Antibodies and antigen-binding fragments thereof that specifically bind pd-l1

Abstract

The application discloses an antibody and an antigen-binding fragment thereof specifically binding to PD-L1. The antibody and the antigen-binding fragment thereof specifically binding to PD-L1 are high in specificity and stability, can enhance secretion of IFN-gamma, have strong T cell function regulation activity, and can significantly inhibit growth of tumors in vivo.

Description

Technical Field

[0001] This invention relates to the field of antibody and antibody humanization research, and more specifically, to an antibody capable of specifically binding to PD-L1 and its antigen-binding fragment. Background Technology

[0002] Programmed death-1 (PD-1) is an immune checkpoint molecule that mainly participates in the regulation of T cell activation and can regulate the strength and duration of the immune response. Under normal circumstances, PD-1 can mediate and maintain the body's self-immune tolerance, prevent the immune system from being over-activated and damaging its own tissues during the inflammatory response, and play a positive role in avoiding the occurrence of autoimmune diseases. Under pathological conditions, it participates in the development of tumor immunity and various autoimmune diseases (Anticancer Agents MedChem. 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 is inducibly expressed on the surface of various immune cells, including T cells, B cells, monocytes, macrophages, dendritic cells (DCs), endothelial cells, and epidermal cells. PD-L2, on the other hand, is only inducibly expressed on some 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, kidney cancer, breast cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, gastric cancer, pancreatic cancer, and colorectal cancer. PD-L2 is highly expressed in B-cell lymphoma. Tumor cells, through the high expression of PD-L1 or PD-L2, bind to PD-1 on T cells, transmitting immunosuppressive signals, which leads to immune tolerance to tumor cells, thus promoting tumor cell growth and metastasis. High expression of PD-1 ligand is closely related to poor prognosis and drug resistance in cancer patients (Hematol Oncol Stem Cell Ther. 2014 Mar; 7(1):1-17.). Further research has found that upregulated expression of PD-1 on the surface of T cells, especially T cells infiltrating tumor cells, is also closely related to poor prognosis (Trends Mol Med. 2015 Jan; 21(1):24-33.).

[0005] Multiple studies have demonstrated that antibodies blocking the PD-1 / PD-Ls signaling pathway have anti-tumor effects. Clinically, PD-1 / PD-Ls blocking antibodies have the following characteristics: First, their efficacy is not limited to a specific tumor type, but rather exhibits strong and sustained anti-tumor effects across a broad spectrum of tumors; second, PD-1 / PD-Ls blocking antibodies have good safety profiles, avoiding common side effects of chemotherapy and targeted therapies such as fatigue, leukopenia, baldness, diarrhea, and rashes, and instead producing only some immune-related side effects. PD-1 antibody nivolumab is marketed for the treatment of advanced melanoma, non-small cell lung cancer, renal cell carcinoma, and lymphoma. Pembrolizumab is marketed for the treatment of advanced melanoma, non-small cell lung cancer, and lymphoma. PD-L1 antibody atezolizumab is marketed for the treatment of advanced non-small cell lung cancer and urothelial carcinoma. Durvalumab is marketed for the treatment of advanced non-small cell lung cancer and urothelial carcinoma. Avelumab is marketed for the treatment of advanced Merkel cell carcinoma.

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

[0007] A first aspect of the invention relates to an isolated antibody against human programmed death ligand 1 (PD-L1), its antigen-binding fragment, or a variant thereof, wherein the antibody or its antigen-binding fragment comprises a light chain variable region and / or a heavy chain variable region, wherein

[0008] The amino acid sequences of the light chain variable regions LCDR1, LCDR2, and LCDR3 are shown in SEQ ID No. 20-22, respectively; and / or

[0009] The amino acid sequence of HCDR1 in the heavy chain variable region is shown in SEQ ID No. 23; the amino acid sequence of HCDR2 in the heavy chain variable region has at least about 94% identity with any one of SEQ ID No. 24 or 45-47; the amino acid sequence of HCDR3 in the heavy chain variable region is shown in SEQ ID No. 25; or

[0010] The amino acid sequence of the CDR in the light chain variable region or heavy chain variable region shown has at least 70% identity with the sequence shown in SEQ ID No. 20-25 or 45-47 and retains the biological activity of the corresponding parent sequence, for example, at least 75%, 80%, 85%, 90%, 95% or higher identity; or

[0011] The amino acid sequences of the CDRs of the light chain variable region or the heavy chain variable region are variant sequences that retain the biological activity of the corresponding parent sequence after the sequences shown in SEQ ID NO: 20-25 or 45-47 are deleted, replaced and / or added by one or more amino acid residues. For example, one, two, three or more of these variant sequences are used.

[0012] The variants are selected from one of chimeric antibodies, humanized antibodies, or fully human antibodies.

[0013] In some embodiments, the heavy chain constant region sequence of the antibody is selected from the constant region sequence of one 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 the κ chain or the 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 light chain κ chain.

[0014] In some embodiments, the amino acid sequence of the light chain variable region of the PD-L1 chimeric antibody and its antigen-binding fragment is as shown in SEQ ID NO. 18; or has at least 70% identity with the sequence shown in SEQ ID No. 18 and retains the biological activity of the corresponding parent sequence, for example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity; or is a variant sequence of the sequence shown in SEQ ID NO. 18 obtained by deleting, substituting and / or adding one or more amino acid residues, retaining the biological activity of the corresponding parent sequence, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more; and / or the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 19; or is similar to SEQ ID NO. 18. The sequence shown in SEQ ID NO. 19 has at least 70% identity and retains the biological activity of the corresponding parent sequence, for example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity; or is a variant sequence of the sequence shown in SEQ ID NO. 19 obtained by deleting, substituting and / or adding one or more amino acid residues, retaining the biological activity of the corresponding parent sequence, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more;

[0015] The amino acid sequences of the light chain constant region and heavy chain constant region of the PD-L1 chimeric antibody and its antigen-binding fragment are as shown in SEQ ID NO.26 and SEQ ID NO.27, respectively, or have at least 70% identity with the sequences shown in SEQ ID NO.26 and SEQ ID NO.27 and retain the biological activity of the corresponding parent sequences, for example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.4%, 99.7% or higher identity; or are variant sequences that retain the biological activity of the corresponding parent sequences obtained by deleting, substituting and / or adding one or more amino acid residues to the sequences shown in SEQ ID NO.26 and SEQ ID NO.27, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more.

[0016] In some embodiments, the light chain variable region backbone of the anti-human PD-L1 antibody, its antigen-binding fragment, or variants thereof includes FR-L1, FR-L2, FR-L3, and FR-L4, and the heavy chain variable region backbone includes FR-H1, FR-H2, FR-H3, and FR-H4, wherein...

[0017] The amino acid sequence of FR-L1 is shown in SEQ ID NO.54;

[0018] The amino acid sequence of FR-L2 is shown in SEQ ID NO.55, or is a further amino acid sequence obtained by one or any combination of the following substitutions:

[0019] The second amino acid Y is replaced with I.

[0020] The third amino acid, Q, is replaced with H.

[0021] The 9th amino acid, A, is replaced with S;

[0022] The amino acid sequence of FR-L3 is shown in SEQ ID NO.56;

[0023] The amino acid sequence of FR-L4 is shown in SEQ ID NO.57;

[0024] The amino acid sequence of FR-H1 is shown in SEQ ID NO.58, or is a further amino acid sequence obtained by one or a combination of the following substitutions:

[0025] The first amino acid Q is replaced with E.

[0026] The 23rd amino acid, K, is replaced with T.

[0027] The amino acid sequence of FR-H2 is shown in SEQ ID NO.59, or is an amino acid sequence obtained by the following substitutions:

[0028] The 13th amino acid M is replaced with I;

[0029] The amino acid sequence of FR-H3 is as shown in SEQ ID NO.60, or is an amino acid sequence obtained by one or any combination of the following substitutions:

[0030] The second amino acid, V, is replaced with A.

[0031] The 8th amino acid, E, is replaced with T.

[0032] The 11th amino acid, S, is replaced with N.

[0033] The 31st amino acid A is replaced with G; and / or

[0034] The amino acid sequence of FR-H4 is shown in SEQ ID NO.61.

[0035] In some embodiments, the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID No. 38, 39, or 44, and / or the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID No. 30-37, 40-43, or 48-53; or, a variant sequence having at least 70% identity with the amino acid sequence shown in SEQ ID No. 30-44 or 48-53 and retaining the biological activity of the corresponding parent sequence, for example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher identity; or, a variant sequence that retains the biological activity of the corresponding parent sequence obtained by deleting, substituting, and / or adding one or more amino acid residues to the sequence shown in SEQ ID No. 30-44 or 48-53, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more.

[0036] 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 NO. 30-37; or

[0037] 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. 30-37 or 48-50, or

[0038] The light chain variable region sequence has an amino acid sequence as shown in SEQ ID NO.44, and / or the heavy chain variable region sequence has an amino acid sequence selected from SEQ ID NO.40-43 or 51-53;

[0039] 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,

[0040] The light chain variable region sequence has an amino acid sequence as 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;

[0041] 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.

[0042] As is known in the art, when referring to identity, since the length of an amino acid sequence can only be a natural number, the actual calculated identity value may not be a finite percentage like 95%, but rather a number close to a percentage like 95%. For example, for a variable region sequence of 117 amino acid residues, when only one amino acid residue changes, the corresponding identity percentage is actually close to 99.15%, but for convenience, such a number is simply labeled as 99% in this article.

[0043] In some embodiments, the antigen-binding fragment is selected from one or more of F(ab')2, Fab', Fab, Fd, Fv, scFv, bispecific antibodies, camel antibodies, CDR, and antibody minimum recognition unit (dAb). Preferably, the antigen-binding fragment is Fab, F(ab')2, or scFv.

[0044] A second aspect of the invention relates to an isolated nucleic acid molecule selected from:

[0045] (1) DNA or RNA encoding the anti-human PD-L1 antibody described in the first aspect, its antigen-binding fragment or a variant thereof;

[0046] (2) Nucleic acids that are completely complementary to DNA or RNA as defined in (1).

[0047] A third aspect of the invention relates to a vector comprising a nucleic acid molecule effectively linked according to the second aspect, preferably an expression vector.

[0048] A fourth aspect of the invention relates to a host cell comprising the nucleic acid molecule described in the second aspect or the vector described in the third aspect.

[0049] The fifth aspect of the invention relates to a composition comprising the anti-human PD-L1 antibody described in the first aspect, its antigen-binding fragment or a variant thereof, the nucleic acid molecule described in the second aspect, the carrier described in the third aspect or the host cell described in the fourth aspect, and a pharmaceutically acceptable carrier, diluent or excipient.

[0050] A sixth aspect of the present invention relates to a method for producing the anti-human PD-L1 antibody described in the first aspect, its antigen-binding fragment, or a variant thereof, comprising the steps of:

[0051] The host cells described in the fourth aspect are expressed under culture conditions suitable for the expression of the anti-human PD-L1 antibody, its antigen-binding fragment or a variant thereof, and optionally, the resulting product is isolated and purified.

[0052] The seventh aspect of the invention relates to the use of the anti-human PD-L1 antibody described in the first aspect, its antigen-binding fragment or a variant thereof, the nucleic acid molecule described in the second aspect, the vector described in the third aspect, or the host cell described in the fourth aspect in the preparation of a medicament for the prevention and / or treatment of PD-L1-mediated diseases or conditions such as autoimmune diseases, immune responses to grafts, allergic reactions, infectious diseases, neurodegenerative diseases, and tumors.

