Antibodies against human programmed death ligand-1 (PD-L1) and their applications

Abstract

The present invention discloses an antibody or antigen-binding fragment that specifically binds to human programmed death ligand-1 (PD-L1), wherein the antibody or antigen-binding fragment can enhance the function of T cells, up-regulate T cell-mediated immune responses, and is used for treating diseases associated with abnormal PD-L1 expression and / or abnormal T cell function, such as tumors.

Description

Technical Field

[0001] The present invention relates to an antibody against human programmed death ligand-1 (PD-L1) or an antigen-binding fragment thereof, its encoding nucleic acid, expression vector and expression cell, preparation method, pharmaceutical composition, and their uses for enhancing the function of T cells, upregulating T cell-mediated immune response, and treating diseases related to abnormal PD-L1 expression and abnormal T cell function, such as tumors. Background Art

[0002] Immunotherapy has become one of the most rapidly developing and most promising research fields in cancer treatment. The use of immune checkpoint inhibitors, such as anti-PD-1 / PD-L1 monoclonal antibodies and anti-CTLA-4 monoclonal antibodies, is a revolutionary treatment method for cancer immunotherapy, which has greatly improved the survival period of patients with malignant tumors.

[0003] T cell-mediated immune responses are strictly regulated by co-stimulatory and co-inhibitory mechanisms, maintaining an optimal balance between antigen immune responses and self-tolerance. This balance involves multiple activating and inhibitory proteins. Inhibitory proteins, also known as immune checkpoint proteins, regulate the activation and effector functions of cytotoxic T lymphocytes (CTLs) to maintain self-tolerance. Immune checkpoint inhibitory proteins play a key role in the regulatory pathways of tumors. One important immune checkpoint protein, PD-1, after binding to its ligand PD-L1, transmits immunosuppressive signals and reduces the activity of T cells. At the same time, tumor cells can also inhibit the activation and proliferation of T cells by expressing PD-L1 on the cell surface, thereby evading the attack and killing of CTLs. By using anti-PD-1 or anti-PD-L1 monoclonal antibodies to block the binding and interaction of PD-1 / PD-L1, the function of T cells can be partially restored, thus enhancing the ability to kill tumor cells.

[0004] In 2011, the first immune checkpoint inhibitor, ipilimumab, an anti-CTLA-4 monoclonal antibody, became a successful tumor immunotherapy for treating melanoma. So far, many patients treated with it have achieved a better 5-year survival rate compared with traditional treatment methods. Subsequently, the FDA has successively approved 3 PD-1 monoclonal antibodies and 3 PD-L1 monoclonal antibodies, which have been successfully used in immunotherapy for more than a dozen tumors other than melanoma and have become the first-line treatment for various cancers, such as non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), and bladder or urothelial cancer. In China, 2 imported PD-1 antibodies and 4 domestic PD-1 antibodies have been approved for marketing so far, but no domestic PD-L1 antibody has been approved. Moreover, considering the differences in the treatment mechanisms of PD-L1 antibodies and PD-1 antibodies, as well as the current clinical trial combination drugs and applicable indications, the research and development of new PD-L1 monoclonal antibodies and PD-L1-based bispecific antibodies still have great social and economic significance. Summary of the Invention

[0005] The present invention provides a chimeric antibody or humanized antibody or antigen-binding fragment, multi-specific antigen-binding molecule, chimeric antigen receptor, immune effector cell, nucleic acid fragment, vector, cell, composition, preparation method, pharmaceutical use, and disease treatment method that specifically binds to PD-L1.

[0006] In some embodiments, the heavy chain variable region and the light chain variable region of the isolated antibody or antigen-binding fragment that specifically binds to human programmed death ligand-1 (PD-L1) have the sequences shown in SEQ ID NO:3 and SEQ ID NO:4, respectively, or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity compared with the sequences shown in SEQ ID NO:3 and SEQ ID NO:4.

[0007] In a preferred embodiment, the antibody or antigen-binding fragment of the present invention has a dissociation constant (KD) for binding to human programmed death ligand-1 (PD-L1) of no greater than 2.1×10 -9 M, and a dissociation constant (KD) for binding to cynomolgus monkey programmed death ligand-1 (PD-L1) of no greater than 1.2×10 -9 M.

