing and / or treating a PD-L1-mediated disease or condition, such as an autoimmune disease, immune response to a transplant, allergic reaction, infectious disease, neurodegenerative disease, or tumor, comprising the step of administering to a subject in need thereof an anti-human PD-L1 antibody, antigen-binding fragment thereof, or variant thereof according to the first aspect, a nucleic acid molecule according to the second aspect, a vector according to the third aspect, or a host cell according to the fourth aspect.

[0029] In some embodiments, the autoimmune disease is one or more selected from arthritis, rheumatoid arthritis, psoriasis, multiple sclerosis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus, glomerulonephritis, dilated cardiomyopathy, Sjögren's syndrome, atopic and contact dermatitis, polymyositis, scleroderma, periarteritis nodosa, rheumatic fever, vitiligo, insulin-dependent diabetes mellitus, Behçet's disease, and chronic thyroiditis. Preferably, the autoimmune disease is one or more selected from arthritis, rheumatoid arthritis, psoriasis, multiple sclerosis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus, glomerulonephritis, rheumatic fever, vitiligo, insulin-dependent diabetes mellitus, and chronic thyroiditis. More preferably, the autoimmune disease is one or more selected from rheumatoid arthritis, psoriasis, multiple sclerosis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus, insulin-dependent diabetes mellitus, and chronic thyroiditis.

[0030] In some embodiments, the immune response to the transplant includes, for example, graft-versus-host disease.

[0031] In some embodiments, the allergic reaction is one or more selected from urticaria, eczema, angioneurotic edema, allergic rhinitis, bronchial asthma, laryngeal edema, food allergic gastroenteritis, and anaphylactic shock. Preferably, the allergic reaction is one or more selected from urticaria, eczema, allergic rhinitis, bronchial asthma, and anaphylactic shock. More preferably, the allergic reaction is one or more selected from allergic rhinitis, bronchial asthma, and anaphylactic shock.

[0032] In some embodiments, the infectious disease refers to local tissue and systemic inflammatory responses caused by the invasion of pathogens, such as viruses, bacteria, fungi, and parasites, and certain toxins into the human body. Examples of pathogenic viruses include HIV, hepatitis viruses (A, B, and C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, and Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arbovirus. Pathogenic bacteria include, for example, Treponema pallidum, Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Neisseria meningitidis and Neisseria gonorrhoeae (conococci), Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, Bacillus subtilis, Vibrio cholerae, Clostridium tetani, Clostridium botulinum, Bacillus anthrax, Yersinia pestis, Leptospira, and Borrelia burgdorferi.Pathogenic fungi include, for example, Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucorales (mucor, Absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, and the like. dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum. Pathogenic parasites include, for example, Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba species, Giardia lambia, Cryptosporidium species, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis. brasiliensis).

[0033] In some embodiments, the neurodegenerative disease is one or more selected from Parkinson's disease, Huntington's disease, Machado-Joseph disease, amyotrophic lateral sclerosis, and Creutzfeldt-Jakob disease. Preferably, the neurodegenerative disease is one or more selected from Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. More preferably, the neurodegenerative disease is one or more selected from Parkinson's disease and Huntington's disease.

[0034] In some embodiments, the tumor is one or more selected from leukemia, lymphoma, myeloma, brain tumor, squamous cell carcinoma of the head and neck, non-small cell lung cancer, small cell lung cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, pancreatic adenocarcinoma, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, urothelial carcinoma, renal cell carcinoma, osteosarcoma, melanoma, and Merkel cell carcinoma. Preferably, the tumor is one or more selected from lymphoma, myeloma, squamous cell carcinoma of the head and neck, non-small cell lung cancer, small cell lung cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, liver cancer, colorectal cancer, breast cancer, cervical cancer, endometrial cancer, prostate cancer, urothelial carcinoma, renal cell carcinoma, osteosarcoma, melanoma, and Merkel cell carcinoma. More preferably, the tumor is one or more selected from lymphoma, head and neck squamous cell carcinoma, non-small cell lung cancer, gastric cancer, liver cancer, colorectal cancer, cervical cancer, urothelial carcinoma, renal cell carcinoma, melanoma, and Merkel cell carcinoma.

[0035] In some embodiments, the subject is selected from mammals, including, but not limited to, humans and / or other primates, including commercially relevant mammals such as cows, pigs, horses, goats, cats, dogs, mice, and / or rats.

[0036] In some embodiments, the anti-human PD-L1 antibodies, antigen-binding fragments or variants thereof, nucleic acid molecules, vectors, or host cells of the invention are administered by methods commonly used in the art, for example, parenteral routes, intravenous administration.

[0037] The anti-PD-L1 monoclonal antibodies of the present invention have high specificity, good stability, potent T cell function-regulating activity, favorable pharmacokinetic properties, and can significantly inhibit tumor growth in vivo. Furthermore, the anti-PD-L1 monoclonal antibodies of the present invention have stronger blocking activity against PD-L1 / PD-1 binding than atezolizumab (see Figure 1B).

[0038] In order to more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the drawings that need to be used in the description of the specific embodiments or the prior art will be briefly introduced below. Please note that the drawings in the following description only represent some embodiments of the present invention. Those skilled in the art can obtain other drawings from these drawings without any creative work. [Brief explanation of the drawings]

[0039] [Figure 1] Figure 1 shows the in vitro activity of anti-human PD-L1 mouse monoclonal antibodies secreted by clones 34-35 in Example 1 of the present invention. Here, A shows the binding activity of the mouse monoclonal antibody to PD-L1. B shows the blocking activity of the mouse monoclonal antibody against PD-L1 / PD-1 binding. [Figure 2] Figure 1 shows the binding activity of anti-human PD-L1 chimeric monoclonal antibodies to human PD-L1 in Example 3 of the present invention. [Figure 3] Figure 1 shows the binding specificity of the anti-human PD-L1 chimeric monoclonal antibody in Example 4 of the invention, where A is the species binding specificity and B is the target binding specificity. [Figure 4] 1 shows the blocking activity of anti-human PD-L1 chimeric monoclonal antibodies against PD-L1 / PD-1 binding in Example 5 of the present invention. [Figure 5] 1 shows the regulatory activity of anti-human PD-L1 chimeric monoclonal antibodies on T cell function in Example 6 of the present invention. [Figure 6]1 shows the blood drug concentration-time curves after a single intraperitoneal administration of an anti-human PD-L1 humanized monoclonal antibody in Example 9 of the present invention to mice. [Figure 7] 1 shows the in vivo anti-tumor effect of an anti-human PD-L1 humanized monoclonal antibody in Example 10 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0040] definition The term human "PD-L1" refers to programmed death ligand-1, also known as CD274 and B7H1, and refers to any naturally occurring PD-L1 from any vertebrate, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats).

[0041] As used herein, the terms "anti-PD-L1 antibody," "anti-PD-L1," "PD-L1 antibody," or "antibody that binds PD-L1" refer to an antibody that can bind to the PD-L1 protein or a fragment thereof with sufficient affinity. In some embodiments, the anti-PD-L1 antibody binds to a conserved PD-L1 epitope in a heterologous PD-L1.

[0042] The term "antibody" refers to an immunoglobulin molecule or a fragment of an immunoglobulin molecule capable of binding to an epitope of an antigen. Naturally occurring antibodies typically comprise a tetramer and are usually composed of at least two heavy (H) chains and at least two light (L) chains. Immunoglobulins include the isotypes IgG, IgA, IgM, IgD, and IgE, with the corresponding heavy chains being γ, α, μ, δ, and ε, respectively. Ig molecules of the same class can also be divided into different subclasses based on differences in the amino acid composition of the hinge region and the number and location of disulfide bonds in the heavy chain. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4 subtypes, and IgA can be divided into IgA1 and IgA2 subtypes. Light chains are divided into κ and λ chains based on the constant region.

[0043] As used herein, the term "antibody" is used in the broadest sense to refer to a protein that contains an antigen-binding site and includes natural and artificial antibodies of various structures, including, but not limited to, complete antibodies and antigen-binding fragments of antibodies.

[0044] A "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to its antigen. Each heavy chain of an antibody is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH), and the heavy chain constant region is usually composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The heavy and light chain variable regions are typically responsible for antigen recognition, while the heavy and light chain constant regions can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells), Fc receptors, and the first component (C1q) of the classical complement system. The heavy and light chain variable regions contain binding regions that interact with antigens. The VH and VL regions are further divided into hypervariable regions (HVRs) called "complementarity-determining regions (CDRs)," with more conserved regions called "framework regions" (FRs) intervening between them. Each VH and VL is composed of three CDR domains and four FR domains, arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the amino terminus to the carboxyl terminus.

[0045] The terms "complementarity determining region" or "CDR region" or "CDR" (which may be used interchangeably herein with hypervariable region "HVR") refer to the regions of an antibody variable domain that form highly variable sequence and structurally determined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). The CDRs are primarily responsible for binding to an epitope of an antigen. Herein, the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3, and the three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3.

[0046] It should be noted that the CDR boundaries of the variable regions of the same antibody obtained based on different numbering schemes (e.g., IMGT®, Kabat, Chothia) may be different. That is, the CDR sequences of the variable regions of the same antibody defined by different numbering schemes will be different. Therefore, when antibodies are defined using specific CDR sequences defined in the present invention, the scope of said antibodies also includes antibodies whose variable region sequences contain said specific CDR sequences, but whose so-called CDR boundaries differ from the specific CDR boundaries defined in the present invention by applying a different scheme (e.g., the rules or combinations of different numbering schemes).