[0053] The eighth aspect of the present invention relates to the anti-human PD-L1 antibody described in the first aspect, its antigen-binding fragment or a variant thereof, the nucleic acid molecule described in the second aspect, the vector described in the third aspect or the host cell described in the fourth aspect, for the prevention and / or treatment of PD-L1-mediated diseases or conditions such as autoimmune diseases, immune responses to grafts, allergic reactions, infectious diseases, neurodegenerative diseases and tumors.

[0054] The ninth aspect of the present invention relates to a method for preventing and / or treating PD-L1-mediated diseases or conditions such as autoimmune diseases, immune responses to grafts, allergic reactions, infectious diseases, neurodegenerative diseases, and tumors, comprising administering to a subject in need an anti-human PD-L1 antibody as described in the first aspect, an antigen-binding fragment thereof, or a variant thereof, a nucleic acid molecule as described in the second aspect, a vector as described in the third aspect, or a host cell as described in the fourth aspect.

[0055] In some embodiments, the autoimmune disease is selected from one or more of arthritis, rheumatoid arthritis, psoriasis, multiple sclerosis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus, glomerulonephritis, dilated cardiomyopathy-like disease, Sjögren's syndrome, allergic contact dermatitis, polymyositis, scleroderma, periarteritis, rheumatic fever, vitiligo, insulin-dependent diabetes mellitus, Behcet's syndrome, and chronic thyroiditis; preferably, the autoimmune disease is selected from one or more of 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 selected from one or more of rheumatoid arthritis, psoriasis, multiple sclerosis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus, insulin-dependent diabetes mellitus, and chronic thyroiditis.

[0056] In some implementations, the immune response against the graft includes, for example, graft-versus-host disease.

[0057] In some embodiments, the allergic reaction is selected from one or more of urticaria, eczema, angioedema, allergic rhinitis, bronchial asthma, laryngeal edema, food allergic gastroenteritis, and anaphylactic shock; preferably, the allergic reaction is selected from one or more of urticaria, eczema, allergic rhinitis, bronchial asthma, and anaphylactic shock; more preferably, the allergic reaction is selected from one or more of allergic rhinitis, bronchial asthma, and anaphylactic shock.

[0058] In some implementations, the infectious disease refers to a local and systemic inflammatory response caused by pathogens such as viruses, bacteria, fungi, and parasites, as well as specific toxins, invading the human body. Examples of pathogenic viruses include, for example, HIV, hepatitis A, B, and C viruses, herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, EB virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, Coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and vector-borne encephalitis virus. Pathogenic bacteria include, for example, syphilis, chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, diphtheria bacillus, Salmonella, Bacillus, cholera bacillus, tetanus bacillus, botulinum toxin, anthrax bacillus, plague bacillus, leptospirosis bacillus and Lyme bacillus. Pathogenic fungi include, for example, Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc., Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucorales (Mucor, Bsidia, Rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.Pathogenic parasites include, for example, Entamoeba histolytica, Balantidium coli, Naegleria fowleri, species of Acanthamoebasp., Giardialambia, species of Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.

[0059] In some embodiments, the neurodegenerative disease is selected from one or more of Parkinson's disease, Huntington's disease, Machado-Joseph disease, amyotrophic lateral sclerosis (ALS), and Kreutzfeldt-Jacob's disease; preferably, the neurodegenerative disease is selected from one or more of Parkinson's disease, Huntington's disease, and ALS; more preferably, the neurodegenerative disease is selected from one or more of Parkinson's disease and Huntington's disease.

[0060] In some embodiments, the tumor is selected from one or more of leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, small cell lung cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, pancreatic cancer, 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 selected from lymphoma, myeloma, head and neck squamous cell carcinoma, and other similar tumors. The tumor is selected from one or more of the following: squamous cell carcinoma, 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 selected from one or more of the following: 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.

[0061] In some implementations, the subjects are selected from mammals, including but not limited to humans and / or other primates; mammals include commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice and / or rats.

[0062] In some embodiments, the anti-human PD-L1 antibody of the present invention, its antigen-binding fragment or a variant thereof, nucleic acid molecule, vector or host cell is used in accordance with conventional administration methods in the art, such as parenteral route, such as intravenous injection.

[0063] The anti-PD-L1 monoclonal antibody of this invention exhibits high specificity, good stability, strong T-cell function regulation activity, and favorable pharmacokinetic characteristics, and can significantly inhibit tumor growth in vivo. Furthermore, the anti-PD-L1 monoclonal antibody of this invention demonstrates stronger blocking activity against PD-L1 / PD-1 binding than atezolizumab (see...). Figure 1 B). Attached Figure Description

[0064] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0065] Figure 1 This represents the in vitro activity of the anti-human PD-L1 murine monoclonal antibody secreted by clones 34-35 in Example 1 of this invention. Wherein A represents the binding activity of the murine monoclonal antibody to PD-L1; and B represents the blocking activity of the murine monoclonal antibody against PD-L1 / PD-1 binding.

[0066] Figure 2 The binding activity of the anti-human PD-L1 chimeric monoclonal antibody to human PD-L1 in Example 3 of this invention.

[0067] Figure 3 This represents the binding specificity of the anti-human PD-L1 chimeric monoclonal antibody in Example 4 of this invention. Wherein A represents species binding specificity; B represents target binding specificity.

[0068] Figure 4 This refers to the blocking activity of the anti-human PD-L1 chimeric monoclonal antibody against PD-L1 / PD-1 binding in Example 5 of this invention.

[0069] Figure 5 This refers to the T-cell function regulation activity of the anti-human PD-L1 chimeric monoclonal antibody in Example 6 of the present invention.

[0070] Figure 6This is the drug-time curve of mice after a single intraperitoneal injection of the humanized anti-human PD-L1 monoclonal antibody in Example 9 of the present invention.

[0071] Figure 7 This invention relates to the in vivo antitumor efficacy of the humanized anti-PD-L1 monoclonal antibody in Example 10 of the present invention. Detailed Implementation

[0072] definition

[0073] The term “PD-L1” refers to programmed death ligand-1, also known as CD274 and B7H1, which means any natural PD-L1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats).

[0074] As used herein, the terms “anti-PD-L1 antibody,” “anti-PD-L1,” “PD-L1 antibody,” or “PD-L1-binding antibody” refer to antibodies capable of binding to the PD-L1 protein or fragments thereof with sufficient affinity. In some embodiments, anti-PD-L1 antibodies bind to conserved PD-L1 epitopes in PD-L1 from different species.

[0075] The term "antibody" refers to an immunoglobulin molecule or a segment of an immunoglobulin molecule that has the ability to bind to an epitope of an antigen. Naturally occurring antibodies typically consist of tetramers, usually composed of at least two heavy (H) chains and at least two light (L) chains. Immunoglobulins include the following isotypes: IgG, IgA, IgM, IgD, and IgE, with their corresponding heavy chains being γ, α, μ, δ, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of their hinge regions and the number and position of disulfide bonds in their heavy chains, different subclasses can be distinguished. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4 subtypes, and IgA into IgA1 and IgA2 subtypes. Light chains are classified into κ and λ chains based on their constant regions.

[0076] In this article, the term “antibody” is used in the broadest sense to refer to a protein that contains an antigen-binding site, encompassing natural and artificial antibodies of various structures, including but not limited to complete antibodies and antigen-binding fragments of antibodies.

[0077] "Variable regions" or "variable domains" are structural domains in the heavy or light chains of an antibody that participate in the binding of the antibody to its antigen. Each heavy chain of an antibody consists of a heavy chain variable region (VH) and a heavy chain constant region (CH), the latter typically consisting of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The heavy and light chain variable regions are typically responsible for antigen recognition, while the heavy and light chain constant domains 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 the antigen. The VH and VL regions can be further subdivided into hypervariable regions (HVR) called "complementarity-determining regions (CDRs)," interspersed with more conserved regions called "backbone regions" (FRs). Each VH and VL consists of three CDR domains and four FR domains, arranged in the following order from the amino terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0078] The terms “complementarity-determining region” or “CDR region” (which may be used interchangeably with “HVR” in this document) refer to regions within the antibody variable domain that are sequence-hypervariant and form structurally defined loops (“hypervariant loops”) and / or contain antigen contact residues (“antigen contact sites”). CDRs are primarily responsible for binding to antigen epitopes. In this document, 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.

[0079] It should be noted that, based on different assignment systems (such as...), The boundaries of the CDRs (Continuous Dependent Lines) of the variable region of the same antibody obtained by Kabat and Chothia may differ. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems may differ. Therefore, when referring to antibodies defined by the specific CDR sequence of this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from the specific CDR boundaries defined by this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0080] The terms "monoclonal antibody," "monoclonal antibody," or "monoclonal antibody composition" refer to a single-molecule composition of antibody molecules, obtained from a group of substantially homogeneous antibodies, meaning that the group of individual antibodies is identical except for the possible small number of naturally occurring mutations. Conventional monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope. In some embodiments, a monoclonal antibody may consist of more than one Fab domain, thereby increasing specificity for more than one target. The terms "monoclonal antibody" or "monoclonal antibody composition" are not limited to any specific method of production (e.g., recombinant, transgenic, hybridoma, etc.).

[0081] The terms "bispecific antibody," "bifunctional antibody," "bispecific antibody," or "BsAb (bispecific antibody)" refer to antibodies that have two different antigen-binding sites. They can bind to two target antigens simultaneously, and while exerting antibody targeting, they also mediate another specific function. The mediated specific function effector molecule can also be a toxin, enzyme, cytokine, radionuclide, etc. The two arms of a bispecific antibody binding antigen can be derived from Fab, Fv, ScFv, or dSFv, etc.

[0082] The term "polyclonal antibody" refers to a preparation that includes different antibodies against different determinants ("epitopes").

[0083] The term "antigen-binding fragment of an antibody" refers to a fragment, portion, region, or domain of an antibody capable of binding to an epitope (e.g., obtainable via cleavage, recombination, synthesis, etc.). An antigen-binding fragment may contain 1, 2, 3, 4, 5, or all six CDR domains of that class of antibody, and may exhibit different specificities, affinities, or selectivity despite being able to bind to the epitope. Preferably, the antigen-binding fragment contains all six CDR domains of the antibody. An antigen-binding fragment of an antibody may be part of or comprise a single polypeptide chain (e.g., scFv), or may be part of or comprise two or more polypeptide chains (each having an amino terminus and a carboxyl terminus) (e.g., biantibody, Fab fragment, F(ab')2 fragment, etc.).

[0084] Examples of antigen-binding fragments included in this invention include (a) Fab' or Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (b) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds in a hinge domain; (c) Fd fragments consisting of a VH region and a CH1 domain; (d) Fv fragments consisting of the VL and VH regions of an antibody arm; (e) single-chain antibodies (scFv), recombinant proteins formed by linking antibodies VH and VL via a linker peptide using genetic engineering methods; (f) dAb fragments (Ward et al., Nature, 341, 544-546 (1989)), which are essentially composed of the VH region and are also called domain antibodies (Holt et al., Trends Biotechnol., 2i(ll):484-90); and (g) camel or nanobodies (Revets et al., Expert Opinion Biol.). Ther., 5(l):111-24) and (h) separated complementarity-determining regions (CDRs).