[0008] In a preferred embodiment, the antibody or antigen-binding fragment of the present invention is chimeric or humanized or fully human.

[0009] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises the sequence of any one of the constant regions of human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably comprises the sequence of the constant region of human or murine antibodies IgG1, IgG2, IgG3 or IgG4; or the sequence of the constant region of human or murine antibodies IgG1, IgG2, IgG3 or IgG4 carrying mutations.

[0010] In a preferred embodiment, the antigen-binding fragment of the present invention is selected from one or more of F(ab)2, Fab’, Fab, Fv, scFv, bispecific antibody, nanobody and antibody minimal recognition unit.

[0011] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention can competitively bind to PD-L1 with the antibody numbered 156, and has the following characteristics:

[0012] 1) Specifically bind to the PD-L1 recombinant protein and cells expressing PD-L1;

[0013] 2) Block the binding of PD-L1 to the PD-1 protein;

[0014] 3) Inhibit the binding of PD-1 to PD-L1 expressed on the cell surface;

[0015] 4) Enhance T cell activity; or / and

[0016] 5) Inhibit tumor growth.

[0017] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention is further conjugated with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioactive isotopes, chemotherapeutic drugs or immunomodulators, and the tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents or photosensitizers.

[0018] In a preferred embodiment, the present invention further provides a multispecific antigen-binding molecule; preferably, the multispecific antigen-binding molecule comprises a first antigen-binding module and a second antigen-binding module, the first antigen-binding module comprises the antibody or antigen-binding fragment described in any one of the above, and the second antigen-binding module specifically binds to an antigen other than PD-L1 or binds to a PD-L1 antigen epitope different from the first antigen-binding module;

[0019] Preferably, the other antigen is selected from PD-1, TNFR2, CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOS-L, GITR, GITR-L, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM, BTLA, CD47 or CD73;

[0020] Preferably, the multispecific antibody is "bispecific", "trispecific" or "tetraspecific".

[0021] In a preferred embodiment, the present invention provides a chimeric antigen receptor (CAR); preferably, the chimeric antigen receptor comprises at least an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain, and the extracellular antigen-binding domain comprises the PD-L1 antibody or antigen-binding fragment described in any one of the above.

[0022] In a preferred embodiment, the present invention provides an immune effector cell; preferably, the immune effector cell comprises the chimeric antigen receptor described above or a nucleic acid fragment comprising the chimeric antigen receptor described above;

[0023] Preferably, the immune effector cell is selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), monocytes, macrophages, dendritic cells or mast cells; the T cells can be selected from inflammatory T cells, cytotoxic T cells, regulatory T cells (Tregs) or helper T cells;

[0024] Preferably, the immune effector cell is an allogeneic immune effector cell or an autologous immune cell.

[0025] In a preferred embodiment, the present invention provides an isolated nucleic acid molecule encoding the nanobody, antigen-binding fragment, or any combination thereof, the multispecific antigen-binding molecule, or the chimeric antigen receptor described in any one of the above of the present invention.

[0026] In some embodiments, the present invention provides an expression vector comprising the isolated nucleic acid molecule described above of the present invention.

[0027] In some embodiments, the present invention provides a host cell comprising the isolated nucleic acid molecule or expression vector described above of the present invention.

[0028] In a preferred embodiment, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli, and / or Bacillus subtilis; more preferably, the host cell is selected from Chinese hamster ovary cells (CHO).

[0029] In some embodiments, the present invention provides a method for preparing an antibody, an antigen-binding fragment, or a multispecific antigen-binding molecule, comprising culturing the host cell described above in the present invention under appropriate conditions and isolating the antibody, the antigen-binding fragment, or the multispecific antigen-binding molecule.

[0030] In some embodiments, the present invention provides a method for preparing an immune effector cell, comprising introducing the nucleic acid fragment of the CAR described above into the immune effector cell. Preferably, the method further comprises initiating the expression of the CAR described above in the immune effector cell.

[0031] In some embodiments, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment, the multispecific antigen-binding molecule, the chimeric antigen receptor, the immune effector cell, the isolated nucleic acid molecule, the expression vector, the cell, or the product prepared by the method described above (such as an antibody and an antigen-binding fragment) in the present invention, and a pharmaceutically acceptable carrier.