[0047] The terms "monoclonal antibody," "monoclonal antibody," or "monoclonal antibody composition" refer to an antibody obtained from a substantially homogeneous antibody population as a preparation of antibody molecules of a single molecular composition, i.e., a population comprising individual antibodies that are identical except for minor, possibly naturally occurring mutations. A typical monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. In some embodiments, a monoclonal antibody may be composed of two or more Fab domains, thereby enhancing specificity for two or more targets. The terms "monoclonal antibody" or "monoclonal antibody composition" are not limited to any particular method of production (e.g., recombinant, transgenic, hybridoma, etc.).

[0048] The terms "dual antibody," "bifunctional antibody," "bispecific antibody," or "BsAb (bispecific antibody)" refer to an antibody that has two different antigen-binding sites and can simultaneously bind to two target antigens, exerting its targeting ability while also mediating the action of another specialized functional effector molecule. The mediated specialized functional effector molecule may be a toxin, enzyme, cytokine, radionuclide, etc., and the two antigen-binding arms of a bispecific antibody may each be derived from Fab, Fv, ScFv, dSFv, etc.

[0049] The term "polyclonal antibody" refers to a preparation of different antibodies directed against different antigenic determinants ("epitopes").

[0050] The term "antigen-binding fragment of an antibody" refers to a fragment, portion, region, or domain of an antibody (which may be obtained, for example, by truncation, recombinantly, synthetically, etc.) that is capable of binding to an epitope. An antigen-binding fragment may comprise one, two, three, four, five, or all six CDR domains of such an antibody, and may exhibit different specificities, affinities, or selectivities while still being capable of binding to the epitope. Preferably, the antigen-binding fragment comprises all six CDR domains of the antibody. An antigen-binding fragment of an antibody may be part of or comprises a single polypeptide chain (e.g., an scFv), or may be part of or comprises two or more polypeptide chains (each having an amino terminus and a carboxyl terminus) (e.g., a bibody, an Fab fragment, an F(ab')2 fragment, etc.).

[0051] Examples of antigen-binding fragments included in the present invention include: (a) Fab' or Fab fragments, which are monovalent fragments consisting of the VL, VH, CL, and CH1 domains; (b) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by a disulfide bond at the hinge domain; (c) Fd fragments, which consist of the VH and CH1 domains; (d) Fv fragments, which consist of the VL and VH domains of one arm of an antibody; (e) single-chain antibodies (single-chain Fv, scFv), which are recombinant proteins in which antibody VH and VL are linked by a connecting peptide segment using genetic engineering techniques; (f) dAb fragments (Ward et al., Nature, 341, 544-546 (1989)), which are essentially composed of the VH domain and are also called domain antibodies (Holt et al., Trends Biotechnol., 2i(ll):484-90); (g) camelid or nanoantibodies (Revets et al., Expert Opin Biol. Ther., 5(l):111-24) and (h) isolated complementarity determining regions (CDRs).

[0052] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, and to fragments of such an antibody, so long as the desired biological activity is exhibited. The present invention provides variable region antigen-binding sequences derived from human antibodies. Therefore, the chimeric antibodies primarily focused on herein include antibodies having one or more human antigen-binding sequences (e.g., CDRs) and containing one or more sequences derived from a non-human antibody, such as FR or C region sequences. Note that the chimeric antibody described herein refers to an antibody containing human variable region antigen-binding sequences of one antibody class or subclass and other sequences, such as FR or C region sequences, derived from another antibody class or subclass.

[0053] The term "humanized antibody" refers to an antibody in which CDR sequences from another mammalian species, such as a murine species, have been grafted onto human framework sequences in which additional framework region modifications can be made.

[0054] The term "human antibody" or "fully human antibody" ("humAb" or "HuMab") includes antibodies having variable and constant regions derived from human species-based immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human species-based immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro, or during gene rearrangement, or by somatic mutation in vivo).

[0055] Mutant antibodies are also included within the scope of the present invention. Therefore, variants of the sequences listed herein are also included within the scope of the present invention. Other variants of antibody sequences with improved affinity can be obtained using methods known in the art, and these variants are also included within the scope of the present invention. For example, amino acid substitution can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of nucleotide sequences can be used to improve the translation efficiency of expression systems for antibody production.

[0056] The sequences of such variant antibodies have 70% or more (e.g., 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity with the sequences listed herein, where such sequence identity is calculated over the full length of the reference sequence (i.e., the sequence listed herein).

[0057] The amino acid residues herein are numbered according to IMGT® (the international ImMunoGeneTics information system®) or Kabat, EA, Wu, TT, Perry, HM, Gottesmann, KS & Foeller, C., (1991), Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication No. 91-3242, United States Department of Health and Human Services; Chothia, C. & Lesk, AM, (1987), Canonical Structures For The Hypervariable Domains Of Immunoglobulins., J. Mol. Biol., 196, 901-917. Unless otherwise specified, the amino acid residues herein are numbered according to the Kabat EU index numbering system.

[0058] An antibody or antigen-binding fragment thereof "specifically" binds to a region of another molecule (i.e., an epitope) if it reacts or binds to that epitope more frequently, more rapidly, with a longer duration, and / or with greater affinity than it does to other epitopes. In some embodiments, the antibodies or antigen-binding fragments thereof of the invention bind to at least 10 -7 M, e.g. 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 The antibody or antigen-binding fragment thereof binds to human PD-L1 with an affinity of M or better. Preferably, the antibody or antigen-binding fragment thereof binds under physiological conditions (e.g., in vivo). Thus, specifically binding to PD-L1 refers to the antibody or antigen-binding fragment thereof's ability to bind to PD-L1 with the above specificity and / or under such conditions. Suitable methods for determining such binding are known in the art.

[0059] In the context of antibody binding to a designated antigen, the term "binding" typically refers to binding of an antibody to a designated antigen within about 10 -6 M or lower KD This means that the molecule binds with an affinity corresponding to K D is at least 10-fold, such as at least 100-fold, at least 1,000-fold lower than the affinity of the antibody's binding to a nonspecific antigen other than the designated antigen or a closely related antigen (e.g., BSA, casein).

[0060] As used herein, the term "k d " (sec-1 or 1 / s) refers to the dissociation rate constant of a particular antibody-antigen interaction. off Also called value.

[0061] As used herein, the term "k a " (M-1 x sec-1 or 1 / Msec) refers to the association rate constant of a particular antibody-antigen interaction.

[0062] As used herein, the term "K D " (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, and k d k a This is obtained by dividing by

[0063] As used herein, the term "K A " (M-1 or 1 / M) refers to the binding equilibrium constant of a particular antibody-antigen interaction, and k a k d This is obtained by dividing by

[0064] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention may be humanized such that only a portion of the CDRs (i.e., the subgroup of CDR residues necessary for binding, called SDRs) are combined. Based on previous studies, such as those described in Gonzales, N.R. et al. (2004), SDR Grafting of a Murine Antibody Using Multiple Human GermLine Templates to Minimize Its Immunogenicity, Mol. Immunol., 41:863-872, CDR residues that do not contact the relevant epitope and are not located in the SDRs can be identified from Kabat CDR regions located outside the Chothia hypervariable loops (e.g., residues H60-H65 in CDR H2 are typically dispensable) (see Kabat et al. (1992), Sequences of Proteins of Immunological Interest, National Institutes of Health, Publication No. 91-3242; Chothia, C. et al. (1987), Canonical Structures for the Hypervariable Regions of Immunoglobulins, J. Mol. Biol., 196:901-917). In such humanized antibodies, at positions where one or more donor CDR residues are absent or where the entire donor CDR is omitted, the amino acid occupying this position may be the amino acid occupying the corresponding position (numbered according to Kabat) in the recipient antibody sequence. Such substitutions are potentially advantageous in reducing the number of murine amino acids in the humanized antibody, thereby reducing potential immunogenicity. However, substitutions may also result in changes in affinity, and it is desirable to avoid significant decreases in affinity. The substitution positions within the CDRs and the amino acids to be substituted can also be selected empirically.

[0065] The fact that changing a single amino acid in a CDR residue results in loss of functional binding (Rudikoff, S. et al. (1982), Single Amino Acid Substitution Altering Antigen-binding Specificity, Proc. Natl. Acad. Sci. (USA)) 79(6):1979-1983) can be used to systematically identify alternative functional CDR sequences. In a preferred method for obtaining such mutant CDRs, the polynucleotide encoding the CDR is mutated (e.g., by random or site-directed mutagenesis) to generate a CDR with a substituted amino acid residue. The substitution score of this substituted BLOSUM62.iij can be determined by comparing the identity of the relevant residue in the original (functional) CDR sequence with that of the substituted (non-functional) mutant CDR sequence. The BLOSUM system provides amino acid substitution matrices created by analyzing sequence databases and used to compare reliability (Eddy, SR, (2004), Where Did The BLOSUM62 Alignment Score Matrix Come From?, Nature Biotech., 22(8):1035-1036; Henikoff, JG, (1992), Amino acid substitution matrices from protein blocks), Proc. Natl. Acad. Sci. (USA), 89:10915-10919; Karlin, S. et al., (1990), Methods For Assessing The Statistical Significance Of Molecular Sequence Features By Using General Scoring Schemes), PNAS, 87:2264-2268; Altschul, SF, (1991), Amino Acid Substitution Matrices From An Information Theoretic Perspective, J. Mol. Biol., 219, 555-565.Currently, the most advanced BLOSUM database is the BLOSUM62 database (BLOSUM62.iij). Table 1 shows the BLOSUM62.iij substitution scores (the higher the score, the more conservative the substitution and the more likely it is that the substitution will not affect function). For example, if the resulting antigen-binding fragment containing the CDR cannot bind to PD-L1, the BLOSUM62.iij substitution score is considered not sufficiently conservative, and new candidate substitutions with higher substitution scores are selected and generated. Thus, for example, if the original residue is glutamic acid (E) and the non-functional replacement residue is histidine (H), the BLOSUM62.iij substitution score is 0, and more conservative changes (e.g., aspartic acid, asparagine, glutamine, or lysine) are preferred.