[0085] The term "chimeric antibody" refers to an antibody whose heavy chain and / or light chain contains a portion of a sequence identical or homologous to the corresponding sequence of an antibody from a specific species or belonging to a specific antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody from another species or belonging to another antibody class or subclass, or fragments of such antibodies, provided they exhibit the desired biological activity. This invention provides variable region antigen-binding sequences from human antibodies. Therefore, chimeric antibodies of primary interest herein include antibodies having one or more human antigen-binding sequences (such as CDRs) and containing one or more sequences from non-human antibodies, such as FR or C region sequences. Furthermore, the chimeric antibodies described herein are antibodies comprising a human variable region antigen-binding sequence of one antibody class or subclass and another sequence from another antibody class or subclass, such as an FR or C region sequence.

[0086] The term "humanized antibody" refers to an antibody in which a CDR sequence derived from another mammalian species, such as a mouse, has been grafted onto a human frame sequence. Additional frame region modifications can be performed within the human frame sequence.

[0087] The term "human antibody" or "fully human antibody" ("humAb" or "HuMab") refers to an antibody that includes variable and constant domains derived from human immunoglobulin sequences. The human antibodies of this invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-specific mutagenesis, or during gene rearrangement, or in vivo by somatic mutations).

[0088] Variant antibodies are also included within the scope of this invention. Therefore, variants of the sequences listed in this application are also included within the scope of this 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 this invention. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can be used to improve the translation efficiency of the expression system in antibody production.

[0089] Such variant antibody sequences have 70% or more (e.g., 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher) sequence homology with the sequences listed in the application. Such sequence homology is calculated relative to the full length of the reference sequence (i.e., the sequence listed in the application).

[0090] The amino acid residues in this invention are numbered according to (the internationalImMunoGeneTics information (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, U.S. 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 in this invention are numbered according to the Kabat numbering system.)

[0091] An antibody or its antigen-binding fragment "specifically" binds to a region of another molecule (i.e., an epitope) meaning that it reacts or binds to that epitope more frequently, more rapidly, for a longer duration, and / or with greater affinity or cohesion than to another epitope. In some embodiments, the antibody or its antigen-binding fragment of the present invention binds to at least 10 -7 M has an affinity for human PD-L1, for example 10 -8 M, 10 -9 M, 10 -10 M, 10 -11M or higher. Preferably, the antibody or its antigen-binding fragment binds under physiological conditions (e.g., in vivo). Therefore, specific binding to PD-L1 refers to the ability of the antibody or its antigen-binding fragment to bind to PD-L1 with the specificity described above and / or under such conditions. Suitable methods for determining said binding are known in the art.

[0092] In the context of antibody binding to a designated antigen, the term "binding" generally refers to binding to an antigen corresponding to approximately 10-10. -6 M or smaller K D The affinity combination of this K D The antibody has an affinity for binding to non-specific antigens (e.g., BSA, casein) other than the specified antigen or closely related antigens, that is at least 10-fold lower, such as at least 100-fold lower or at least 1,000-fold lower.

[0093] As used in this article, the term "k" d (sec⁻¹ or 1 / s) refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also known as k. off value.

[0094] As used in this article, the term "k" a "(M-1x sec-1 or 1 / Msec) refers to the binding rate constant of a specific antibody-antigen interaction."

[0095] As used in this article, the term "K" D (M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction and is expressed by k. d Divide by k a get.

[0096] As used in this article, the term "K" A (M-1 or 1 / M) refers to the binding equilibrium constant of a specific antibody-antigen interaction and is expressed by k. a Divide by k d get.

[0097] In some embodiments, the antibody or its antigen-binding fragment of the present invention requires only a portion of the CDR (i.e., the subset of CDR residues required for binding, referred to as SDR) to remain bound in the humanized antibody. CDR residues that do not contact the relevant epitope and are not in the SDR can be identified by molecular modeling and / or empirically, or as described in Gonzales, NR et al., (2004), SDR Grafting Of A Murine Antibody Using Multiple Human Germline Templates To Minimize Its Immunogenicity, Mol. Immunol., 41:863-872, based on previous studies (e.g., residues H60-H65 in CDR H2 are generally not needed). These residues can be identified from the Kabat CDR region located outside the Chothia hypervariable ring (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 the entire donor CDR is omitted, the amino acid occupying that position can be an amino acid that occupies the corresponding position in the recipient antibody sequence (by Kabat numbering). Such substitutions are potentially advantageous in reducing the number of mouse amino acids in the humanized antibody and thus reducing potential immunogenicity. However, substitutions can also cause changes in affinity, and a significant reduction in affinity is preferably avoided. The substitution positions within the CDR and the amino acids to be substituted can also be selected empirically.

[0098] The fact that a single amino acid alteration of a CDR residue can lead to 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) allows for the systematic identification of alternative functional CDR sequences. In a preferred method for obtaining such variant CDRs, the polynucleotide encoding the CDR is mutagenized (e.g., via random mutagenesis or by site-directed methods) to produce a CDR with substituted amino acid residues. The BLOSUM62.iij substitution score can be identified by comparing the identity of the relevant residues in the original (functional) CDR sequence with the identity of the substituted (non-functional) variant CDR sequence. The BLOSUM system provides amino acid substitution matrices created by analyzing sequence databases for reliable alignment (Eddy, SR, (2004), Where Did The BLOSUM62 AlignmentScore 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 presents the substitution scores of BLOSUM62.iij (the higher the score, the more conservative the substitution, and therefore the more likely that the substitution will not affect functionality).For example, if the antigen-binding fragment containing the resulting CDR cannot bind to PD-L1, the BLOSUM62.iij substitution score is considered insufficiently conserved, and new candidate substitutions with higher substitution scores are selected and generated. Therefore, for example, if the original residue is glutamic acid (E) and the nonfunctional substituted residue is histidine (H), the BLOSUM62.iij substitution score will be 0, and more conserved variations (such as to aspartic acid, asparagine, glutamine, or lysine) are preferred.

[0099]

[0100]

[0101] This invention therefore considers the use of random mutagenesis for identifying improved CDRs. In the context of this invention, conservative substitutions can be defined by substitutions within amino acid classes reflected in one or more of the following three tables:

[0102] Classes of conserved substituted amino acid residues:

[0103]

[0104] Category of alternative conserved amino acid residue substitutions:

[0105]

[0106]

[0107] Classification of amino acid residues by physical and functional substitution:

[0108]

[0109] More conservative substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0110] In some implementations, the hydrophilic amino acid is selected from Arg, Asn, Asp, Gln, Glu, His, Tyr, and Lys.

[0111] Other amino acid groups can also be formulated using principles described, for example, Creighton (1984), Proteins: Structure and Molecular Properties (WH Freeman and Company).

[0112] Therefore, the sequence of the CDR variant of the contained antibody or its antigen-binding fragment can differ from the sequence of the parent antibody's CDR by substitution; for example, substitution of 4, 3, 2, or 1 amino acid residues. According to embodiments of the invention, the amino acids in the CDR region can be substituted with conserved substitutions, as defined in the three tables above.

[0113] "Homology" or "sequence identity" refers to the percentage of residues in a polynucleotide or polypeptide sequence variant that are identical to those in a non-variant sequence after sequence alignment and nick introduction. In specific embodiments, the 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 polynucleotide or polypeptide described herein.

[0114] Such variant polypeptide sequences have 70% or more (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity with the sequences listed in the application. In other embodiments, the invention provides polypeptide fragments comprising consecutive extensions of various lengths of the amino acid sequences disclosed herein. For example, where applicable, the peptide sequences provided by the invention comprise at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150 or more of one or more sequences disclosed herein, as well as all intermediate length peptides therebetween.

[0115] The term "treatment" refers to improving, slowing, reducing, or reversing the progression or severity of a disease or condition, or improving, slowing, reducing, or reversing one or more symptoms or side effects of such a disease or condition. In this invention, "treatment" also refers to a method for obtaining a beneficial or desired clinical outcome, wherein "beneficial or desired clinical outcome" includes, but is not limited to, symptom relief, reduction of symptoms or disease severity, stabilization (i.e., no worsening) of a disease or condition, delay or slowing of the progression of a disease or condition, improvement or reduction of a disease or condition, and remission of a disease or condition, whether partial or complete, detectable or undetectable.

[0116] The term "prevention" refers to the application of the antibodies and antigen-binding fragments of the present invention to prevent or inhibit the development of at least one symptom of a disease or condition. The term also includes treatment of subjects in remission to prevent or inhibit relapse.

[0117] The antibodies of the present invention can have any isotype. The choice of isotype is generally determined by the desired effector function (e.g., ADCC induction). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either the human light chain constant domain κ or λ can be used. If desired, the class of the anti-PD-L1 antibody of the present invention can be converted by known methods. For example, the antibody of the present invention, which is originally IgG, can be class-converted to the IgM antibody of the present invention. Furthermore, class-conversion techniques can be used to convert one IgG subclass to another, for example, from IgG1 to IgG2. Thus, the effector function of the antibody of the present invention can be changed by isotype switching to, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies for various therapeutic uses. In some embodiments, the antibody of the present invention is an IgG2 antibody, such as IgG2a. An antibody belongs to a specific isotype if its amino acid sequence is substantially homologous to that isotype relative to other isotypes.

[0118] In some embodiments, the antibody of the present invention is a full-length antibody, preferably an IgG antibody. In other embodiments, the antibody of the present invention is an antibody-antigen binding fragment or a single-chain antibody.

[0119] In some embodiments, the anti-PD-L1 antibody is a monovalent antibody, preferably a monovalent antibody with a hinge region deletion as described in WO 2007059782 (which is incorporated herein by reference in its entirety). Therefore, 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 a light chain of the monovalent antibody, the construct comprising a nucleotide sequence encoding a VL region encoding a selected antigen-specific anti-PD-L1 antibody and a nucleotide sequence encoding a constant CL region encoding Ig, wherein the nucleotide sequence encoding the VL region of the selected antigen-specific antibody and the nucleotide sequence encoding the CL region are effectively linked together, and wherein, in the case of the IgG1 subtype, the nucleotide sequence encoding the CL region has been modified such that, in the presence of polyclonal human IgG or when administered to an animal or human, the CL region does not contain any amino acids capable of forming disulfide or covalent bonds with other peptides containing the same amino acid sequence as the CL region; ii) providing a nucleic acid construct encoding a heavy chain of the monovalent antibody, the construct comprising encoding a... The nucleotide sequence of the VH region of the selected antigen-specific antibody and the nucleotide sequence of the constant CH region encoding human Ig, wherein the nucleotide sequence encoding the CH region has been modified such that, in the presence of polyclonal human IgG or when administered to an animal, the regions corresponding to the hinge region and other regions of the CH region (such as the CH3 region, as required by the Ig subtype) do not contain any amino acid residues that participate in forming disulfide bonds or covalent or stable non-covalent heavy chain bonds with other peptides that form a consistent amino acid sequence of the CH region of human Ig, wherein the nucleotide sequence encoding the VH region of the selected antigen-specific antibody and the nucleotide sequence encoding the CH region of said Ig are effectively linked together; iii) providing a cell expression system for producing a monovalent antibody; iv) producing said monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in the cells of the cell expression system of (iii).

[0120] Similarly, in some implementations, the anti-PD-L1 antibody is a monovalent antibody that comprises:

[0121] (i) the variable region or antigen-binding portion of the antibody of the present invention as described herein, and

[0122] (ii) The CH region of an immunoglobulin or a domain containing CH2 and CH3 domains, wherein the CH region or its domains have been modified such that the domains corresponding to the hinge region and (if the immunoglobulin is not IgG4 subtype) other domains of the CH region (such as 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 heavy chain bonds with the same CH region in the presence of polyclonal human IgG.