[0032] In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or adjuvant; more preferably, the pharmaceutical composition further comprises an additional anti-tumor agent.

[0033] In some embodiments, the present invention provides a method for preventing and / or treating a disease associated with abnormal PD-L1 expression and / or abnormal T cell function, comprising administering to a patient in need thereof the antibody or antigen-binding fragment, the multispecific antigen-binding molecule, the chimeric antigen receptor, the immune effector cell, the isolated nucleic acid molecule, the expression vector, the cell, the product prepared by the method described above, or the pharmaceutical composition described above in the present invention; the disease is preferably a tumor.

[0034] Preferably, the tumor is selected from lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, testicular cancer, salivary gland cancer, thyroid cancer, thymic cancer, epithelial cancer, pancreatic cancer, colon cancer, rectal cancer, malignant hematological diseases, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body cancer, and osteosarcoma.

[0035] In some embodiments, the present invention provides the use of the antibody or antigen-binding fragment described above, the multi-specific antigen-binding molecule described above, the chimeric antigen receptor described above, the immune effector cell described above, the isolated nucleic acid molecule described above, the expression vector described above, the cell described above, the product prepared by the method described above (such as antibody and antigen-binding fragment), or the pharmaceutical composition described above in the preparation of a drug for preventing and / or treating a disease associated with abnormal PD-L1 expression and / or abnormal T cell function, preferably a tumor;

[0036] Preferably, the tumor is selected from lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, testicular cancer, salivary gland cancer, thyroid cancer, thymic cancer, epithelial cancer, pancreatic cancer, colon cancer, rectal cancer, malignant hematological diseases, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body cancer, and osteosarcoma.

[0037] In some embodiments, the present invention provides the use of the antibody or antigen-binding fragment described above, the multi-specific antigen-binding molecule described above, the chimeric antigen receptor described above, the immune effector cell described above, the isolated nucleic acid molecule described above, the expression vector described above, the cell described above, the product prepared by the method described above (such as antibody and antigen-binding fragment), or the pharmaceutical composition described above for preventing and / or treating a disease associated with abnormal PD-L1 expression and / or abnormal T cell function; preferably a tumor;

[0038] Preferably, the tumor is selected from lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, testicular cancer, salivary gland cancer, thyroid cancer, thymic cancer, epithelial cancer, pancreatic cancer, colon cancer, rectal cancer, malignant hematological diseases, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body cancer, and osteosarcoma.

[0039] In some embodiments, the present invention provides a kit comprising the antibody or antigen-binding fragment described above in the present invention, the multispecific antigen-binding molecule described above in the present invention, the chimeric antigen receptor described above in the present invention, the immune effector cell described above in the present invention, the isolated nucleic acid molecule described above in the present invention, the expression vector described above in the present invention, the cell described above in the present invention, or a product prepared by the method described above in the present invention (such as an antibody and an antigen-binding fragment), or the pharmaceutical composition described above in the present invention, and instructions for use.

[0040] Term Definitions and Explanations

[0041] As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to a target antigen or has immunoreactivity, including polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies (including but not limited to chimeric antibodies, humanized antibodies, fully human antibodies, heteroconjugate antibodies (such as bispecific, trispecific, and tetra-specific antibodies, diabodies, triabodies, and tetra-bodies, antibody conjugates) and antigen-binding fragments of antibodies (including, for example, Fab’, F(ab’)2, Fab, Fv, rIgG, and scFv fragments). In addition, unless otherwise specified, the term "monoclonal antibody" (mAb) means including a complete antibody molecule capable of specifically binding to a target protein and an incomplete antibody fragment (such as Fab and F(ab’)2 fragments, which lack the Fc fragment of the complete antibody (cleared more quickly from the animal circulation), and thus lack Fc-mediated effector function (see Wahl et al., J. Nucl. Med. 24:316, 1983; the content of which is incorporated herein by reference).

[0042] As used herein, "antibody" can be derived from any animal, including but not limited to human and non-human animals, and the non-human animals can be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, rheas, alpacas, sheep, rabbits, mice, rats, or Chondrichthyes (such as sharks).