[0066] [Table 1]

[0067] Thus, the present invention contemplates the use of random mutagenesis in identifying improved CDRs. In the context of the present invention, conservative substitutions may be defined by substitutions within one or more of the amino acid categories in the following three tables:

[0068] Types of amino acid residues to be conservatively substituted: [Table 2]

[0069] Alternative conservative amino acid residue substitution types: [Table 3]

[0070] Classification of physical and functional alternatives of amino acid residues: [Table 4]

[0071] More conservative substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0072] In some embodiments, the hydrophilic amino acids are selected from Arg, Asn, Asp, Gln, Glu, His, Tyr, and Lys.

[0073] Additionally, other amino acid groups can be generated using the principles described, for example, in Creighton, (1984), Proteins: Structure and Molecular Properties, WH Freeman and Company.

[0074] Thus, the sequences of the CDR variants of the included antibodies or antigen-binding fragments thereof can differ from the sequences of the CDRs of the parent antibody by substitutions, for example, by substitutions of 4, 3, 2 or 1 amino acid residues. According to embodiments of the invention, amino acids in the CDR regions may be substituted with conservative substitutions, as defined in the three tables above.

[0075] "Homology" or "sequence identity" refers to the percentage of identical residues between a polynucleotide or polypeptide sequence variant and a non-variant sequence after aligning the sequences and introducing gaps. In specific embodiments, polynucleotide and polypeptide variants have at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% polynucleotide or polypeptide homology with the polynucleotides or polypeptides described herein.

[0076] Such variant polypeptide sequences have 70% or more (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity to the sequences set forth herein. In other embodiments, the present invention provides polypeptide fragments comprising contiguous stretches of various lengths of the amino acid sequences disclosed herein. For example, where applicable, the peptide sequences provided herein include at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150, or more consecutive peptides of one or more of the sequences disclosed herein, as well as all peptides of intermediate lengths therebetween.

[0077] The term "treatment" refers to ameliorating, alleviating, attenuating, or reversing the progression or severity of a disease or condition, or ameliorating, alleviating, attenuating, or reversing one or more symptoms or side effects of such a disease or condition. In the present invention, "treatment" also refers to an approach for obtaining a beneficial or promising clinical result, where "beneficial or promising clinical result" includes, but is not limited to, alleviation of symptoms, reduction in the condition or extent of disease, stabilized (i.e., not worsening) state of the disease or condition, delaying or alleviating the progression of the condition of the disease or condition, improvement or palliation of the condition of the disease or condition, and remission of the disease or condition, whether partial or total, detectable or undetectable.

[0078] The term "prevention" refers to preventing or inhibiting the development of at least one symptom of a disease or condition by administering the antibodies and functional fragments thereof of the present invention. This term further includes treating a subject in remission to prevent or inhibit recurrence.

[0079] Antibodies of the present invention may have any isotype. The choice of isotype is typically determined by the desired effector function (e.g., ADCC induction). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either the kappa or lambda human light chain constant region can be used. If necessary, the class of the anti-PD-L1 antibodies of the present invention can be converted using known methods. For example, an initial IgG antibody of the present invention can be class converted to an IgM antibody of the present invention. Note that class conversion techniques can also be used to convert an IgG subclass to another subclass, for example, IgG1 to IgG2. Thus, the effector function of the antibodies of the present invention can be converted by isotype switching, for example, to an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic applications. In some embodiments, antibodies of the present invention are IgG2 antibodies, e.g., IgG2a. An antibody belongs to a particular isotype if its amino acid sequence is nearly identical to that of another isotype.

[0080] In some embodiments, the antibodies of the invention are full-length antibodies, preferably IgG antibodies, hi other embodiments, the antibodies of the invention are antibody-antigen binding fragments or single chain antibodies.

[0081] In some embodiments, the anti-PD-L1 antibody is a monovalent antibody, preferably a monovalent antibody with a deletion in the hinge region, such as those described in WO2007059782, which is incorporated herein by reference in its entirety. Thus, in some embodiments, the antibody is a monovalent antibody, wherein the anti-PD-L1 antibody is constructed by: i) providing a nucleic acid construct encoding the light chain of the monovalent antibody, wherein the construct comprises a nucleotide sequence encoding the VL region of a selected antigen-specific anti-PD-L1 antibody and a nucleotide sequence encoding an Ig constant CL region, wherein the nucleotide sequence encoding the VL region of the selected antigen-specific antibody and the nucleotide sequence encoding the Ig CL region are operatively linked, and in the case of the IgG1 subclass, the nucleotide sequence encoding the CL region has been modified so that the CL region does not contain any amino acids that can form disulfide bonds or covalent bonds with other peptides comprising the matching amino acid sequence of the CL region when in the presence of polyclonal human IgG or when administered to an animal or human; ii) providing a nucleic acid construct encoding the heavy chain of the monovalent antibody, wherein the construct comprises a nucleotide sequence encoding the heavy chain of a selected antigen-specific anti-PD-L1 antibody the nucleotide sequence encoding the VH region of an antibody and the nucleotide sequence encoding the constant CH region of a human Ig, wherein the nucleotide sequence encoding the CH region has already been modified so that, when administered to an animal or a human in the presence of polyclonal human IgG or to an animal or a human, the region corresponding to the hinge region and other regions of the CH region (e.g., the CH3 region) (e.g., required for an Ig subclass) do not contain any amino acid residues involved in forming disulfide bonds or covalent or stable non-covalent inter-heavy chain bonds with another peptide containing the identical amino acid sequence of the CH region of a human Ig, wherein the nucleotide sequence encoding the VH region of a selected antigen-specific antibody and the nucleotide sequence encoding the CH region of the Ig are operatively linked; iii) providing a cell expression system for producing a monovalent antibody; and iv) co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii) to produce the monovalent antibody.

[0082] Similarly, in some embodiments, the anti-PD-L1 antibody is a monovalent antibody, (i) a variable region or an antigen-binding portion of said domain of an antibody of the invention as described herein; (ii) a CH region of an immunoglobulin or a domain comprising the CH2 and CH3 domains thereof; Here, this CH region or a domain thereof has already been modified so that the hinge region and (if this immunoglobulin is not of the IgG4 subclass) the domain corresponding to another domain of the CH region (e.g., the CH3 domain) do not contain any amino acid residues that can form disulfide bonds with the same CH region or other covalent or stable non-covalent inter-heavy chain bonds with the same CH region in the presence of polyclonal human IgG.

[0083] In some other embodiments, the heavy chain of the monovalent antibody is modified to delete the entire hinge region.

[0084] In other embodiments, the sequence of the monovalent antibody is modified so that it does not contain any acceptor sites for N-linked glycosylation.

[0085] The present invention further includes "bispecific antibodies" in which the anti-PD-L1 binding region (e.g., the PD-L1 binding region of an anti-PD-L1 monoclonal antibody) is part of a bivalent or multivalent bispecific framework that targets more than one epitope (e.g., the second epitope can include an epitope of an active transport receptor, thereby providing the bispecific antibody with improved cell translocation across biological barriers (e.g., the blood-brain barrier), or the second epitope is an epitope that targets another protein of interest). Thus, in another embodiment, a monovalent Fab of an anti-PD-L1 antibody can be linked to a Fab or scfv that targets another, different protein, to produce a bispecific antibody. Bispecific antibodies can have dual functions, for example, a therapeutic function conferred by the anti-PD-L1 binding region and the ability to bind to a receptor molecule and enhance transport function across biological barriers (e.g., the blood-brain barrier).

[0086] The antibodies and antigen-binding fragments thereof of the present invention further include single-chain antibodies. Single-chain antibodies are peptides in which the Fv domains of the heavy and light chains are linked. In some embodiments, the present invention provides single-chain Fvs (scFvs), in which the heavy and light chains in the Fv of an anti-PD-L1 antibody of the invention are linked by a flexible peptide (typically about 10, 12, 15 or more amino acid residues) to form a single peptide chain. Methods for producing such antibodies are described, for example, in US 4,946,778; Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore ed., Springer-Verlag, New York, pages: 269-315 (1994); Bird et al., Science, 242, 423-426 (1988); Huston et al., PNAS USA 85, 5879-5883 (1988) and McCafferty et al., Nature, 348, 552-554 (1990). Single-chain antibodies are monovalent if only a single VH and VL are used, bivalent if two VH and VL are used, or multivalent if two or more VH and VL are used.

[0087] Antibodies of the present invention can be produced by any technique known in the art, including, but not limited to, any chemical, biological, genetic, or enzymatic technique, which may be used alone or in combination. Typically, the amino acid sequence of the desired sequence is known, and one of skill in the art can readily produce the antibody using standard techniques for producing polypeptides. For example, these antibodies can be synthesized by known solid-phase methods, preferably using commercially available peptide synthesizers (e.g., those manufactured by Applied Biosystems, Foster City, California) according to the manufacturer's instructions. Alternatively, antibodies of the present invention can be synthesized by recombinant DNA techniques known in the art. For example, a DNA sequence encoding the antibody can be incorporated into an expression vector and the vector introduced into a suitable eukaryotic or prokaryotic host for expression of the desired antibody, resulting in the antibody as a DNA expression product, which can then be isolated from the host using known techniques.