[0123] In some other implementations, the heavy chain of the monovalent antibody is modified to remove the entire hinge region.

[0124] In another embodiment, the sequence of the monovalent antibody is modified so that it does not contain any receptor sites for N-linked glycosylation.

[0125] The present invention also includes "bispecific antibodies" wherein 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 targeting more than one epitope (e.g., the second epitope may include an epitope of an active transport receptor, thus enabling the bispecific antibody to exhibit improved transcellular transport across biological barriers (e.g., the blood-brain barrier), or the second epitope is an epitope targeting another target protein). Therefore, in another embodiment, the monovalent Fab of the anti-PD-L1 antibody may be linked to another Fab or scfv targeting a different protein to generate a bispecific antibody. The bispecific antibody may have dual functions, such as therapeutic functions conferred by the anti-PD-L1 binding region and transport functions that can bind to receptor molecules to enhance transcellular transport across biological barriers (e.g., the blood-brain barrier).

[0126] The antibodies and antigen-binding fragments of the present invention also include single-chain antibodies. A single-chain antibody is a peptide in which the heavy and light chain Fv domains are linked. In some embodiments, the present invention provides a single-chain Fv (scFv) wherein the heavy and light chains in the Fv of the anti-PD-L1 antibody of the present invention are linked into a single peptide chain by flexible peptide linkers (typically about 10, 12, 15 or more amino acid residues). Methods for generating 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., PNASUSA 85, 5879-5883 (1988); and McCafferty et al., Nature, 348, 552-554 (1990). If only a single VH and VL are used, the single-chain antibody is monovalent; if two VHs and VLs are used, it is bivalent; or if more than two VHs and VLs are used, it is polyvalent.

[0127] The antibodies of the present invention can be generated using any technique known in the art, such as, but not limited to, any chemical, biological, genetic, or enzymatic techniques, either alone or in combination. Generally, knowing the amino acid sequence of the desired sequence, those skilled in the art can readily generate the antibodies using standard techniques for generating peptides. For example, these antibodies can be synthesized using known solid-phase methods, preferably using commercially available peptide synthesis equipment (such as those manufactured by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, the antibodies of the present invention can be synthesized using recombinant DNA techniques well known in the art. For example, after incorporating the DNA sequence encoding the antibody into an expression vector and introducing these vectors into a suitable eukaryotic or prokaryotic host for expressing the desired antibody, antibodies can be obtained as DNA expression products, which can then be isolated from the host using known techniques.

[0128] The antibodies and their antigen-binding fragments of the present invention can be modified by including any “suitable” number of modified amino acids and / or by conjugating substituents. In this case, “suitable” is generally determined by the ability to at least substantially retain the PD-L1 selectivity and / or PD-L1 specificity associated with the non-derived parental anti-PD-L1 antibody. Including one or more modified amino acids can be advantageous in, for example, increasing the serum half-life of the peptide, reducing the antigenicity of the peptide, or increasing the storage stability of the peptide. Modification of one or more amino acids can be performed, for example, concurrently with or after translation during recombinant production (e.g., N-linked glycosylation on the NXS / T motif during mammalian cell expression), or by synthetic means. Non-limiting examples of modified amino acids include glycosylated amino acids, sulfated amino acids, isoprene-modified (e.g., farnesylation, geranyl-geranylization) amino acids, acetylated amino acids, acylated amino acids, polyethylene glycol-modified amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, etc. References on amino acid modification are commonplace in the field; see, for example, Walker, (1998), Protein Protocols On CD-ROM, Humana Press, Totowa, New Jersey. Modified amino acids can be, for example, selected from glycosylated amino acids, polyethylene glycol-modified amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids coupled to lipid moieties, or amino acids coupled to organic derivatives.

[0129] The antibodies and their antigen-binding fragments of the present invention can also be chemically modified by covalent coupling to polymers to increase their cycling half-life. Exemplary polymers and methods of linking them to peptides are described, for example, in US 4,766,106; US 4,179,337; US 4,495,285 and US 4,609,546. Other exemplary polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., PEG with a molecular weight between about 1,000 and 40,000 D, such as PEG with a molecular weight between about 2,000 and 20,000 D, such as PEG with a molecular weight between about 3,000 and 12,000 D).

[0130] The term "subject" refers to a warm-blooded animal, preferably a mammal (including humans, livestock and farm animals, zoo animals, sporting animals, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.), and more preferably a human. In one embodiment, the subject may be a "patient," i.e., a warm-blooded animal, more preferably a human, who is awaiting or receiving medical care or will be the subject of a medical procedure or disease development monitoring. In one embodiment, the subject is an adult (e.g., a subject aged 18 years or older). In another embodiment, the subject is a child (e.g., a subject under 18 years of age). In one embodiment, the subject is male. In another embodiment, the subject is female.

[0131] In one embodiment of the invention, the sample is a biological sample. Examples of biological samples include, but are not limited to, tissue lysates and extracts prepared from diseased tissues, 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 fluid, and alveolar macrophages.

[0132] In one embodiment of the invention, the term "sample" is intended to refer to a sample taken from an individual prior to any analysis.

[0133] Therefore, in some embodiments, the anti-PD-L1 antibody and its antigen-binding fragment of the present invention include intact antibodies, such as IgG (IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (IgA1 and IgA2 subtypes), IgD, IgM, and IgE; antigen-binding fragments such as SDR, CDR, Fv, dAb, Fab, Fab2, Fab', F(ab')2, Fd, scFv, camel antibodies, or nanobodies; and variant sequences of the antibody or its antigen-binding fragment, such as variant sequences having at least 80% sequence identity with the aforementioned antibody or its antigen-binding fragment. In some embodiments, the present invention also includes derivatives comprising anti-PD-L1 or its antigen-binding fragment, such as chimeric antibodies derived from intact antibodies, humanized antibodies, fully human antibodies, recombinant antibodies, and bispecific antibodies.

[0134] In another aspect, the present invention relates to expression vectors encoding one or more polypeptide chains of the antibody or antigen-binding fragment thereof of the present invention. Such expression vectors can be used for recombinant generation of the antibody or antigen-binding fragment thereof of the present invention.

[0135] In this invention, the expression vector can be any suitable DNA or RNA vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences containing a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, bacteriophage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and bacteriophage DNA, and viral nucleic acid (RNA or DNA) vectors. In some implementations, the nucleic acid encoding the anti-PD-L1 antibody is contained in a naked DNA or RNA vector, including, for example, linear expression elements (as described in, for example, Sykes and Johnston, Nat Biotech, 12, 355-59 (1997)), compact nucleic acid vectors (as described in, for example, US 6,077,835 and / or WO 00 / 70087), plasmid vectors (such as pBR322, pUC 19 / 18, or pUC 118 / 119), minimal-size nucleic acid vectors (as described in, for example, Schakowski et al., MoI Ther, 3, 793-800 (2001)), or as a precipitation-type nucleic acid vector construct, such as a CaPO4 precipitation construct (as described in, for example, 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 use are well known in the art (see, for example, US 5,589,466 and US 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).

[0136] In some implementations, the vector is suitable for expressing anti-PD-L1 antibodies or their antigen-binding fragments in bacterial cells. Examples of such vectors include, for example, BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem, 264, 5503-5509 (1989)), pET vectors (Novagen, Madison, Wisconsin), etc.

[0137] The expression vector can also be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system can be used. Suitable vectors include, for example, those containing constitutive or inducible promoters (such as α-factors, alcohol oxidases, and PGH) (reviewed in: 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 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); 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)).

[0138] In the expression vector of the present invention, the nucleic acid encoding the anti-PD-L1 antibody may contain or be bound to any suitable promoter, enhancer, and other elements that facilitate expression. Examples of such elements include strongly expressive promoters (e.g., the human CMV IE promoter / enhancer, as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), efficient poly(A) termination sequences, origins of replication for plasmid production in *E. coli*, antibiotic resistance genes as selective markers, and / or convenient cloning sites (e.g., multi-linker). The nucleic acid may also contain an inducible promoter as opposed to a constitutive promoter (such as CMV IE).

[0139] In another aspect, the present invention relates to recombinant eukaryotic or prokaryotic host cells (such as transfected tumors) that produce the antibodies of the present invention or antigen-binding fragments thereof, or bispecific molecules of the present invention. Examples of host cells include yeast, bacteria, and mammalian cells (such as CHO or HEK cells). For example, in some embodiments, the present invention provides cells comprising nucleic acids stably integrated into the cell genome containing nucleic acid sequences encoding the anti-PD-L1 antibody of the present invention or antigen-binding fragments thereof. In other embodiments, the present invention provides cells comprising non-integrated nucleic acids (such as plasmids, granules, phage particles, or linear expression elements) containing sequences encoding the anti-PD-L1 antibody of the present invention or antigen-binding fragments thereof.

[0140] The antibodies and their antigen-binding fragments of the present invention can be generated in different cell lines, such as human cell lines, non-human mammalian cell lines, and insect cell lines, such as CHO cell line, HEK cell line, BHK-21 cell line, mouse cell lines (e.g., myeloma cell lines), fibrosarcoma cell lines, PER.C6 cell line, HKB-11 cell line, CAP cell line, and HuH-7 human cell line (Dumont et al., 2015, Crit Rev Biotechnol., Sep. 18, 1-13., the contents of which are incorporated herein by reference).

[0141] The antibodies of the present invention are appropriately separated from the culture medium using conventional immunoglobulin purification methods, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0142] The present invention further relates to compositions comprising, consisting of, or substantially consisting of the antibodies of the present invention.

[0143] As used herein, with respect to a composition, "consistently of..." means that at least one of the present invention antibodies as described above is the only biologically active therapeutic agent or reagent in the composition.

[0144] In one embodiment, the composition of the present invention is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

[0145] The term "pharmaceuticalally acceptable carrier" refers to an excipient that, when administered to animals, preferably humans, does not produce adverse, allergic, or other adverse reactions. This includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents, etc. For human use, the formulation should meet the sterility, pyrogenicity, general safety, and purity standards required by regulatory agencies (such as the FDA office or EMA).

[0146] The present invention further relates to pharmaceuticals comprising, being composed of, or substantially composed of the antibodies of the present invention.