[0043] As used herein, the term "monospecific" refers to having one or more binding sites, where each binding site binds the same epitope of the same antigen.

[0044] As used herein, the term "multispecific" refers to having at least two antigen-binding sites, and each of the at least two antigen-binding sites binds a different epitope of the same antigen or a different epitope of a different antigen. Thus, terms such as "bispecific", "trispecific", "tetraspecific", etc. refer to the number of different epitopes that an antibody / antigen-binding molecule can bind.

[0045] As used herein, the terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably and refer to an antibody having a structure that is substantially similar to the structure of a native antibody.

[0046] As used herein, the term "antigen-binding fragment" refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Antibody fragments can be Fab, F(ab’)2, scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments that encompass the "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (V) dAb comprising VH and VL domains; (vi) dAb fragment consisting of a VH domain (Ward et al., Nature 341:544-546, 1989); (vii) dAb consisting of a VH or VL domain; (viii) isolated complementarity-determining region (CDR); and (ix) a combination of two or more isolated CDRs, which may optionally be linked by a synthetic linker. In addition, although the two domains VL and VH of an Fv fragment are encoded by separate genes, these two domains can be joined using recombinant methods by a linker that enables them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some embodiments, by chemical peptide synthesis procedures known in the art.

[0047] As used herein, the term "PD-L1" refers to programmed death ligand-1, also known as CD279 (cluster of differentiation 279), which is an important immunosuppressive molecule. The PD-L1 is preferably human PD-L1.

[0048] As used herein, terms such as "anti-programmed death ligand-1 antibody", "programmed death ligand-1 antibody", "anti-PD-L1 antibody", "PD-L1 antibody", "anti-PD-L1 antibody portion" and / or "anti-PD-L1 antibody fragment" refer to any protein- or peptide-containing molecule that includes at least a portion of an immunoglobulin molecule capable of specifically binding to PD-L1 (e.g., but not limited to, at least one complementarity-determining region (CDR) of a heavy or light chain or its ligand-binding portion, a variable region of a heavy or light chain, a constant region of a heavy or light chain, a framework region or any portion thereof). PD-L1 antibodies also include antibody-like protein scaffolds (such as the tenth fibronectin type III domain (10Fn3)), which contain BC, DE, and FG structural loops that are structurally and solvent-accessibly similar to antibody CDRs. The tertiary structure of the 10Fn3 domain is similar to that of the variable region of an IgG heavy chain, and those skilled in the art can graft, for example, the CDRs of a PD-L1 monoclonal antibody onto the fibronectin scaffold by replacing the residues of the BC, DE, and FG loops of 10Fn3 with residues from the CDR-H1, CDR-H2, or CDR-H3 regions of a PD-L1 monoclonal antibody.

[0049] As used herein, the term "bispecific antibody" refers to an antibody that has monoclonal binding specificity for at least two different antigens, which are typically human or humanized antibodies. In the present invention, one of the binding specificities can be detected against an epitope of PD-L1, and the other can be detected against another epitope of PD-L1 or any other antigen other than PD-L1, such as against a cell surface protein, receptor, receptor subunit, tissue-specific antigen, virus-derived protein, virus-encoded envelope protein, bacterium-derived protein, or bacterium surface protein, etc.

[0050] As used herein, the term "chimeric" antibody refers to an antibody that has variable sequences of immunoglobulins derived from one source organism (such as a rat or mouse) and constant regions of immunoglobulins derived from a different organism (such as a human). Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, Bio Techniques 4:214-221; Gillies et al., 1985 J Immunol Methods 125:191-202; the above are incorporated herein by reference.

[0051] As used herein, the term "complementary determining region" (CDR) refers to the hypervariable regions found in both the light and heavy chain variable domains. The more conserved portions of the variable domains are called framework regions (FR). As understood in the art, the amino acid positions that represent the hypervariable regions of an antibody can vary depending on the context and the various definitions known in the art. Some positions within the variable domain can be considered hybrid hypervariable positions because these positions can be considered to be within the hypervariable regions under one set of criteria (such as IMGT or Kabat), while being considered outside the hypervariable regions under a different set of criteria (such as Kabat or IMGT). One or more of these positions can also be found in the extended hypervariable regions. The present invention includes antibodies that contain modifications at these hybrid hypervariable positions. The variable domains of the native heavy and light chains each contain four framework regions that predominantly adopt a sheet configuration, which are connected by three CDRs (CDR1, CDR2, and CDR3), which form loops that connect the sheet structures and, in some cases, form part of the sheet structure. The CDRs in each chain are held tightly together in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 by the FR regions, and together with the CDRs from the other antibody chain contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; which is incorporated herein by reference).