[0088] The antibodies and antigen-binding fragments thereof of the present invention can be modified by including any "suitable" number of modified amino acids and / or by attaching coupling substituents. In such cases, "suitable" is generally determined by the ability to retain at least essentially the PD-L1 selectivity and / or specificity associated with the underivatized parent anti-PD-L1 antibody. The inclusion of one or more modified amino acids can contribute, for example, to increasing the serum half-life of the polypeptide, reducing polypeptide antigenicity, or improving the storage stability of the polypeptide. Modifications to one or more amino acids can be made, for example, co-translationally during recombinant production, post-translationally (e.g., N-linked glycosylation at NXS / T sequences during expression in mammalian cells), or by synthetic means. Non-limiting examples of modified amino acids include glycosylated amino acids, sulfated amino acids, isoprenated (e.g., farnesylated, geranyl-geranylated) amino acids, acetylated amino acids, acylated amino acids, pegylated amino acids, biotin-acylated amino acids, carboxylated amino acids, phosphorylated amino acids, etc. References for making amino acid modifications are well known in the art, see, for example, Walker, (1998), Protein Protocols On CD-Rom, Humana Press, Totowa, New Jersey. Modified amino acids may be selected from, for example, glycosylated amino acids, pegylated amino acids, farnesylated amino acids, acetylated amino acids, biotin-acylated amino acids, amino acids conjugated to a lipid moiety, or amino acids conjugated to an organic derivatizing agent.

[0089] The antibodies and antigen-binding fragments thereof of the present invention can also be chemically modified by covalent conjugation to a polymer to increase their circulating half-life. Exemplary polymers and methods for linking them to peptides are described in, e.g., U.S. Pat. Nos. 4,766,106, 4,179,337, 4,495,285, and 4,609,546. Exemplary polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., PEG having a molecular weight of about 1,000-40,000 D, e.g., about 2,000-20,000 D, e.g., about 3,000-12,000 D).

[0090] The term "subject" refers to a warm-blooded animal, preferably a mammal (human, domestic and farm animals, zoo animals, sporting or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc.), more preferably a human. In one embodiment, the subject may be a "patient," i.e., a warm-blooded animal, more preferably a human, awaiting admission, receiving medical care, or the subject of a medical program or disease progression monitoring. In one embodiment, the subject is an adult (e.g., a subject 18 years of age or older). In another embodiment, the subject is a child (e.g., a subject under the age of 18). In one embodiment, the subject is male. In another embodiment, the subject is female.

[0091] In one embodiment of the present invention, the sample is a biological sample, examples of which include, but are not limited to, diseased tissues and body fluids, preferably blood, more preferably serum, plasma, synovial fluid, bronchoalveolar lavage fluid, sputum, lymph, ascites, urine, amniotic fluid, peritoneal fluid, cerebrospinal fluid, pleural fluid, pericardial effusion, and tissue digests and extracts prepared from alveolar macrophages.

[0092] In one embodiment of the present invention, the term "sample" refers to a sample taken from an individual prior to any analysis.

[0093] Thus, in some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof of the invention include whole antibodies, such as IgG (of subclasses IgG1, IgG2, IgG3, and IgG4), IgA (of subclasses IgA1 and IgA2), IgD, IgM, and IgE; antigen-binding fragments, such as SDRs, CDRs, Fvs, dAbs, Fabs, Fab2, Fab', F(ab')2, Fds, scFvs, camelids, or nanobodies; and variant sequences of antibodies or antigen-binding fragments thereof, such as variant sequences that share at least 80% sequence identity to the above-mentioned antibodies or antigen-binding fragments thereof. In some embodiments, the invention further includes derivatives comprising anti-PD-L1 or antigen-binding fragments thereof, such as chimeric antibodies, humanized antibodies, fully human antibodies, recombinant antibodies, and bispecific antibodies derived from the whole antibodies.

[0094] In another aspect, the present invention relates to expression vectors encoding one or more polypeptide chains of an antibody or antigen-binding fragment thereof of the invention, which can be used to recombinantly produce the antibody or antigen-binding fragment thereof of the invention.

[0095] In the present invention, the expression vector may be any suitable DNA or RNA vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (containing a set of appropriate nucleic acid sequences for expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In some embodiments, the nucleic acid encoding the anti-PD-L1 antibody is contained in a naked DNA or RNA vector, such as a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech, 12, 355-59 (1997)), a small nucleic acid vector (e.g., as described in US 6,077,835 and / or WO 00 / 70087), a plasmid vector (e.g., pBR322, pUC19 / 18, or pUC118 / 119), a minimal size nucleic acid vector (e.g., as described in Schakowski et al., Mol Ther, 3, 793-800 (2001)), or a precipitated nucleic acid vector construct, such as a CaPO4 precipitated construct (e.g., as described in WO 00 / 46147; Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986); Wigler et al., Cell, 14, 725 (1978) and Coraro and Pearson, Somatic Cell Genetics, 2,603 ​​(1981). Such nucleic acid vectors and their uses are well known in the art (see, e.g., US Pat. Nos. 5,589,466 and 5,973,972). In some embodiments, the expression vector is X0GC (derived from patent WO2008 / 048037), pCDNA3.1 (ThermoFisher, catalog number V79520), or pCHO1.0 (ThermoFisher, catalog number R80007).

[0096] In some embodiments, the vector is suitable for expressing the anti-PD-L1 antibody or antigen-binding fragment thereof in bacterial cells. Examples of such vectors include, for example, BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem, 264, 5503-5509 (1989)), and pET vectors (Novagen, Madison, Wisconsin).

[0097] The expression vector may be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be employed. Suitable vectors include, for example, vectors containing constitutive or inducible promoters (e.g., α-factor, alcohol oxidase, and PGH) (for reviews, see F. Ausubel et al., ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience, New York (1987); Grant et al., Methods in Enzymol, 153, 516-544 (1987); Mattanovich, D. et al., Methods in Enzymol, 153, 516-544 (1987)). Mol. Biol., 824, 329-358 (2012); Celik, E. et al., Biotechnol. Adv., 30(5), 1108-1118 (2012); Li, P. et al., Appl. Biochem. Biotechnol., 142(2), 105-124 (2007); Boer, E. et al., Appl. Microbiol. Biotechnol., 77(3), 513-523 (2007); van der Vaart, JM, Methods Mol. Biol., 178, 359-366 (2002) and Holliger, P., Methods Mol. Biol., 178, 349-357 (2002).

[0098] In the expression vectors of the invention, the nucleic acid encoding the anti-PD-L1 antibody can include any suitable promoter, enhancer, and other expression-contributing elements, or a combination thereof. Examples of such elements include a strong promoter (e.g., the human CMV IE promoter / enhancer and the RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), an efficient poly(A) terminator sequence, an origin of replication for producing the plasmid in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid can also include an inducible promoter for a constitutive promoter (e.g., CMV IE).

[0099] According to another aspect, the invention relates to recombinant eukaryotic or prokaryotic host cells (e.g., transfectomas) that produce the antibodies or antigen-binding fragments thereof, or bispecific molecules of the invention. Exemplary host cells include yeast, bacteria, and mammalian cells (e.g., CHO or HEK cells). For example, in some embodiments, the invention provides cells that comprise a nucleic acid stably integrated into the cellular genome, where the genome comprises a nucleic acid sequence encoding an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention. In other embodiments, the invention provides cells that comprise a non-integrated nucleic acid (e.g., a plasmid, cosmid, phagemid, or linear expression element), where the nucleic acid comprises a sequence encoding an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention.

[0100] The antibodies and antigen-binding fragments thereof of the present invention can be produced in different cell lines, such as human cell lines, non-human mammalian cell lines, and insect cell lines, such as CHO cell lines, HEK cell lines, BHK-21 cell lines, murine cell lines (e.g., myeloma cell lines), fibrosarcoma cell lines, PER.C6 cell lines, HKB-11 cell lines, CAP cell lines, and HuH-7 human cell lines (Dumont et al., 2015, Crit Rev Biotechnol., Sep. 18, 1-13, the contents of which are incorporated herein by reference).

[0101] Antibodies of the invention and culture medium are suitably isolated by conventional immunoglobulin purification methods, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0102] The present invention further relates to compositions comprising, consisting of or consisting essentially of an antibody of the invention.

[0103] As used herein, with respect to a composition, "consisting essentially of" means that at least one antibody of the invention, as described above, is the only biologically active therapeutic agent or reagent in the composition.

[0104] In one embodiment, the composition of the present invention is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier, excipient or diluent.

[0105] The term "pharmaceutically acceptable carrier" refers to an excipient that does not produce any adverse, allergic, or other untoward reaction when administered to animals, preferably humans. It includes any and all solvents, dispersion media, coating layers, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. For human administration, formulations should meet sterility, pyrogenicity, general safety and purity standards required by regulatory agencies (e.g., FDA office and EMA).

[0106] The present invention further relates to a medicament comprising, consisting of or consisting essentially of an antibody of the invention.