[0147] In some embodiments, the glycosylation of the antibodies of the present invention is modified. For example, glycosylated-free antibodies (i.e., antibodies lacking glycosylation) can be prepared. Glycosylation can be altered to, for example, increase the antibody's affinity for the antigen or alter the antibody's ADCC activity. Such carbohydrate modification 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, resulting in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such glycosylation can increase the antibody's affinity for the antigen. This method is further described in detail in U.S. Patents Nos. 5,714,350 and 6,350,861 (incorporated herein by reference). Alternatively or additionally, antibodies with altered glycosylation types can be prepared, such as low-fucosylated or non-fucosylated antibodies with reduced amounts or no fucosylation residues, or antibodies with increased bipartite GlcNac structures. Such altered fucosylation patterns have been shown to increase the ADCC ability of antibodies. This carbohydrate modification can be achieved, for example, by expressing antibodies in host cells with altered glycosylation mechanisms. Cells with altered glycosylation mechanisms have been described in the art and can be used as host cells in which the recombinant antibodies of the present invention are expressed, thereby producing antibodies with altered glycosylation. For example, EP1176195 by Hang et al. (incorporated herein by reference) describes a cell line with a functionally disrupted FUT8 gene encoding fucosyltransferase, such that antibodies expressed in such cell lines exhibit low fucosylation or lack of fucosyl residues. Therefore, in some embodiments, the human antibodies (preferably monoclonal antibodies) of the present invention can be produced by recombinant expression in cell lines exhibiting low or non-fucosylation patterns, for example, mammalian cell lines with expression of the FUT8 gene lacking fucosyltransferase. Presta's PCT publication WO 03 / 035835 (incorporated hereby by reference) describes a variant CHO cell line, Lecl3 cells, which has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, resulting in hypofucosylation of antibodies expressed in this host cell (see also Shields, RL et al. 2002 J. Biol. Chem. 277:26733-26740). Umana et al.'s PCT publication WO 99 / 54342 (incorporated hereby by reference) describes engineered cell lines to express glycoprotein-modified glycosyltransferases (e.g., β(1,4)-N-acetylglucosamine aminotransferase III (GnTIII)), resulting in antibodies expressed in engineered cell lines exhibiting increased bipartite GlcNac structures, leading to increased ADCC activity of the antibodies (see also Umana et al. 1999 Nat. Biotech. 17:176-180).Eureka Therapeutics further describes genetically engineered CHO mammalian cells capable of producing antibodies with a mammalian glycosylation pattern exhibiting a deficiency of 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 that are used for mammalian-like glycosylation patterns and are capable of producing antibodies lacking fucose as a glycosylation pattern (see, for example, EP1297172B1).

[0148] The antibody of this invention acts on PD-L1, which participates in the regulation of T cell activation and can modulate the strength and duration of the immune response. Under normal circumstances, PD-L1 can mediate and maintain the body's autoimmune tolerance, preventing excessive activation of the immune system during inflammatory responses and protecting the body's own tissues, thus playing a positive role in avoiding autoimmune diseases. Under pathological conditions, it participates in tumor immunity and the development of various autoimmune diseases. Clinical evidence shows that PD-L1 is highly expressed on the surface of various tumor cells, including melanoma, lung cancer, kidney cancer, breast cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, gastric cancer, pancreatic cancer, and colorectal cancer. Tumor cells, through high expression of PD-L1, bind to PD-1 or CD80 on T cells, transmitting immunosuppressive signals, leading to immune tolerance to tumor cells, which is conducive to tumor cell growth and metastasis. High expression of PD-L1 is closely related to poor prognosis and drug resistance in cancer patients. The PD-L1 antibody of the present invention enhances the anti-tumor immune response by blocking the PD-L1 / PD-1 and PD-L1 / CD80 signaling pathways, thereby exerting a broad-spectrum anti-tumor effect, including non-small cell lung cancer, urothelial carcinoma, Merkel cell carcinoma, melanoma, renal cell carcinoma, lymphoma, head and neck cancer, colorectal cancer, liver cancer, gastric cancer, etc.

[0149] When range values ​​are provided, it should be understood that, unless expressly stated otherwise, each interpolated value, one-tenth of a unit to the lower limit, the range between the upper and lower limits, and any other value or interpolated value within the range are included within the scope of this invention. The upper and lower limits of these smaller ranges, which may be independently included, are also included in this invention, provided that any limits specifically excluded from the range are removed. When the range includes one or both limits, the range excluding one or both of the included limits is also included in this invention.

[0150] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any similar or equivalent methods and materials described herein may also be used in the practice or testing of this invention, preferred methods and materials are now disclosed. All publications mentioned herein are incorporated herein by reference in their entirety.

[0151] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. One aspect of the embodiments provided by the present invention is to describe the antibody preparation process of the present invention. This preparation process is merely illustrative of the relevant methods and is not restrictive. Those skilled in the art will understand that many modifications can be made to the present invention without departing from its spirit, and such modifications also fall within the scope of the present invention. Another aspect of the embodiments provided by the present invention is to illustrate the features and advantages of the antibodies of the present invention, but the present invention is not limited to these features and advantages.

[0152] Unless otherwise specified, the experimental methods described below are conventional methods. Where specific conditions are not mentioned, they should be performed according to conventional conditions or the manufacturer's recommendations. Unless otherwise specified, the experimental materials used can be readily obtained from commercial companies. All antibodies used in the following embodiments of the present invention are derived from commercially available standard antibodies.

[0153] Example

[0154] Example 1: Preparation of mouse-derived anti-human PD-L1 monoclonal antibody

[0155] 1.1 Animal Immunization

[0156] Female BALB / c mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd. Mice were immunized one week after acclimatization. The initial immunization used 50 μg of recombinant human PD-L1-Fc protein (prepared by Beijing Hanmi Pharmaceutical Co., Ltd., where the human PD-L1 amino acid sequence is derived from Genebank #: NP_001254635.1) mixed thoroughly with Freund's complete adjuvant (Sigma-Aldrich, catalog number F5881) to form an emulsion, which was then injected intraperitoneally into the mice. Two weeks later, a booster immunization was performed. The booster immunization used 25 μg of recombinant human PD-L1-Fc protein mixed thoroughly with Freund's incomplete adjuvant (Sigma-Aldrich, catalog number F5806) to form an emulsion, which was then injected intraperitoneally into the mice. Booster immunizations were performed every 2 weeks using the same method, for a total of 3 booster immunizations. On day 10 after the final immunization, blood was collected from the retro-orbital venous plexus of the mice, and serum was separated by centrifugation. Antibody titers were determined by ELISA. Mice with high titers were selected for hybridoma fusion. Three days prior to fusion, 50 μg of recombinant human PD-L1-Fc protein (without adjuvant) was injected intraperitoneally. On the day of fusion, the spleen was aseptically harvested and a single-spleen cell suspension was prepared for fusion.

[0157] 1.2 Preparation of hybridoma cells

[0158] Log-growing myeloma cells SP2 / 0 were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in incomplete DMEM medium (Gibco, cat No. 11965) for counting. The desired number of cells were collected, and the cells were washed twice with the same incomplete culture medium. Simultaneously, an immunosuppressed spleen cell suspension was prepared and washed twice with incomplete culture medium. Myeloma cells and spleen cells were mixed at a ratio of 1:10 or 1:5, and washed once with incomplete culture medium in a 50 ml plastic centrifuge tube at 1200 rpm for 8 minutes. The supernatant was discarded, and any remaining liquid was aspirated with a dropper. The bottom of the centrifuge tube was gently tapped on the palm of the hand to loosen and evenly distribute the precipitated cells; the tube was then preheated in a 40°C water bath. Using a 1ml pipette, add 1ml of 45% PEG-4000 (pH 8.0, Sigma, CAT No. P7181) preheated to 40℃ over approximately 1 minute (optimal time is 45 seconds), stirring gently while adding. Visible particles should appear visually. Using a 10ml pipette, add 20-30ml of incomplete culture medium preheated to 37℃ over 90 seconds to terminate the PEG reaction; incubate at 20-37℃ for 10 minutes. Stir at 1000rpm for 5 minutes, discarding the supernatant. Add 5ml of HAT medium (DMEM+HAT, Sigma, CAT No. 1H0262-10VL), gently aspirate the precipitated cells (do not blow forcefully to avoid breaking up confluent cells), resuspend and mix, then add HAT medium to 80-100ml (to achieve a spleen cell concentration of 1-2 × 10⁶ cells / mL). 6 / ml). Dispense 0.1ml into each well of a 96-well cell culture plate; dispense 1.0–1.5ml into each well of a 24-well plate; then incubate the plates at 37°C in a 6% CO2 incubator. Generally, six 96-well plates are used. After 5 days, replace half of the medium with HAT medium. After another 7–10 days, replace the HAT medium with HT medium (DMEM+HT, Sigma cat No. H0137-10VL). Regularly observe the growth of hybridoma cells; when they reach more than 1 / 10 of the bottom area of ​​the well, aspirate the supernatant for antibody detection. Expand the culture of positive clones and freeze them.

[0159] 1.3 Clone Screening and Identification

[0160] ELISA was used to screen for anti-human PD-L1 antibodies in hybridoma culture supernatants. Recombinant human PD-L1 (purchased from Beijing Yiqiao Shenzhou) was coated onto 96-well high-adsorption ELISA plates (Corning, catalog number 42592) with carbonate buffer (pH 9.6) at a concentration of 1 μg / mL, at a volume of 100 μL per well, overnight at 4°C. The plates were washed five times with PBST. Blocking was performed with 300 μL / well of PBST containing 1% BSA, and incubation was carried out at 25°C for 1 hour. The plates were washed five times with PBST. Culture supernatant samples and positive serum controls (positive serum from immunized mice (immunized with human PD-L1-Fc protein)) were added to each well at 100 μL, and incubation was carried out at 25°C for 1 hour. The plates were washed five times with PBST. Then, add 100 μL of horseradish peroxidase-labeled anti-mouse IgG antibody (Abcam, catalog number Ab7068) diluted 1:10000 in PBST containing 1% BSA to each well and incubate at 25°C for 1 hour. Wash 5 times with PBST. Add 100 μL of TMB substrate per well and incubate at room temperature for 10 minutes. Add 100 μL of 1M H2SO4 per well to stop the color development. Read the absorbance at 450 nm on a microplate reader. Select positive clones that secrete anti-human PD-L1 binding antibody based on the intensity of OD450 nm.

[0161] ELISA was used to determine whether anti-human PD-L1 antibodies secreted by positive clones could block PD-L1 / PD-1 binding. Recombinant human PD-L1-Fc was coated with 100 μL of carbonate buffer (pH 9.6) per well in a 96-well high-adsorption ELISA plate at a concentration of 1 μg / mL, and the coating was performed overnight at 4°C. The plates were washed five times with PBST. Blocking was achieved with 300 μL of PBST containing 1% BSA per well, and incubation was performed at 25°C for 1 hour. The plates were washed five times with PBST. Anti-human PD-L1 antibody sample and positive control atezolizumab were added to each well (50 μL each), along with 50 μL of biotin-labeled PD-1-Fc (Beijing Hanmei Pharmaceutical Co., Ltd.) at a concentration of 40 nM (final concentration 20 nM), and incubated at 25°C for 90 minutes. The plates were washed five times with PBST. Then add Streptavidin-HRP (BD Pharmingen, catalog number 554066) diluted 1:1000 in PBST containing 1% BSA, 100 μL per well, and incubate at 25°C for 1 hour. Wash 5 times with PBST. Add 100 μL of the colorimetric substrate TMB per well and incubate at room temperature for 10 minutes. Add 100 μL of 1M H2SO4 per well to stop the color development. Read the absorbance at 450 nm on a microplate reader. Anti-human PD-L1 antibodies that can inhibit the binding of human PD-L1-Fc / biotin-labeled PD-1-Fc have neutralizing activity. Positive clones that secrete anti-human PD-L1 neutralizing antibodies are selected based on the strength of their blocking ability.

[0162] The results are as follows Figure 1 As shown in Figure A, clones 34-35 exhibit strong human PD-L1 binding activity, according to... Figure 1 As shown in Figure B, clones 34 and 35 exhibit strong human PD-L1 / PD-1 binding blocking activity, which is slightly stronger than that of atezolizumab.

[0163] 1.4 Determination of Monoclonal Antibody Sequence

[0164] Clones exhibiting both antigen-binding and antigen-neutralizing activities were selected and their antibody DNA sequences were determined. First, cellular mRNA was extracted using the RNAprep Pure kit (Tiangen, DP430) according to the manufacturer's instructions. Then, the first strand of cDNA was synthesized using the QuantScript RT kit (Tiangen, KR103). The first strand of cDNA generated by reverse transcription was used in subsequent PCR reactions.