[0052] As used herein, the term "monoclonal antibody" refers to an antibody that is derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), and is not limited to the method by which the antibody is produced.

[0053] As used herein, the term "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody (including the heavy chain of an Fv, scFv, or Fab). The term "VL" refers to the variable region of the immunoglobulin light chain (including the light chain of an Fv, scFv, dsFv, or Fab).

[0054] As used herein, the term "heavy chain constant region" refers to the carboxyl-terminal portion of an antibody heavy chain that does not directly participate in antibody-antigen binding but exhibits effector functions, such as interaction with Fc receptors, and has a more conserved amino acid sequence relative to the variable domains of the antibody. The "heavy chain constant region" comprises at least one of the following: CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof. The "heavy chain constant region" includes "full-length heavy chain constant region" and "heavy chain constant region fragment", the former having a structure substantially similar to the native antibody constant region, while the latter only includes "a part of the full-length heavy chain constant region". Exemplarily, a typical "full-length antibody heavy chain constant region" consists of CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it further includes the CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. Exemplarily, typical "heavy chain constant region fragments" can be selected from CH1, Fc, or CH3 domains.

[0055] As used herein, the term "light chain constant region" refers to the carboxyl-terminal portion of an antibody light chain that does not directly participate in antibody-antigen binding, and the light chain constant region can be selected from the constant κ domain or the constant λ domain.

[0056] As used herein, the term "Fc" refers to the carboxyl-terminal portion of an antibody obtained by papain digestion of a whole antibody, and typically, it comprises the CH3 and CH2 domains of the antibody. The Fc region includes, for example, native sequence Fc region, recombinant Fc region, and variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain, and thus, the Fc region may or may not include Lys447.

[0057] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase its homology with the sequence of a human antibody. Generally, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., variable region FR and / or constant region) are derived from a human immunoglobulin (receptor antibody). A humanized antibody generally retains or partially retains the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, the ability to enhance immune response, etc.

[0058] As used herein, the term "percent (%) sequence identity" refers to the percentage of amino acid (or nucleotide) residues in a candidate sequence that are identical to the amino acid (or nucleotide) residues in a reference sequence after aligning the sequences to achieve maximum percent sequence identity and introducing gaps (if necessary) (e.g., for optimal alignment, gaps may be introduced in one or both of the candidate and reference sequences, and non-homologous sequences may be disregarded for comparison purposes). For purposes of determining percent sequence identity, the alignment can be accomplished in various ways well known to those of skill in the art, such as using publicly available computer software, such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those of skill in the art can determine the appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison to a candidate sequence can show that the candidate sequence exhibits sequence identity of from 50% to 100% over the full length of the candidate sequence or over a selected portion of the contiguous amino acid (or nucleotide) residues of the candidate sequence. The length of a candidate sequence aligned for comparison purposes can be, for example, at least 30% of the length of the reference sequence (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). Molecules are identical at that position when the position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence.

[0059] As used herein, the term "Kabat numbering system" generally refers to the immunoglobulin alignment and numbering system developed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0060] As used herein, the term "specifically binds" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules, such as those specifically recognized by an antibody or an antigen-binding fragment thereof. An antibody or an antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. For example, an antibody or an antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (e.g., between 1 pM and 100 nM). An antibody or an antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or its epitope will exhibit a KD for that particular antigen or its epitope of greater than 100 nM (e.g., greater than 500 nM, 1 μM, 100 μM, 500 μM, or 1 mM). A variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein or carbohydrate. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with a protein or carbohydrate. See, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), which describe immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0061] As used herein, the term "antibody conjugate" refers to a conjugate formed by chemically bonding an antibody molecule directly or through a linker to another molecule. For example, an antibody-drug conjugate (ADC), where the drug molecule is the other molecule.