[0107] In some embodiments, the glycosylation of the antibodies of the invention is modified. For example, an aglycosylated antibody (i.e., the antibody is not glycosylated) can be produced. Altering glycosylation can, for example, increase the affinity of the antibody for an antigen or alter the ADCC activity of the antibody. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. Such aglycosylation can improve the affinity of the antibody for an antigen. U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al. (incorporated herein by reference) describe such methods in more detail. Alternatively, antibodies with an altered type of glycosylation can be produced, such as hypofucosylated or nonfucosylated antibodies with reduced amounts or no fucosyl residues, or antibodies with added bisecting GlcNac structures. Such an altered fucosylation mode has been shown to improve the ADCC ability of antibodies. Such carbohydrate modifications may be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and may be used as host cells to produce antibodies with altered glycosylation by expressing a recombinant antibody of the invention in the host cell. For example, Hang et al., in EP 1176195 (incorporated herein by reference), describe a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in such a cell line exhibit hypofucosylation or an absence of fucosyl residues.Thus, in some embodiments, human antibodies (preferably monoclonal antibodies) of the invention may be produced by recombinant expression in a cell line exhibiting a hypofucosylated or nonfucosylated mode, e.g., a mammalian cell line lacking expression of the FUT8 gene encoding fucosyltransferase. Presta's PCT disclosure WO 03 / 035835 (incorporated herein by reference) describes a mutant CHO cell line, Lecl3 cells, which have a reduced ability to attach fucose to Asn(297)-linked carbohydrates, resulting in hypofucosylation of antibodies expressed in these host cells (see also Shields, R.L. et al., 2002 J. Biol. Chem. 277:26733-26740). Umana et al., PCT Publication WO 99 / 54342 (incorporated herein by reference), describes that by expressing a glycoprotein-modified glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) in an engineered cell line, antibodies expressed in the engineered cell line display an added bisecting GlcNac structure, resulting in improved ADCC activity of the antibody (see also Umana et al., 1999 Nat. Biotech. 17:176-180). Eureka Therapeutics further described genetically engineered CHO mammalian cells capable of producing antibodies with an altered mammalian glycosylation mode lacking fucose residues (http: / / www.eurekainc.com / a&boutus / companyoverview.html). Alternatively, the human antibodies (preferably monoclonal antibodies) of the present invention can be produced in yeast or filamentous fungi, which are used in a mammalian-like glycosylation mode and can produce antibodies lacking fucose as a glycosylation mode (see, e.g., EP1297172B1).

[0108] The antibodies of the present invention act on PD-L1, which is involved in regulating T cell activation and can regulate the intensity and duration of immune responses. Under normal circumstances, PD-L1 mediates and maintains self-tolerance in living tissues, preventing the immune system from overactivating and damaging self-tissues during inflammatory responses and playing a key role in preventing the development of autoimmune diseases. Under pathological conditions, PD-L1 is involved in tumor immunity and the development and progression of various autoimmune diseases. Clinical evidence has shown that PD-L1 is highly expressed on the surface of various tumor cells, including melanoma, lung cancer, renal cancer, breast cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, gastric cancer, pancreatic adenocarcinoma, and intestinal cancer. Tumor cells bind to PD-1 or CD80 on T cells via highly expressed PD-L1, transmitting immunoinhibitory signals and inducing immune tolerance to tumor cells, which is beneficial to tumor cell growth and metastasis. High PD-L1 expression is closely associated with poor prognosis and drug resistance in tumor patients. The PD-L1 antibodies of the present invention enhance anti-tumor immune responses by blocking the PD-L1 / PD-1 and PD-L1 / CD80 signaling pathways, thereby exerting anti-tumor effects against a wide range of cancers, including non-small cell lung cancer, urothelial carcinoma, Merkel cell carcinoma, melanoma, renal cell carcinoma, lymphoma, head and neck cancer, colorectal cancer, liver cancer, and gastric cancer.

[0109] Where ranges of values ​​are provided, unless otherwise stated herein, it is to be understood that each intervening value, tenth of a unit up to the lower limit, between the upper and lower limits of this range, and any other such value or intervening value within any said range, is included within the scope of the invention. Except as specifically excluded limits, the upper and lower limits of these smaller ranges, which may independently be included in smaller ranges, are also included within the invention, provided that they exclude any specifically excluded boundary within the range. Where a range includes one or two limits, ranges excluding one or both included boundaries are also included within the invention.

[0110] Unless otherwise defined, all technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the presently preferred methods and materials are disclosed. All publications mentioned herein are incorporated by reference in their entirety.

[0111] Hereinafter, embodiments of the present invention will be described in detail with reference to examples. However, those skilled in the art will understand that the following examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention. Meanwhile, the examples provided herein are intended to illustrate the manufacturing process of the antibody of the present invention. This manufacturing process is merely intended to illustrate related methods and is not limiting. Those skilled in the art will recognize that various modifications can be made to the present invention without departing from the spirit of the present invention. Such modifications are also within the scope of the present invention. Meanwhile, the examples provided herein are intended to illustrate the features and advantages of the antibody of the present invention, but the present invention is not limited to these features and advantages.

[0112] Unless otherwise specified, the following experimental methods are all conventional methods, and unless specific conditions are specified, they are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the experimental materials used are readily available from commercial companies. The antibodies used in the following examples of the present invention are all commercially available standard antibodies. [Example]

[0113] Example 1: Preparation of mouse-derived anti-human PD-L1 monoclonal antibodies 1.1 Animal Immunization Experimental animals used were 6-8 week-old female BALB / c mice purchased from HFK Bioscience. After one week of adaptation, the mice were immunized. For the first immunization, 50 μg of recombinant human PD-L1-Fc protein (Beijing Hanmi Pharm, in which the human PD-L1 amino acid sequence is derived from genebank#:NP_001254635.1) and Freund's complete adjuvant (Sigma-Aldrich, catalog number F5881) were thoroughly mixed to form an emulsion and administered intraperitoneally to the mice. Two weeks later, booster immunizations were performed. For the second immunization, 25 μg of recombinant human PD-L1-Fc protein and Freund's incomplete adjuvant (Sigma-Aldrich, catalog number F5806) were thoroughly mixed to form an emulsion and administered intraperitoneally to the mice. Booster immunizations were performed every two weeks in the same manner, for a total of three times. Ten days after the final immunization, blood was collected from the orbital plexus of the mice, centrifuged to separate the serum, and antibody titers were measured by ELISA. Mice with high antibody titers were selected for hybridoma production by fusion. Three days before fusion, 50 μg of adjuvant-free recombinant human PD-L1-Fc protein was intraperitoneally administered. On the day of fusion, the spleens were aseptically removed, and a single spleen cell suspension was prepared for fusion.

[0114] 1.2. Preparation of hybridoma cells SP2 / 0 myeloma cells in the logarithmic growth phase were harvested and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were suspended in incomplete DMEM medium (Gibco, cat. No. 11965) and counted. The required number of cells was taken and washed twice with the above incomplete medium. Simultaneously, a spleen immune cell suspension was prepared and washed twice with incomplete medium. Myeloma cells and spleen cells were mixed at a ratio of 1:10 or 1:5 and washed once with incomplete medium at 1200 rpm for 8 minutes in a 50 mL plastic centrifuge tube. The supernatant was discarded, and the remaining liquid was aspirated with a dropper. The bottom of the centrifuge tube was gently tapped with the palm of the hand to loosen and homogenize the precipitated cells. The tube was then placed in a 40°C water bath for preheating. Using a 1 mL pipette, 1 mL of 45% PEG-4000 (pH 8.0, Sigma, cat No. P7181) preheated to 40°C was added over approximately 1 minute (optimal time: 45 seconds) while gently stirring (by pipette). Visual observation revealed visible particles. To terminate the action of PEG, 20–30 mL of incomplete medium preheated to 37°C was added over 90 seconds using a 10 mL pipette. The mixture was left to stand at 20–37°C for 10 minutes. The mixture was then stirred at 1000 rpm for 5 minutes, after which the supernatant was discarded. Five mL of HAT medium (DMEM+HAT, Sigma, cat No. 1 H0262-10VL) was added, and the precipitated cells were suspended and mixed uniformly by gentle blowing (without blowing too hard to avoid breaking up the fused cells). The HAT medium was then replenished to 80–100 mL (spleen cell concentration: 1–2 × 10 6 (The total volume was 1 mL / mL.) 0.1 mL / well was dispensed into a 96-well cell culture plate. 1.0–1.5 mL / well was dispensed into a 24-well plate. The culture plate was then placed in a 37°C, 6% CO2 incubator and incubated. Typically, six 96-well plates were seeded. After 5 days, half of the medium was replaced with HAT medium. After another 7–10 days, the HAT medium was replaced with HT medium (DMEM+HT, Sigma cat. No. H0137-10VL). The hybridoma cell growth was constantly monitored, and when the cells grew to occupy more than 1 / 10 of the bottom area of ​​the well, the supernatant was aspirated and subjected to antibody detection. Cells of positive clones were expanded and cryopreserved.

[0115] 1.3. Screening and identification of clones ELISA was used to screen for anti-human PD-L1 antibodies in hybridoma culture supernatants. Recombinant human PD-L1 (purchased from Beijing Sino Biological Co., Ltd.) was coated onto a 96-well high-binding ELISA plate (Corning, catalog number 42592) at a coating concentration of 1 μg / mL in a coating volume of 100 μL per well using carbonate buffer solution, pH 9.6. Coating was performed overnight at 4°C. The plate was washed five times with PBST. Blocking was performed with 300 μL per well of PBST containing 1% BSA and incubated for 1 hour at 25°C. The plate was washed five times with PBST. Culture supernatant samples and a positive serum control (positive serum from immunized mice (immunized with human PD-L1-Fc protein)) were added at 100 μL per well and incubated for 1 hour at 25°C. The plate was then washed five times with PBST. Next, 100μL / well of horseradish peroxidase-conjugated anti-mouse IgG antibody (Abcam, catalog no. Ab7068) diluted 1:10,000 in PBST containing 1% BSA was added and incubated at 25℃ for 1 hour. The plate was then washed five times with PBST. 100μL / well of the colorimetric substrate TMB was added and allowed to develop at room temperature for 10 minutes. Color development was stopped by adding 100μL / well of 1M H2SO4. The absorbance at 450nm was read using a microplate reader. Positive clones capable of secreting anti-human PD-L1 binding antibodies were selected based on the intensity at OD450nm.