[0165] The primers used in the PCR reaction are shown in Table 5.

[0166] Table 5 PCR Primers

[0167]

[0168]

[0169] When using primers, any upstream primer in the heavy chain variable region primer (VH primer) can be used with any downstream primer; similarly, any upstream primer in the light chain variable region primer (VL primer) can be used with any downstream primer. The target band obtained from PCR amplification is cloned into the pGEM-T vector. Single clones are picked for DNA sequencing.

[0170] Example 2: Preparation of chimeric anti-human PD-L1 monoclonal antibody

[0171] The amino acid sequences of the variable region of the antibody light chain and the variable region of the antibody heavy chain were obtained by PCR amplification as shown in SEQ ID NO. 18 and SEQ ID NO. 19, respectively. The amino acid sequences of the three complementarity-determining regions (CDR1, LCDR2, and LCDR3) of the light chain are shown in SEQ ID NO. 20, 21, and 22, respectively; the amino acid sequences of the three CDR1, HCDR2, and HCDR3 of the heavy chain are shown in SEQ ID NO. 23, 24, and 25, respectively. The variable region sequences encoding the above light and heavy chains were cloned into the eukaryotic cell expression vector X0GC, which contains the encoding sequences of the constant regions of the antibody light and heavy chains, respectively. The amino acid sequence of the constant region of the antibody light chain is shown in SEQ ID NO. 26, and the sequence of the constant region of the antibody heavy chain is shown in SEQ ID NO. 27. Expression vectors containing the coding sequences of the full-length antibody heavy chain (obtained by linking the variable region of the antibody heavy chain with SEQ ID NO. 27) and the full-length antibody light chain (obtained by linking the variable region of the antibody light chain with SEQ ID NO. 26) were transfected into ExpiCHO cells (ExpiCHO cells). TM Cells (catalog number A29127, Invitrogen). Cells were seeded one day before transfection at a seeding density of 35 x 10⁻⁶ cells. 5 Cells / mL. On the day of transfection, use fresh ExpiCHO expression medium (ExpiCHO). TM Expression Medium (catalog number A29100-01, invitrogen) was used to dilute the cells to a density of 60 x 10⁻⁶. 5 Cells / mL. Take the plasmid according to the transfection volume; the final plasmid concentration is 0.5 μg / mL. Use OptiPRO. TM SFM medium (OptiPRO) TM SFM (catalog number 12309-019, Invitrogen) was diluted to 4% of the transfection volume and mixed by inversion. 6.4 times the amount of plasmid was taken from ExpiFectamine.TM Transfection reagent (ExpiFectamine) TM CHO Transfection Kit (item number A29129, invitrogen), using OptiPRO TM Dilute the transfection reagent to 4% of the transfection volume in SFM medium and mix thoroughly by inverting. Add the diluted transfection reagent to the diluted plasmid, mix gently, and incubate at room temperature for 1–5 minutes. Slowly add the solution dropwise to the cells. Then place the cells in a cell culture incubator (8% CO2) and incubate at 37°C with a shaker at 120 rpm for 20 hours. Slowly add 0.006 times the transfection volume of ExpiCHO to the cells. TM Enhancer (ExpiFectamine) TM CHO Transfection Kit (catalog number A29129, invitrogen) and 0.24 times the transfection volume of ExpiCHO TM Feed(ExpiCHO TM Feed (product number A29101-02, Invitrogen). Incubate on a shaker at 120 rpm at 32°C. Centrifuge to collect the cell culture supernatant after 10 days of transfection.

[0172] Expression levels were determined by ELISA. Precipitation was removed by filtration through a 0.2 μm filter before column chromatography. This step was performed at 4 °C.

[0173] Example 3: Binding activity and binding kinetic constant of anti-human PD-L1 chimeric monoclonal antibody to human PD-L1

[0174] The binding activity of the anti-human PD-L1 chimeric monoclonal antibody to its antigen, human PD-L1, was determined by ELISA. Recombinant human PD-L1 (purchased from Sinopharm) was coated onto 96-well high-adsorption ELISA plates with carbonate buffer (pH 9.6) at a concentration of 1 μg / mL, at a volume of 100 μL per well, overnight at 4°C. The plates were washed five times with PBST. Blocking was performed with 300 μL / well of PBST containing 1% BSA, and incubation was carried out at 25°C for 1 hour. The plates were washed five times with PBST. Sequence-diluted anti-human PD-L1 chimeric monoclonal antibody and positive control atezolizumab were added to each well at 100 μL, and incubation was carried out at 25°C for 1 hour. The plates were washed five times with PBST. Then add 100 μL of horseradish peroxidase-labeled anti-human IgG antibody (Chemicon, catalog number AP309P) diluted 1:10000 in PBST containing 1% BSA to each well and incubate at 25°C for 1 hour. Wash 5 times with PBST. Add 100 μL of TMB substrate per well and incubate at room temperature for 10 minutes. Add 100 μL of 1M H₂SO₄ per well to stop the color development. Read the absorbance at 450 nm on a microplate reader.

[0175] The results are as follows Figure 2 As shown, the anti-human PD-L1 chimeric monoclonal antibody has good human PD-L1 binding affinity, similar to the binding activity of atezolizumab.

[0176] The Biacore X100 instrument was used to detect the kinetic constants of the binding between the anti-human PD-L1 chimeric monoclonal antibody and its antigen human PD-L1. This instrument utilizes optical surface plasmon resonance (SPR) technology to detect the binding and dissociation between the molecule conjugated on the biochip and the analyte. The binding and dissociation kinetic constants were analyzed and calculated using Biacore X100 evaluation software. The binding, dissociation, and equilibrium constants of the anti-human PD-L1 chimeric antibody are shown in Table 6. The data indicate that, compared to atezolizumab, the anti-human PD-L1 chimeric monoclonal antibody binds to PD-L1 more quickly, while the dissociation rate is comparable.

[0177] Table 6. Kinetic constants of binding between anti-human PD-L1 chimeric monoclonal antibodies and human PD-L1

[0178]

[0179] Example 4: Species binding specificity and target binding specificity of anti-human PD-L1 chimeric monoclonal antibody

[0180] 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 Sinopharm) were coated onto 96-well hyperabsorbent ELISA plates with carbonate buffer (pH 9.6) at a concentration of 1 μg / mL and a coating volume of 100 μL per well. Coating was performed overnight at 4°C. The plates were washed five times with PBST. Blocking was performed with 300 μL / well of PBST containing 1% BSA, and incubation was carried out at 25°C for 1 hour. The plates were washed five times with PBST. Sequence-diluted anti-human PD-L1 chimeric monoclonal antibody samples (100 μL per well) were added, and the plates were incubated at 25°C for 1 hour. The plates were washed five times with PBST. Then add 100 μL of horseradish peroxidase-labeled anti-human IgG antibody (Chemicon, catalog number AP309P) diluted 1:10000 in PBST containing 1% BSA to each well and incubate at 25°C for 1 hour. Wash 5 times with PBST. Add 100 μL of TMB substrate per well and incubate at room temperature for 10 minutes. Add 100 μL of 1M H2SO4 per well to stop the color development. Read the absorbance at 450 nm on a microplate reader.

[0181] The target binding specificity of the anti-human PD-L1 chimeric monoclonal antibody was determined by ELISA. Recombinant human PD-1, CD28, CTLA4, ICOS, BTLA, PD-L1, PD-L2, CD80, CD86, and B7-H2 (all purchased from Sinocare) were coated onto 96-well hyperabsorbent ELISA plates with a coating concentration of 1 μg / mL and a coating volume of 100 μL per well. Coating was performed overnight at 4°C. The plates were washed five times with PBST. Blocking was performed with 300 μL / well of PBST containing 1% BSA, and incubation was carried out at 25°C for 1 hour. The plates were washed five times with PBST. The anti-human PD-L1 chimeric monoclonal antibody samples and controls, diluted in PBST containing 1% BSA, were added to each well at 100 μL, and incubated at 25°C for 1 hour. The plates were washed five times with PBST. Then add 100 μL of horseradish peroxidase-labeled anti-human IgG antibody (Chemicon, catalog number AP309P) diluted 1:10000 in PBST containing 1% BSA to each well and incubate at 25°C for 1 hour. Wash 5 times with PBST. Add 100 μL of TMB substrate per well and incubate at room temperature for 10 minutes. Add 100 μL of 1M H2SO4 per well to stop the color development. Read the absorbance at 450 nm on a microplate reader.

[0182] The results are as follows Figure 3 As shown in Figure A, the anti-human PD-L1 chimeric monoclonal antibody can bind to both human PD-L1 and monkey PD-L1 with similar affinity, but it does not bind to rat or mouse PD-L1, exhibiting species-specific binding. Meanwhile, as... Figure 3As shown in Figure B, the anti-human PD-L1 chimeric monoclonal antibody also has strong target binding specificity, binding only to PD-L1 and not to other members of the B7 family or members of the CD28 family.

[0183] Example 5: The activity of the anti-human PD-L1 chimeric monoclonal antibody in blocking the binding of PD-L1 and PD-1.

[0184] Recombinant human PD-L1-Fc was coated onto a 96-well high-adsorption ELISA plate with a carbonate buffer solution at pH 9.6 at a concentration of 1 μg / mL, in a volume of 100 μL per well, overnight at 4°C. The plate was washed five times with PBST. Blocking was performed with 300 μL of PBST containing 1% BSA per well, and incubation was carried out at 25°C for 1 hour. The plate was washed five times with PBST. Anti-human PD-L1 chimeric antibody sample and positive control were added to each well at 50 μL, followed by 50 μL of biotin-labeled PD-1-Fc at a concentration of 40 nM (final concentration 20 nM), and incubation was carried out at 25°C for 90 minutes. The plate was washed five times with PBST. Then, Streptavidin-HRP (BD Pharmingen, catalog number 554066) diluted 1:1000 in PBST containing 1% BSA was added to each well at 100 μL, and incubation was carried out at 25°C for 1 hour. Wash 5 times with PBST. Add 100 μL of TMB substrate per well and incubate at room temperature for 10 minutes. Add 100 μL of 1 M H2SO4 per well to stop the incubation. Read the absorbance at 450 nm using a microplate reader.

[0185] The results are as follows Figure 4 As shown, the anti-human PD-L1 chimeric monoclonal antibody has similar PD-L1 / PD-1 binding blocking activity to atezolizumab.

[0186] Example 6: T cell function regulation activity of anti-human PD-L1 chimeric monoclonal antibody

[0187] The peripheral blood mononuclear cells (PBMCs) used in the experiment were purchased from Lonza, catalog number CC-2702.