[0062] As used herein, the term "chimeric antigen receptor (CAR)" refers to a recombinant protein that comprises at least (1) an extracellular antigen-binding domain, such as the variable heavy or light chain of an antibody, (2) a transmembrane domain that anchors the CAR into an immune effector cell, and (3) an intracellular signaling domain. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a scFv. The scFv can be derived from the variable regions of a fused antibody. Alternatively or additionally, the scFv can be derived from Fab’s (rather than an antibody, e.g., obtained from a Fab library). In certain embodiments, the scFv is fused to the transmembrane domain and then to the intracellular signaling domain.

[0063] As used herein, the term "nucleic acid" includes any compound and / or substance that comprises a polymer containing nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Generally, a nucleic acid molecule is described by the sequence of its bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented as 5′ to 3′. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and hybrid polymers comprising two or more of these molecules. The nucleic acid molecule can be linear or circular. In addition, the term nucleic acid molecule includes both the sense strand and the antisense strand, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleobases having a derivatized sugar or phosphate backbone linkage or chemically modified residues. The nucleic acid molecule also encompasses DNA and RNA molecules that are suitable as vectors for the direct expression of the antibodies of the present invention in vitro and / or in vivo, e.g., in a host or a patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, such that the mRNA can be injected into a subject to produce an antibody in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP 2 101 823 B1).

[0064] As used herein, the term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition is administered.

[0065] As used herein, the terms "subject", "individual", and "patient" refer to an organism that is being treated for a specific disease or disorder (such as cancer or an infectious disease) as described herein. Examples of individuals and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the bovine family (such as domestic cattle, bison, water buffalo, elk, and yaks, etc.), sheep, and horses, etc., that are being treated for a disease or disorder (e.g., a cell proliferative disorder, such as cancer or an infectious disease).

[0066] As used herein, the term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesired physiological changes or lesions in a subject to be treated, such as the progression of a cell proliferative disorder (such as cancer or an infectious disease). Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the degree of the disease, stabilization of the disease state (i.e., not getting worse), delay or slowing of the progression of the disease, improvement or mitigation of the disease state, and remission (whether partial or complete remission), whether detectable or undetectable. Subjects in need of treatment include those who already have a disorder or disease and those who are predisposed to having a disorder or disease or intend to prevent a disorder or disease. When referring to terms such as slow down, alleviate, reduce, mitigate, and remission, their meanings also include elimination, disappearance, non-occurrence, etc.

[0067] As used herein, the term "effective amount" refers to the amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or alleviating a disease disorder or the progression of the disease. "Effective amount" also refers to the amount of a compound sufficient to alleviate symptoms, such as treating, curing, preventing, or alleviating a related medical disorder, or increasing the rate of treating, curing, preventing, or alleviating these disorders. When the active ingredient is administered alone to an individual, the therapeutically effective dose refers only to that ingredient. When applying a combination, the therapeutically effective dose refers to the combined amount of the active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0068] As used herein, the term "cancer" refers to or describes a physiological condition in a mammal that is typically characterized by unregulated cell growth. This definition includes both benign and malignant cancers.

[0069] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when mentioned herein.

[0070] Beneficial effects

[0071] Compared with the prior art, the technical solution of the present invention has at least one of the following beneficial effects:

[0072] 1. Compared with murine antibodies, the humanized antibodies of the present invention not only have the ability to bind to PD-L1, but also reduce immunogenicity while ensuring that the molecular stability and biological functions meet the requirements, which helps to reduce the risk of immune rejection when used in human subjects.

[0073] 2. The humanized antibody of the present invention exhibits good binding ability to human PD-L1 and / or cynomolgus PD-L1, which is beneficial to improving its therapeutic effect and / or conducting preclinical animal experiments.

[0074] 3. The humanized antibody of the present invention can effectively block the binding of PD-1 and PD-L1 proteins, enhance the function of T cells, and up-regulate the T cell-mediated immune response, which is beneficial to the treatment of diseases related to abnormal PD-L1 expression and / or abnormal T cell function. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Unless otherwise defined in the present invention, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0076] Figure 1 Results of SEC-HPLC for detecting the purity of the purified humanized PD-L1 antibody.