[0116] The ability of anti-human PD-L1 antibodies secreted by positive clones to block PD-L1 / PD-1 binding was determined by ELISA. Recombinant human PD-L1-Fc was coated onto a 96-well high-binding ELISA plate at a coating concentration of 1 μg / mL in a coating volume of 100 μL / well using carbonate buffer solution, pH 9.6. Coating was performed overnight at 4°C. The plate was washed five times with PBST. Blocking was performed with 300 μL / well of PBST containing 1% BSA and incubated for 1 hour at 25°C. The plate was washed five times with PBST. 50 μL / well of anti-human PD-L1 antibody samples and the positive control atezolizumab were added, and 50 μL / well of 40 nM (final concentration: 20 nM) biotin-labeled PD-1-Fc (Beijing Hanmi Pharm Co., Ltd.) was added and incubated for 90 minutes at 25°C. After washing five times with PBST, 100μL / well of Streptavidin-HRP (BD Pharmingen, catalog no. 554066) diluted 1:1000 in PBST containing 1% BSA was added and incubated at 25℃ for 1 hour. After washing five times with PBST, 100μL / well of the chromogenic substrate TMB was added and allowed to develop at room temperature for 10 minutes. Color development was stopped by adding 100μL / well of 1M H2SO4. The absorbance at 450nm was read using a microplate reader. Anti-human PD-L1 antibodies capable of inhibiting the binding of human PD-L1-Fc / biotin-labeled PD-1-Fc possess neutralizing activity. Positive clones secreting anti-human PD-L1 neutralizing antibodies were selected based on their blocking ability.

[0117] The results are shown in Figure 1A. Clone numbers 34-35 had strong human PD-L1 binding activity. As shown in Figure 1B, clone numbers 34-35 also had strong blocking activity against human PD-L1 / PD-1 binding, which was slightly stronger than atezolizumab.

[0118] 1.4. Measurement of monoclonal antibody sequences Clones with both antigen-binding and antigen-neutralizing activity obtained through screening were subjected to antibody DNA sequencing. Cellular mRNA was extracted using the RNAprep Pure Kit (Tiangen, DP430) according to the manufacturer's instructions. The first strand of cDNA was then synthesized using the QuantScript RT Kit (Tiangen, KR103). The first strand of cDNA produced by reverse transcription was used in the subsequent PCR reaction.

[0119] The primers used in the PCR reactions are shown in Table 5.

[0120] [Table 5]

[0121] When primers were used, any upstream primer of the heavy chain variable region primer (VH primer) could be used in combination with any downstream primer. Similarly, any upstream primer of the light chain variable region primer (VL primer) could be used in combination with any downstream primer. The target band obtained by PCR amplification was cloned into the pGEM-T vector. Monoclonal antibodies were isolated and subjected to DNA sequencing.

[0122] Example 2: Preparation of anti-human PD-L1 chimeric monoclonal antibody The amino acid sequence of the antibody light chain variable region obtained by PCR amplification is shown in SEQ ID NO. 18, and the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO. 19. The amino acid sequences of the three light chain complementarity determining regions, LCDR1, LCDR2, and LCDR3, are shown in SEQ ID NOs. 20, 21, and 22, respectively. The amino acid sequences of the three heavy chain complementarity determining regions, HCDR1, HCDR2, and HCDR3, are shown in SEQ ID NOs. 23, 24, and 25, respectively. The variable region sequences encoding the above light and heavy chains were cloned into the eukaryotic expression vector X0GC, linked to the antibody light chain constant region and heavy chain constant region sequences, respectively. The amino acid sequence of the antibody light chain constant region is shown in SEQ ID NO. 26, and the antibody heavy chain constant region sequence is shown in SEQ ID NO. 27. Expression vectors containing the full-length antibody heavy chain sequence (the full-length heavy chain sequence is formed by linking the heavy chain variable region of the antibody to SEQ ID NO. 27) and the full-length antibody light chain sequence (the full-length light chain sequence is formed by linking the light chain variable region of the antibody to SEQ ID NO. 26) were transfected into ExpiCHO cells (ExpiCHO® cells, catalog number A29127, Invitrogen). The day before transfection, the cells were transfected into 35*10 5 On the day of transfection, cells were inoculated at an inoculation density of 60*10 cells / mL in fresh ExpiCHO Expression Medium (ExpiCHO® Expression Medium, Cat. No. A29100-01, Invitrogen). 5The plasmid was diluted to a dilution density of 1000 cells / mL. The plasmid was extracted according to the transfection volume, and the final concentration of the plasmid was adjusted to 0.5 μg / mL. The plasmid was diluted with OptiPRO® SFM medium (OptiPRO® SFM, catalog number 12309-019, Invitrogen) to 4% of the transfection volume and mixed by inversion. A 6.4-fold amount of the plasmid was extracted from ExpiFectamine® transfection reagent (ExpiFectamine® CHO Transfection Kit, catalog number A29129, Invitrogen) and diluted with OptiPRO® SFM medium to 4% of the transfection volume and mixed by inversion. The diluted transfection reagent was added to the diluted plasmid, mixed gently, and left at room temperature for 1-5 minutes before being gradually added dropwise to the cells. The mixture was then placed in a cell incubator (8% CO2) and incubated at 37°C at 120 rpm for 20 hours. 0.006x transfection volume of ExpiCHO® Enhancer (ExpiFectamine® CHO Transfection Kit, Catalog No. A29129, Invitrogen) and 0.24x transfection volume of ExpiCHO® Feed (ExpiCHO® Feed, Catalog No. A29101-02, Invitrogen) were slowly added dropwise to the cells. The cells were incubated at 32°C in a shaker at 120 rpm. The supernatant of the 10-day transfected cell culture was collected by centrifugation.

[0123] The expression level was measured by ELISA. Before purification by chromatography column, the precipitate was removed by filtration through a 0.2 μm filter membrane. This step was performed at 4°C.

[0124] Example 3 Binding activity and binding kinetic constant of anti-human PD-L1 chimeric monoclonal antibodies to human PD-L1 The binding activity of anti-human PD-L1 chimeric monoclonal antibodies to their antigen, human PD-L1, was measured by ELISA. Recombinant human PD-L1 (purchased from Sino Biologicals) was coated onto a 96-well high-binding ELISA plate at a coating concentration of 1 μg / mL in a coating volume of 100 μL / well using carbonate buffer solution, pH 9.6. Coating was performed overnight at 4°C. The plate was washed five times with PBST. Blocking was performed with 300 μL / well of PBST containing 1% BSA and incubated for 1 hour at 25°C. The plate was washed five times with PBST. Serial dilutions of anti-human PD-L1 chimeric monoclonal antibody samples and the positive control atezolizumab, diluted in PBST containing 1% BSA, were added at 100 μL / well and incubated for 1 hour at 25°C. The plate was washed five times with PBST. Next, 100 μL / well of horseradish peroxidase-conjugated anti-human IgG antibody (Chemicon, Cat. No. AP309P) diluted 1:10,000 in PBST containing 1% BSA was added and incubated for 1 hour at 25°C. The plate was washed five times with PBST. 100 μL / well of the colorimetric substrate TMB was added and allowed to develop for 10 minutes at room temperature. Color development was stopped by adding 100 μL / well of 1 M H2SO4. The absorbance at 450 nm was read using a microplate reader.

[0125] The results are shown in Figure 2. The anti-human PD-L1 chimeric monoclonal antibody had excellent human PD-L1 binding affinity, with the binding activity similar to that of atezolizumab.

[0126] The binding kinetic constants of the anti-human PD-L1 chimeric monoclonal antibody to its antigen, human PD-L1, were measured using a Biacore X100 instrument. This instrument uses optical surface plasma resonance technology to measure the binding and dissociation between molecules bound to and coated on a biochip and the test molecule. The binding kinetic constants and dissociation kinetic constants were analyzed and calculated using the Biacore X100 evaluation software. The binding kinetic constants, dissociation kinetic constants, and dissociation equilibrium constants of the anti-human PD-L1 chimeric antibody are shown in Table 6. The data showed that the anti-human PD-L1 chimeric monoclonal antibody can bind to PD-L1 more quickly than atezolizumab, with similar dissociation rates.

[0127] [Table 6]

[0128] Example 4 Species- and target-binding specificities of anti-human PD-L1 chimeric monoclonal antibodies The species binding specificity of anti-human PD-L1 chimeric monoclonal antibodies was determined by ELISA. Recombinant human PD-L1, monkey PD-L1, rat PD-L1, and mouse PD-L1 (all purchased from Sino Biologicals) were coated onto a 96-well high-binding ELISA plate using carbonate buffer solution, pH 9.6, at a coating concentration of 1 μg / mL in a coating volume of 100 μL per well. The plate was coated overnight at 4°C and washed five times with PBST. Blocking was performed with 300 μL / well of PBST containing 1% BSA and incubated for 1 hour at 25°C. The plate was washed five times with PBST. Serially diluted anti-human PD-L1 chimeric monoclonal antibody samples in PBST containing 1% BSA were added at 100 μL / well and incubated for 1 hour at 25°C. The plate was washed five times with PBST. Next, 100 μL / well of horseradish peroxidase-conjugated anti-human IgG antibody (Chemicon, Cat. No. AP309P) diluted 1:10,000 in PBST containing 1% BSA was added and incubated for 1 hour at 25°C. The plate was washed five times with PBST. 100 μL / well of the colorimetric substrate TMB was added and allowed to develop for 10 minutes at room temperature. Color development was stopped by adding 100 μL / well of 1 M H2SO4. The absorbance at 450 nm was read using a microplate reader.