[0188] First, DC cells were induced using PBMCs: PBMCs were resuscitated in complete medium (RPMI 1640 + 10% FBS), washed once with the appropriate serum-free medium, resuspended in serum-free medium, and seeded into cell culture flasks. The flasks were then incubated at 37°C in a 5% CO2 incubator. After 90 minutes, non-adherent cells and the culture medium were removed. Adherent monocytes were cultured in complete medium with 100 ng / mL GM-CSF (granulocyte-macrophage colony-stimulating factor) and 100 ng / mL IL-4 (interleukin-4) (Sinochem). The medium was changed after 3 days, and the cells were cultured for another 3 days. Then, the medium was changed to complete medium with 100 ng / mL GM-CSF, 100 ng / mL IL-4, and 20 ng / mL TNF-α (tumor necrosis factor-α) (Sinochem) and cultured for 1 day, thus completing the DC cell induction. T cells were then isolated from PBMCs from another individual source: T cells were isolated using the Miltenyi Biotech PanT Cell Isolation Kit (catalog number 5150414820), following the manufacturer's instructions. Induced mature dendritic cells (DCs) were seeded into 96-well plates at 10,000 cells per well, followed by the isolated T cells at 100,000 cells per well. Finally, the test sample was added, and the plates were incubated for 120 hours. At the end of incubation, the supernatant was collected, and IFN-γ levels were measured using a RayBiotech ELISA kit.

[0189] The results are as follows Figure 5 As shown, the anti-human PD-L1 chimeric monoclonal antibody can enhance IFN-γ secretion in a mixed lymphocyte culture system and has slightly stronger T cell function regulation activity than atezolizumab.

[0190] Example 7: Preparation of humanized anti-human PD-L1 monoclonal antibody

[0191] The anti-human PD-L1 humanized monoclonal antibody was obtained according to the method of Leung et al. (1995, Molecule Immunol 32:1413-27). Humanized templates with the best sequence match for the variable region of the murine antibody were selected from the Germline database. The template for the light chain variable region was IGKV1-33*01, as shown in SEQ ID NO. 28; the template for the heavy chain variable region was IGHV1-69*01, as shown in SEQ ID NO. 29. The CDR region of the murine antibody was transplanted onto the selected humanized template, replacing the CDR region of the human template, resulting in the transplanted light chain variable region and the transplanted heavy chain variable region of the human antibody. Reverse mutations were then performed on the light chain backbone region at specific sites, yielding the light chain variable region sequences shown in SEQ ID NO. 38, 39, and 44. Similarly, reverse mutations were performed on the heavy chain backbone region at specific sites, yielding the heavy chain variable region sequences shown in SEQ ID NO. 30-37 and 40-43. The NG site on HCDR2, as shown in sequence SEQ ID NO. 24, was mutated to remove possible deamidation sites, resulting in the mutated heavy chain HCDR2, the amino acid sequences of which are shown in SEQ ID NO. 45-47. The mutated heavy chain variable region sequences of HCDR2 targeting the heavy chain variable region shown in SEQ ID NO. 36 are shown in SEQ ID NO. 48-50; the mutated heavy chain variable region sequences of HCDR2 targeting the heavy chain variable region shown in SEQ ID NO. 41 are shown in SEQ ID NO. 51-53. The light chain variable region was linked to the light chain constant region (sequence SEQ ID NO. 26) to obtain the corresponding full-length light chain sequences, and the heavy chain variable region was linked to the heavy chain constant region (sequence SEQ ID NO. 27) to obtain the corresponding full-length heavy chain sequences. The humanized sequences are shown in Table 7. Exemplary combinations of the heavy chain variable region VH and the light chain variable region VL in the humanized antibody are shown in Table 8. The affinity data (dissociation constant) of the humanized antibody are shown in Table 9.

[0192] Table 7. Humanization Sequence

[0193]

[0194]

[0195]

[0196] Table 8. Exemplary combinations of heavy chain variable region VH and light chain variable region VL in antibodies

[0197]

[0198]

[0199] Table 9. Determination of affinity for humanized sequences (dissociation constant)

[0200]

[0201]

[0202] Example 8: Thermal stability of the anti-human PD-L1 humanized monoclonal antibody

[0203] A Waters xbridge BHE200 3.5µm, 7.8mm×30cm column (catalog number: 186007640) was used; the mobile phase was 0.1mol / L phosphate buffer (NaH2PO4-Na2HPO4), 0.1mol / L sodium sulfate buffer, pH 6.7; the flow rate was 0.6mL / min; the column temperature was 25℃; the sample cell temperature was 4℃; the detection wavelength was 280nm; the sample was diluted to 1mg / mL with sample buffer, and the injection volume was 10μL. The experimental results were processed using an Agilent HPLC 1260 system workstation, and the purity was calculated using the area normalization method to determine the proportion of the main peak. To determine the thermal stability of these monoclonal antibodies, the samples were placed at 40℃, and samples were taken at weeks 2 and 4 for SE-HPLC analysis to observe thermal stability. The results are shown in Table 10. The humanized anti-human PD-L1 antibodies, except for VLS10-VHS7 P2, all showed good and considerable stability.

[0204] Table 10. Thermal stability of humanized anti-human PD-L1 monoclonal antibodies at 40℃ as determined by SE-HPLC

[0205]

[0206]

[0207] Example 9: In vitro biological activity of humanized anti-PD-L1 monoclonal antibody

[0208] The in vitro biological activity of humanized anti-human PD-L1 monoclonal antibodies was determined, including their binding activity to human PD-L1, their blocking activity against PD-L1 / PD-1 binding and PD-L1 / CD80, and the kinetic constant of their binding to human PD-L1. The humanized sequences determined included VLS10-VHS7, VLS10-VHS7 P1, VLS10-VHS7 P2, VLS10-VHS7 P3, VLS15-VHS12, VLS15-VHS12 P1, VLS15-VHS12 P2, VLS15-VHS12 P3, as well as the control atezolizumab and the chimeric anti-human PD-L1 monoclonal antibody. The specific experimental methods were the same as those used to determine the in vitro biological activity of the chimeric anti-human PD-L1 monoclonal antibody.

[0209] The experimental results are shown in Tables 11 and 12. Compared with the chimeric anti-human PD-L1 monoclonal antibody, the humanized sequences tested all maintained their activity well, showing strong PD-L1 binding activity and PD-L1 / PD-1 and PD-L1 / CD80 blocking activity, with the VLS15-VHS12-P3 sequence being the best.

[0210] Table 11. Activity of anti-human PD-L1 humanized monoclonal antibodies in binding to PD-L1 and blocking PD-L1 / receptor

[0211]

[0212] Table 12. Kinetic constants of binding between humanized anti-human PD-L1 monoclonal antibodies and human PD-L1

[0213] sample <![CDATA[K on (1 / Ms)]]> <![CDATA[K off (1 / s)]]> <![CDATA[K D (M)]]> Atezolizumab 1.92E+5 3.86E-4 2.01E-9 Chimeric monoclonal antibodies 5.07E+5 5.94E-4 1.17E-9 VLS10-VHS7 3.41E+5 5.84E-4 1.71E-9 VLS10-VHS7 P1 3.41E+5 1.08E-03 3.16E-9 VLS10-VHS7 P2 3.07E+5 9.94E-4 3.23E-9 VLS10-VHS7 P3 2.55E+5 7.47E-4 2.93E-9 VLS15-VHS12 3.42E+5 4.77E-4 1.39E-9 VLS15-VHS12P1 3.26E+5 8.33E-4 2.56E-9 VLS15-VHS12P2 3.72E+5 7.20E-4 1.94E-9 VLS15-VHS12P3 3.38E+5 5.28E-4 1.56E-9

[0214] Example 10: Pharmacokinetic Study of Anti-human PD-L1 Humanized Monoclonal Antibody in Human PD-1 / Human PD-L1 Dual Gene Knock-in Mice

[0215] The experimental materials used were female human PD-1 / human PD-L1 double gene knock-in mice (C57BL / 6 background), aged 6-8 weeks, purchased from Beijing Biocytogen Biotechnology Co., Ltd. One week after acclimatization, the mice were administered the anti-human PD-L1 humanized monoclonal antibody VLS15-VHS12 P3 at a dose of 70 nmol / kg via intraperitoneal injection, as a single dose. At 0:00, blood samples were collected from the orbital sinus at 6, 24, 72, 120, 168, 240, and 312 hours post-administration, with two mice at each sampling point. No anticoagulation was used. Blood samples were left at room temperature for 30 minutes to 1 hour until clotting, followed by centrifugation at 3000 rpm for 10 minutes. The resulting serum samples were frozen and stored at -80°C for later analysis.

[0216] The concentration of humanized anti-human PD-L1 monoclonal antibody in serum was determined by ELISA. Briefly, recombinant human PD-L1 protein was coated onto a high-absorption ELISA plate overnight at 4°C with carbonate buffer (pH 9.6). The plate was washed with PBST. To prevent non-specific binding, the plate was blocked with PBST containing 5% skim milk powder, followed by PBST washing. Then, the serum sample to be tested, diluted with PBST containing 10% mixed mouse serum and 1% BSA, was added and incubated at 25°C for 1 hour, followed by PBST washing. Horseradish peroxidase-labeled anti-human IgG antibody (Chemicon, catalog number AP309P) diluted in PBST containing 5% skim milk powder was added, and the plate was incubated at 25°C for 1 hour, followed by PBST washing. Finally, colorimetric development was performed using the TMB substrate at room temperature for 10 minutes. The colorimetric development was stopped by adding 1M H₂SO₄. The absorbance was read at 450 nm using an ELISA reader.

[0217] The results are as follows Figure 6 As shown, a single intraperitoneal injection of 70 nmol / kg of the anti-human PD-L1 humanized monoclonal antibody exhibited favorable pharmacokinetic and time-dependent 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 are as follows: half-life t... 1 / 2 The duration is 130 hours; the area under the curve (AUC) during drug administration is... 0-312hr It is 77917 nM.hr; apparent volume of distribution V d The concentration was 147 mL / kg; the clearance rate (CL) was 0.78 mL / hr / kg; and the mean residence time (MRT) was... last It lasts for 90 hours.

[0218] Example 11: Antitumor efficacy study of anti-human PD-L1 humanized monoclonal antibody in human PD-1 / human PD-L1 dual gene knock-in mice

[0219] This embodiment examines the growth-inhibiting effect of anti-human PD-L1 humanized monoclonal antibody on MC38 / human PD-L1 tumor grafts in human PD-1 / human PD-L1 double gene knock-in mice.

[0220] The experimental materials used were female human PD-1 / human PD-L1 double gene knock-in mice (C57BL / 6 background, Beijing Biocytogen Biotechnology Co., Ltd.). One week after acclimatization, each mouse was inoculated with 5 x 10⁻⁶ g of the vaccine. 5 One MC38 / human PD-L1 (Beijing Biocytogen Biotechnology Co., Ltd.) is a constructed mouse colon cancer cell line expressing human PD-L1. The tumor volume was increased to approximately 150 mm². 3Mice were divided into two groups of six based on tumor volume: a solvent control group and a group receiving the humanized anti-PD-L1 monoclonal antibody VLS15-VHS12 P3. The dosage was 70 nmol / kg, administered intraperitoneally twice weekly for two weeks. Tumor volume was measured twice weekly from the start of administration, including the major axis (a) and minor axis (b). The tumor volume was calculated using the formula: Tumor volume (mm²) = ... 3 )=(axb 2 ) / 2.

[0221] The results are as follows Figure 7 As shown, the anti-human PD-L1 humanized monoclonal antibody has anti-tumor activity and significantly inhibits the growth of MC38 / human PD-L1 xenografts in human PD-1 / human PD-L1 double gene knock-in mice.