[0129] The target binding specificity of anti-human PD-L1 chimeric monoclonal antibodies was measured by ELISA. Recombinant human PD-1, CD28, CTLA4, ICOS, BTLA, PD-L1, PD-L2, CD80, CD86, and B7-H2 (all purchased from Sino Biologicals) were coated onto a 96-well high-binding ELISA plate at a coating concentration of 1 μg / mL in a coating volume of 100 μL per well using carbonate buffer solution, pH 9.6. Coating was performed overnight at 4°C. The plate was washed five times with PBST. Blocking was performed with 300 μL / well of PBST containing 1% BSA and incubated for 1 hour at 25°C. The plate was washed five times with PBST. Serial dilutions of anti-human PD-L1 chimeric monoclonal antibody samples and controls in PBST containing 1% BSA were added at 100 μL / well and incubated for 1 hour at 25°C. The plate was then washed five times with PBST. Next, 100 μL / well of horseradish peroxidase-conjugated anti-human IgG antibody (Chemicon, Cat. No. AP309P) diluted 1:10,000 in PBST containing 1% BSA was added and incubated for 1 hour at 25°C. The plate was washed five times with PBST. 100 μL / well of the colorimetric substrate TMB was added and allowed to develop for 10 minutes at room temperature. Color development was stopped by adding 100 μL / well of 1 M H2SO4. The absorbance at 450 nm was read using a microplate reader.

[0130] The results are shown in Figure 3A, which shows that the anti-human PD-L1 chimeric monoclonal antibody can bind to human PD-L1 and monkey PD-L1 with similar affinity, but does not bind to rat or mouse PD-L1, demonstrating species binding specificity. At the same time, as shown in Figure 3B, the anti-human PD-L1 chimeric monoclonal antibody also has strong target binding specificity, binding only to PD-L1, but not to other members of the B7 family or CD28 family members.

[0131] Example 5 Blocking activity of anti-human PD-L1 chimeric monoclonal antibodies against the binding of PD-L1 to PD-1 Recombinant human PD-L1-Fc was coated onto a 96-well high-binding ELISA plate at a coating concentration of 1μg / mL in a coating volume of 100μL / well using carbonate buffer solution (pH 9.6). Coating was performed overnight at 4℃. The plate was washed five times with PBST. Blocking was performed with 300μL / well of PBST containing 1% BSA and incubated at 25℃ for 1 hour. The plate was washed five times with PBST. Anti-human PD-L1 chimeric antibody samples and positive controls were added at 50μL / well, and biotin-labeled PD-1-Fc at a concentration of 40nM (final concentration 20nM) was added at 50μL / well and incubated at 25℃ for 90 minutes. The plate was washed five times with PBST. Streptavidin-HRP (BD Pharmingen, Cat. No. 554066) diluted 1:1000 in PBST containing 1% BSA was then added at 100 μL / well and incubated at 25°C for 1 hour. The plate was washed five times with PBST. 100 μL / well of the colorimetric substrate TMB was added and allowed to develop at room temperature for 10 minutes. Color development was stopped by adding 100 μL / well of 1 M H2SO4. Absorbance at 450 nm was read using a microplate reader.

[0132] The results are shown in Figure 4, which shows that the anti-human PD-L1 chimeric monoclonal antibody has blocking activity against PD-L1 / PD-1 binding similar to that of atezolizumab.

[0133] Example 6: Modulation of T cell function by anti-human PD-L1 chimeric monoclonal antibodies Peripheral blood mononuclear cells (PBMCs) used in the experiments were purchased from Lonza, catalog number CC-2702.

[0134] First, DC cells were induced from PBMCs: PBMCs were resuscitated with complete medium (RPMI 1640 + 10% FBS), washed once with the corresponding serum-free medium, resuspended in serum-free medium, seeded into cell culture bottles, and incubated at 37°C in a 5% CO2 incubator. After 90 minutes, non-adherent cells and the medium were removed. The medium was then replaced with complete medium plus 100 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and 100 ng / mL interleukin-4 (IL-4) (Sino Biologicals) for incubation with mononuclear cells. After 3 days, the medium was replaced once and incubated for another 3 days. The medium was then replaced with complete medium plus 100 ng / mL GM-CSF, 100 ng / mL IL-4, and 20 ng / mL tumor necrosis factor α (TNF-α) (Sino Biologicals) for 1 day of incubation to complete DC cell induction. T cells were isolated from PBMCs from another individual using the Pan T Cell Isolation Kit (Miltenyi Biotech, Catalog No. 5150414820). For specific experimental procedures, please refer to the manufacturer's specifications. The induced mature DC cells were seeded into a 96-well plate at 10,000 cells / well, followed by the isolated T cells at 100,000 cells / well. Finally, the test sample was added and incubated for 120 hours. After incubation, the supernatant was collected and the IFN-γ level was detected using an ELISA kit purchased from RayBiotech.

[0135] The results are shown in Figure 5. When the anti-human PD-L1 chimeric monoclonal antibody was added to mixed lymphocyte incubation, it was able to enhance IFN-γ secretion and had a slightly stronger regulatory activity on T cell function than atezolizumab.

[0136] Example 7 Preparation of anti-human PD-L1 humanized monoclonal antibody The anti-human PD-L1 humanized monoclonal antibody was obtained according to the method of Leung et al. (1995, Molecule Immunol 32:1413-27). From the GermLine database, a humanization template that best matched the variable region sequence of the mouse-derived antibody was selected, where the light chain variable region template was IGKV1-33*01, the sequence of which is shown in SEQ ID NO. 28. The heavy chain variable region template was IGHV1-69*01, the sequence of which is shown in SEQ ID NO. 29. The CDR regions of the mouse-derived antibody were grafted into the selected humanization template, replacing the CDR regions of the human template, to obtain the grafted humanized antibody light chain variable region and the grafted humanized antibody heavy chain variable region. Further backmutations at specific sites were performed in the light chain framework regions to obtain light chain variable region sequences shown in SEQ ID Nos. 38, 39, and 44. Further backmutations at specific sites were performed in the heavy chain framework regions to obtain heavy chain variable region sequences shown in SEQ ID Nos. 30-37 and 40-43. Mutations at the NG site in HCDR2 shown in SEQ ID No. 24 were performed to remove deamidable sites, resulting in mutated heavy chain HCDR2s, the amino acid sequences of which are shown in SEQ ID Nos. 45-47. The heavy chain variable region sequences after mutating the HCDR2 of the heavy chain variable region shown in SEQ ID No. 36 are shown in SEQ ID Nos. 48-50. The heavy chain variable region sequences after mutating the HCDR2 of the heavy chain variable region shown in SEQ ID No. 41 are shown in SEQ ID Nos. 51-53. The light chain variable region and light chain constant region (sequence SEQ ID NO. 26) were linked to obtain the corresponding full-length light chain sequence, and the heavy chain variable region and heavy chain constant region (sequence SEQ ID NO. 27) were linked to obtain the corresponding full-length heavy chain sequence. The humanized sequences are shown in Table 7. Exemplary combinations of heavy chain variable region VH and light chain variable region VL in humanized antibodies are shown in Table 8. The affinity data (dissociation constants) of the humanized antibodies are shown in Table 9.

[0137] [Table 7]

[0138] [Table 8]

[0139] [Table 9]

[0140] Example 8 Thermal stability of anti-human PD-L1 humanized monoclonal antibodies A Waters Xbridge BHE200 3.5 μm, 7.8 mm x 30 cm chromatography column (catalog number: 186007640) was used. The mobile phase was 0.1 mol / L phosphate buffer (NaH2PO4-Na2HPO4), 0.1 mol / L sodium sulfate buffer, pH 6.7. Flow rate: 0.6 mL / min; column temperature: 25°C; sample cell temperature: 4°C; detection wavelength: 280 nm; sample was diluted to 1 mg / mL with sample buffer, and the sampling volume was 10 μL. Data processing was performed on the Agilent High-Performance Liquid Chromatography 1260 System Station, and the percentage of the main peak, which represents purity, was calculated using the area normalization method. To determine the thermal stability of these monoclonal antibodies, the above samples were placed at a high temperature of 40°C. Samples were taken at 2 and 4 weeks, and SE-HPLC detection was performed to observe the thermal stability. The results are shown in Table 10. All humanized anti-human PD-L1 antibodies, except for VLS10-VHS7 P2, showed good and comparable stability.

[0141] [Table 10]

[0142] Example 9 In vitro biological activity of anti-human PD-L1 humanized monoclonal antibodies The in vitro biological activities of anti-human PD-L1 humanized monoclonal antibodies were measured, including binding activity to human PD-L1, blocking activity for PD-L1 / PD-1 binding and PD-L1 / CD80, and binding kinetic constants to human PD-L1. The humanized sequences measured included VLS10-VHS7, VLS10-VHS7 P1, VLS10-VHS7 P2, VLS10-VHS7 P3, VLS15-VHS12, VLS15-VHS12 P1, VLS15-VHS12 P2, and VLS15-VHS12 P3, as well as the control atezolizumab and anti-human PD-L1 chimeric monoclonal antibodies. The specific experimental methods were the same as those used to measure the in vitro biological activity of anti-human PD-L1 chimeric monoclonal antibodies.

[0143] The experimental results are shown in Tables 11 and 12. Compared to the anti-human PD-L1 chimeric monoclonal antibodies, all of the tested humanized sequences maintained good activity and showed strong PD-L1 binding activity and PD-L1 / PD-1 and PD-L1 / CD80 blocking activity, with the VLS15-VHS12-P3 sequence being the most potent.

[0144] [Table 11]

[0145] [Table 12]

[0146] Example 10 Pharmacokinetic study of anti-human PD-L1 humanized monoclonal antibody in human PD-1 / human PD-L1 gene knock-in mice Six- to eight-week-old female human PD-1 / human PD-L1 double gene knockin mice (C57BL / 6 background, purchased from Beijing Biocytogen Biotechnology Co., Ltd.) were used. After one week of adaptation, the mice received a single intraperitoneal injection of the anti-human PD-L1 humanized monoclonal antibody VLS15-VHS12 P3 at a dose of 70 nmol / kg. Blood samples were collected from the retro-orbital venous plexus of two mice at each sampling point: 0 h, 6 h, 24 h, 72 h, 120 h, 168 h, 240 h, and 312 h after administration. The blood samples were left at room temperature for 30 to 1 hour without anticoagulation. After clotting, the blood was centrifuged at 3000 rpm for 10 minutes. The resulting serum samples were frozen and stored at -80°C for analysis.

[0147] The concentration of anti-human PD-L1 humanized monoclonal antibody in serum was measured by ELISA. Briefly, human recombinant PD-L1 protein was coated onto a high-binding ELISA plate using a carbonate buffer solution (pH 9.6). Coating was performed overnight at 4°C and washed with PBST. To prevent nonspecific binding, the plate was blocked with PBST containing 5% nonfat dry milk and washed with PBST. Test serum samples diluted with PBST containing 10% mixed mouse serum and 1% BSA were then added and incubated at 25°C for 1 hour, after which the plate was washed with PBST. Horseradish peroxidase-conjugated anti-human IgG antibody (Chemicon, catalog no. AP309P) diluted in PBST containing 5% nonfat dry milk was added and incubated at 25°C for 1 hour, after which the plate was washed with PBST. Finally, color development was performed using the chromogenic substrate TMB for 10 minutes at room temperature. Color development was stopped by adding 1M H2SO4. The absorbance at 450 nm was read on a microplate reader.

[0148] The results are shown in Figure 6. The anti-human PD-L1 humanized monoclonal antibody, administered intraperitoneally at a single dose of 70 nmol / kg, demonstrated excellent in vivo blood drug concentration-time curves and pharmacokinetic characteristics in human PD-1 / human PD-L1 double gene knock-in mice. The pharmacokinetic parameters of the anti-human PD-L1 humanized monoclonal antibody were as follows: half-life t 1 / 2 The area under the blood drug concentration-time curve, AUC 0-312hr was 77917 nM.hr; apparent volume of distribution V d was 147 mL / Kg; clearance CL was 0.78 mL / hr / kg; mean residence time MRT last was 90 hours.

[0149] Example 11 Antitumor Pharmacodynamic Study of Anti-Human PD-L1 Humanized Monoclonal Antibody in Human PD-1 / Human PD-L1 Co-Knockin Mice In this example, we investigated the inhibitory effect of anti-human PD-L1 humanized monoclonal antibodies on the growth of MC38 / human PD-L1 tumor xenografts inoculated into human PD-1 / human PD-L1 double gene knock-in mice.

[0150] Six- to eight-week-old female human PD-1 / human PD-L1 double gene knock-in mice (C57BL / 6 background, Beijing Biocytogen Biotechnology Co., Ltd.) were used as experimental materials. After allowing the mice to adapt to the environment for 1 week, 5 × 10 5 MC38 / human PD-L1 (Beijing Biocytogen Biotechnology Co., Ltd.), a mouse colorectal cancer cell line expressing engineered human PD-L1, was inoculated. Tumor volumes were approximately 150 mm. 3 When tumor volume reached 1000 mcg, mice were divided into groups of 6 mice each based on tumor volume, and assigned to either the vehicle control group or the anti-human PD-L1 humanized monoclonal antibody VLS15-VHS12 P3 treatment group. The mice were intraperitoneally administered at a dose of 70 nmol / kg twice a week for two consecutive weeks. From the day of administration, tumor volume was measured twice a week, and its major axis (a) and minor axis (b) were measured. Tumor volume was calculated using the following formula: tumor volume (mm 3 )=(a×b 2) / 2.

[0151] The results are shown in Figure 7. The anti-human PD-L1 humanized monoclonal antibody had anti-tumor activity and significantly inhibited the growth of MC38 / human PD-L1 xenograft tumors inoculated into human PD-1 / human PD-L1 double gene knock-in mice.

[0152] Finally, the above embodiments are intended to illustrate the technical solutions of the present invention, but are not intended to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments can still be modified or some or all of the technical features can be replaced with equivalents, and such modifications or replacements do not cause the essence of the corresponding technical solutions to depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An isolated anti-human PD-L1 antibody or antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprises an LCDR1 amino acid sequence set forth in SEQ ID No. 20, an LCDR2 amino acid sequence set forth in SEQ ID No. 21, and an LCDR3 amino acid sequence set forth in SEQ ID No. 22; The heavy chain variable region comprises an HCDR1 amino acid sequence set forth in SEQ ID No. 23, an HCDR2 amino acid sequence set forth in SEQ ID No. 24, 45, or 47, and an HCDR3 amino acid sequence set forth in SEQ ID No.

25. An anti-human PD-L1 antibody or an antigen-binding fragment thereof.

2. The anti-human PD-L1 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-human PD-L1 antibody or antigen-binding fragment thereof is a chimeric antibody, a humanized antibody, or a fully human antibody.

3. the heavy chain constant region sequence of the antibody is selected from the constant region sequences of any of human IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, and / or the light chain constant region sequence of the antibody is selected from a κ chain or a λ chain; The anti-human PD-L1 antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. The anti-human PD-L1 antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain constant region sequence is selected from IgG1 or IgG4 constant region sequences, and / or the light chain constant region sequence is selected from κ chain constant region sequences.

5. The anti-human PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the light chain variable region has the amino acid sequence shown in SEQ ID NO: 18, and the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:

19.

6. the light chain variable region framework region of the anti-human PD-L1 antibody or antigen-binding fragment thereof comprises FR-L1, FR-L2, FR-L3, and FR-L4, and the heavy chain variable region framework region comprises FR-H1, FR-H2, FR-H3, and FR-H4; The FR-L1 has the amino acid sequence shown in SEQ ID NO.

54. The FR-L2 has the amino acid sequence shown in SEQ ID NO. 55, or an amino acid sequence obtained by further introducing any one of the following substitutions or any combination thereof: the second amino acid Y is replaced by I, The third amino acid Q is replaced by H, The ninth amino acid, A, is replaced by S; The FR-L3 has the amino acid sequence shown in SEQ ID NO. 56, The FR-L4 has the amino acid sequence shown in SEQ ID NO. 57, The FR-H1 has the amino acid sequence shown in SEQ ID NO. 58, or an amino acid sequence obtained by further introducing any one of the following substitutions or any combination thereof: The first amino acid Q is replaced by E, The 23rd amino acid K is replaced by T, The FR-H2 has the amino acid sequence shown in SEQ ID NO. 59 or an amino acid sequence obtained by the following substitution: The 13th amino acid, M, is replaced with I; The FR-H3 has the amino acid sequence set forth in SEQ ID NO. 60, or an amino acid sequence obtained by any one of the following substitutions or any combination thereof: The second amino acid V is replaced by A, The eighth amino acid, E, is replaced by T; The 11th amino acid S is replaced with N, the 31st amino acid A is substituted with G, and / or The anti-human PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the FR-H4 has the amino acid sequence shown in SEQ ID NO.

61.

7. (a) the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 38; the heavy chain variable region has an amino acid sequence selected from SEQ ID NO: 30-37; or (b) the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 39; the heavy chain variable region has an amino acid sequence selected from SEQ ID NOs. 30-37, 48, or 50; or (c) the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 44; The anti-human PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the heavy chain variable region has an amino acid sequence selected from SEQ ID NOs. 40-43 or 51-53.

8. (a) the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 39; the heavy chain variable region has an amino acid sequence selected from SEQ ID NO. 36, 48, or 50; or (b) the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 44; The anti-human PD-L1 antibody or antigen-binding fragment thereof according to claim 7, wherein the heavy chain variable region has an amino acid sequence selected from SEQ ID NO. 41 or 51-53.

9. The antigen-binding fragment is F(ab') ２ , Fab', Fab, Fd, Fv, scFv, bispecific antibody, camel antibody, CDR, and the smallest antibody recognition unit (dAb), The anti-human PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. The anti-human PD-L1 antibody or antigen-binding fragment thereof according to claim 9, wherein the antigen-binding fragment is Fab, F(ab')2, or scFv.

11. (1) DNA or RNA encoding the anti-human PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10; or (2) A nucleic acid completely complementary to the nucleic acid defined in (1).

12. An expression vector comprising the nucleic acid molecules of claim 11 operatively ligated together.

13. A host cell comprising the nucleic acid molecule of claim 11 or the expression vector of claim 12.

14. A composition comprising the anti-human PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1 to 10, the nucleic acid molecule of claim 11, the expression vector of claim 12, or the host cell of claim 13, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

15. Culturing the host cell of claim 13 under culture conditions suitable for expression of the anti-human PD-L1 antibody or antigen-binding fragment thereof. A method for producing the anti-human PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

16. 16. The method of claim 15, wherein after said culturing, the resulting product is isolated and purified.

17. Use of the PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1 to 10, the nucleic acid molecule of claim 11, the expression vector of claim 12, or the host cell of claim 13 in the manufacture of a medicament for preventing and / or treating a PD-L1-mediated disease or condition, The disease or condition is a tumor.

18. 18. The use according to claim 17, wherein the tumor is one or more selected from leukemia, lymphoma, myeloma, brain tumor, squamous cell carcinoma of the head and neck, non-small cell lung cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, pancreatic adenocarcinoma, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, and melanoma.

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