[0222] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. sequence list <110> Beijing Hanmi Pharmaceutical Co., Ltd. <120> Antibodies that specifically bind to PD-L1 and their antigen-binding fragments <130> LZ2117819CN07 <160> 61 <170> PatentIn version 3.3 <210> 1 <211> 36 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 1 gaggtgaagc tgcaggagtc aggacctagc ctggtg 36 <210> 2 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 2 aggtsmaact gcagsagtcw gg 22 <210> 3 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 3 aggtsmagct gcagsagtcw gg 22 <210> 4 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 4 aggtscagct gcagsagtcw gg 22 <210> 5 <211> 38 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 5 ccaggggcca gtggatagac aagcttgggt gtcgtttt 38 <210> 6 <211> 27 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 6 atagacagat gggggtgtcg ttttggc 27 <210> 7 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 7 cttgaccagg catcctagag tca 23 <210> 8 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 8 aggggccagt ggatagactg atgg 24 <210> 9 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 9 agggaccaag ggatagacag atgg 24 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> PCR primers <220> <221> misc_feature <222> (6)..(6) <223> n is a, c, g, or t <400> 10 sargtnmagc tgsagsagtc 20 <210> 11 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> PCR primers <220> <221> misc_feature <222> (6)..(6) <223> n is a, c, g, or t <400> 11 sargtnmagc tgsagsagtc wgg 23 <210> 12 <211> 32 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 12 ggtgatatcg tgatracmca rgatgaactc tc 32 <210> 13 <211> 32 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 13 ggtgatatcw tgmtgaccca awctccactc tc 32 <210> 14 <211> 32 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 14 ggtgatatcg tkctcacyca rtctccagca at 32 <210> 15 <211> 32 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 15 gggaagatgg atccagttgg tgcagcatca gc 32 <210> 16 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 16 ggatacagtt ggtgcagcat c 21 <210> 17 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 17 gayattgtgm tsacmcarwc tmca 24 <210> 18 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Antibody light chain variable region <400> 18 Asp Ile Val Met Thr Gln Ser His Lys Val Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val His Thr Ala 20 25 30 Val Ala Trp Ile His Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Thr Leu Glu Leu Lys 100 105 <210>19 <211>117 <212>PRT <213>Artificial sequence <220> <223>Variable region of antibody heavy chain <400>19 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Met Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210>20 <211> 11 <212> PRT <213> Artificial sequence <220> <223> LCDR1 <400> 20 Lys Ala Ser Gln Asp Val His Thr Ala Val Ala 1 5 10 <210> twenty one <211> 7 <212> PRT <213> Artificial sequence <220> <223> LCDR2 <400> twenty one Ser Ala Ser Asn Arg Tyr Thr 1 5 <210> twenty two <211> 9 <212> PRT <213> Artificial sequence <220> <223> LCDR3 <400> twenty two Gln Gln His Tyr Ile Thr Pro Leu Thr 1 5 <210> twenty three <211> 10 <212> PRT <213> Artificial sequence <220> <223> HCDR1 <400> twenty three Gly Phe Asn Ile Glu Asp Thr Tyr Ile His 1 5 10 <210> twenty four <211> 17 <212> PRT <213> Artificial sequence <220> <223> HCDR2 <400> twenty four Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln 1 5 10 15 Gly <210> 25 <211> 8 <212> PRT <213> Artificial sequence <220> <223> HCDR3 <400> 25 Gly Leu Gly Ala Trp Phe Ala Tyr 15 <210> 26 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Antibody light chain constant region <400> 26 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210>27 <211>330 <212>PRT <213>Artificial <220> <223>Constant region of antibody heavy chain <400>27 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210>28 <211>95 <212>PRT <213>Artificial <220> <223>Light chain variable region template <400>28 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 8^0 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro 85 90 95 <210>29 <211>98 <212>PRT <213>Artificial sequence <220> <223>Heavy chain variable region template <400>29 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser I 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210>30 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 30 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 31 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400>31 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210>32[[ID=3�]] <211>117 <212>PRT <213>Artificial <220> <223>Heavy chain variable region <400>32 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210>33 <211>117 <212>PRT <213>Artificial <220> <223>Heavy chain variable region <400>33 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210>34<000​​​​​​​​​​​​​​​​​​​​​​​​Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210>35 <211>117 <212>PRT <213>Artificial <220> <223>Heavy chain variable region <400>35 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210>36 <211>117 <212>PRT <213>Artificial <220> <223>Heavy chain variable region <400>36 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210>37 <211>117 <212>PRT <213>Artificial sequence <220> <223>Heavy chain variable region <400>37 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210>38 <211>107 <212>PRT <213>Artificial sequence <220> <223>Light chain variable region <400>38 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val His Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60<^ Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210>39 <211>107 Please note that in the original text, there is a tag which seems to be misspelled as <^ in the provided text. I have translated it as in the result. If this is not correct, please adjust accordingly. <212> PRT <213> Artificial sequence <220> <223> Light chain variable region <400> 39 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val His Thr Ala 20 25 30 Val Ala Trp Ile His Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 40 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 40 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210>41 <211>117 <212>PRT <213>Artificial <220> <223>Heavy chain variable region <400>41 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210>42 <211>117 <212>PRT <213>Artificial <220> <223>Heavy chain variable region <400>42 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210>43 <211>117 <212>PRT <213>Artificial <220> <223>Heavy chain variable region <400>43 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210>44 <211>107 <212>PRT <213>Artificial <220> <223>Light chain variable region <400>44 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val His Thr Ala 20 25 30 Val Ala Trp Ile Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 45 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Mutant HCDR2 <400> 45 Arg Ile Asp Pro Ala Gln Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln 1 5 10 15 Gly <210> 46 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Mutant HCDR2 <400> 46 Arg Ile Asp Pro Ala Ser Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln 1 5 10 15 Gly <210> 47 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Mutant HCDR2 <400> 47 Arg Ile Asp Pro Ala Asn Ala Asn Thr Lys Tyr Asp Pro Lys Phe Gln 1 5 10 15 Gly <210>48 <211>117 <212>PRT <213>Artificial <220> <223>Mutated heavy chain variable region <400>48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Gln Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 49 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Mutated heavy chain variable region <400> 49 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Ser Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 50 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Mutated heavy chain variable region <400> 50 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Ala Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 51 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Mutated heavy chain variable region <400> 51 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Gln Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210>52 <211>117 <212>PRT <213>Artificial <220> <223>Mutated heavy chain variable region <400>52 [[ID=​​​Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Ser Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 1​​​​​​​​​​​​​​​​​​​​​​​​​​​Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Ala Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Gly Leu Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210>54 <211>23 <212>PRT <213>Artificial <220> <223>FR‑L1 <400>54 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20 <210>55 <211>15 <212>PRT <213>Artificial <220> <223>FR‑L2 <400>55 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 151015 <210> 56 <211> 32 <212> PRT <213> artificial sequence(Artificial) <220> <223> FR‑L3 <400> 56 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys 20 25 30 <210> 57 <211> 10 <212> PRT <213> artificial sequence(Artificial) <220> <223> FR‑L4 <400> 57 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 58 <211> 25 <212> PRT <213> artificial sequence(Artificial) <220> <223> FR‑H1 <400> 58 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser 20 25 <210> 59 <211> 14 <212> PRT <213> Artificial sequence <220> <223> FR-H2 <400> 59 Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 <210> 60 <211> 32 <212> PRT <213> Artificial sequence <220> <223> FR-H3 <400> 60 Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 61 <211> 11 <212> PRT <213> Artificial sequence <220> <223> FR-H4 <400> 61 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10

Claims

1. An isolated anti-human PD-L1 antibody or its antigen-binding fragment thereof, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises LCDR1 with the amino acid sequence shown in SEQ ID No. 20, LCDR2 with the amino acid sequence shown in SEQ ID No. 21, and LCDR3 with the amino acid sequence shown in SEQ ID No. 22; and The heavy chain variable region contains HCDR1 with an amino acid sequence as shown in SEQ ID No. 23, HCDR2 with an amino acid sequence as shown in SEQ ID No. 24, 45, 46 or 47, and HCDR3 with an amino acid sequence as shown in SEQ ID No.

25.

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

3. The anti-human PD-L1 antibody or its antigen-binding fragment according to claim 1, wherein the heavy chain constant region sequence of the antibody is selected from the constant region sequence of one 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 the κ chain or the λ chain.

4. The anti-human PD-L1 antibody or its antigen-binding fragment according to claim 3, wherein 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 chain.

5. The anti-human PD-L1 antibody or its antigen-binding fragment according to claim 1, wherein the light chain variable region has an amino acid sequence as shown in SEQ ID No. 18; and / or the heavy chain variable region has an amino acid sequence as shown in SEQ ID No.

19.

6. The anti-human PD-L1 antibody or its antigen-binding fragment according to claim 1, wherein the light chain variable region backbone of the anti-human PD-L1 antibody or its antigen-binding fragment includes FR-L1, FR-L2, FR-L3 and FR-L4, and the heavy chain variable region backbone includes FR-H1, FR-H2, FR-H3 and FR-H4, wherein, 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 substituting one or any combination of the following: The second amino acid Y is replaced with I. The third amino acid, Q, is replaced with H. The 9th amino acid, A, is replaced with 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 substituting one or a combination of the following: The first amino acid Q is replaced with E. The 23rd amino acid, K, is replaced with T. The FR-H2 has the amino acid sequence shown in SEQ ID No. 59, or an amino acid sequence obtained by the following substitutions: The 13th amino acid M is replaced with I; The FR-H3 has the amino acid sequence shown in SEQ ID No. 60, or an amino acid sequence obtained by one or any combination of the following substitutions: The second amino acid, V, is replaced with A. The 8th amino acid, E, is replaced with T. The 11th amino acid, S, is replaced with N. The 31st amino acid A is replaced with G; and / or The FR-H4 has the amino acid sequence shown in SEQ ID No.

61.

7. The anti-human PD-L1 antibody or its antigen-binding fragment according to claim 1, wherein: (a) The light chain variable region has an amino acid sequence as shown in SEQ ID No. 38; and / or 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 an amino acid sequence as shown in SEQ ID No. 39; and / or The heavy chain variable region has an amino acid sequence selected from SEQ ID No. 30-37 or 48-50; or (c) The light chain variable region has an amino acid sequence as shown in SEQ ID No. 44; and / or The heavy chain variable region has an amino acid sequence selected from SEQ ID No. 40-43 or 51-53.

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

9. The anti-human PD-L1 antibody or its antigen-binding fragment according to claim 1, wherein the antigen-binding fragment is selected from one or more of F(ab')2, Fab', Fab, Fv, scFv and bispecific antibodies.

10. The anti-human PD-L1 antibody or its antigen-binding fragment according to claim 1, wherein the antigen-binding fragment is Fab, F(ab')2 or scFv.

11. An isolated nucleic acid molecule selected from: (1) DNA or RNA encoding the anti-human PD-L1 antibody or its antigen-binding fragment as described in any one of claims 1-10; (2) Nucleic acids that are completely complementary to the nucleic acids defined in (1).

12. An expression vector comprising a effectively linked nucleic acid molecule of claim 11.

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

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

15. A method for producing the anti-human PD-L1 antibody or its antigen-binding fragment according to any one of claims 1-10, comprising the steps of: The host cells of claim 13 are cultured under culture conditions suitable for the expression of the anti-human PD-L1 antibody or its antigen-binding fragment, and optionally, the resulting product is isolated and purified.

16. The use of the anti-human PD-L1 antibody or its antigen-binding fragment according to any one of claims 1-10, the nucleic acid molecule according to claim 11, the expression vector according to claim 12, or the host cell according to claim 13 in the preparation of a medicament for the prevention and / or treatment of PD-L1-mediated diseases or conditions, wherein the disease or condition is a tumor, and the tumor is selected from one or more of leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, pancreatic cancer, 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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