[0077] Figure 2 Results of the humanized PD-L1 antibody blocking the binding of PD-L1 and PD-1, where the positive control is Avelumab.

[0078] Figure 3 Results of FACS for measuring the binding ability of the humanized PD-L1 antibody to PD-L1 at the cellular level, where the positive control is Avelumab.

[0079] Figure 4 Results of testing the PD-L1 / PD-1 blocking ability of the humanized PD-L1 antibody using the Jurkat-PD-1 / CHO-PD-L1-NFAT system, where the positive control is Avelumab.

[0080] Figure 5 Results of the humanized anti-PD-L1 antibody promoting the secretion of IFN-γ in the mixed lymphocyte reaction, where the negative control is anti-Hel antibody and the positive control is Avelumab.

[0081] Figure 6 Results of the humanized PD-L1 antibody inhibiting the growth of A375 human melanoma in vivo.

[0082] Figure 7 Results of body weight monitoring for in vivo pharmacodynamic evaluation of the humanized PD-L1 antibody. DETAILED DESCRIPTION OF THE INVENTION

[0083] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0084] The embodiments of the present invention are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements all fall within the protection scope of the present invention.

[0085] Example 1. Antibody Humanization

[0086] First, the classical "CDRs grafting" method is used for antibody humanization, that is, the human antibody with the highest homology is selected through sequence to provide the antibody framework region (FRs), and the antigen-binding fragment complementarity-determining regions (CDRs) of the target antibody based on the Kabat nomenclature method are grafted into the former to form a humanized antibody. Secondly, to better maintain the antibody activity and affinity, based on the antibody structure modeling analysis by MOE software: 1). Select the amino acid residues at the VH-VL interface of the antibody framework region, close to or having direct interaction with the CDRs for back mutation. Such amino acid residues are mostly important for maintaining the conformation of the CDRs region; 2). Considering immunogenicity, try to select the amino acids buried inside the protein for back mutation; 3). Considering antibody stability and expression level, priority is given to mutations that reduce molecular energy. By testing the affinity of the humanized antibodies with different mutations for human PD-L1 and the binding to cells expressing PD-L1 on the surface, humanized antibodies with affinity, antibody characterization, and activity functions equivalent to or better than those of the murine PD-L1 antibody are screened.

[0087] Among them, the amino acid information of the heavy and light chain variable regions of the preferred candidate antibody molecule 156-BM12 after humanization of the murine PD-L1 antibody PDL1-156 is shown in Table 1 below.

[0088] Table 1. Specific sequence information of murine and humanized anti-PD-L1 antibody heavy chain variable region and light chain variable region

[0089]

[0090] Example 2. Expression and Purification of Humanized Antibody

[0091] 2.1 Expression of Humanized Antibody

[0092] One day before transfection, ExpiCHO-S cells (Thermo fisher, A29127) were seeded at 2.5×10 6 -4×10 6Cells were inoculated at a density of cells / mL into fresh ExpiCHO Expression Medium (Thermo Fisher Scientific, A29100-01) and cultured overnight in a shaker. On the day of transfection, the ExpiCHO-S cell suspension from overnight culture was counted. The cell viability was >95%, and the density was between 7×10 6 -10×10 6 viable cells / mL. The required cell suspension was taken and diluted to a density of 6×10 6 cells / mL with ExpiCHO Expression Medium (Thermo Fisher Scientific, A29100-01) and placed in a shaker for standby. The prepared humanized antibody expression plasmid was added to the medium for dilution, and the centrifuge tube was gently shaken to mix it evenly. Then it was added to OptiPROTMSFM-DNA Dilution (Thermo Fisher Scientific, 12309-019), and the centrifuge tube was gently shaken to mix it evenly and then left standing at room temperature for 1-5 mins. The above plasmid complex was slowly dropped into the cell suspension to be transfected, and the shaker flask was shaken during the dropping process. After transfection, the cells were placed in a shaker for overnight culture. On the first day after transfection, ExpiFectamineTMCHO Enhancer (Thermo Fisher Scientific, A29129) equivalent to 0.6% of the cell volume and 16% of ExpiCHOTM Feed (Thermo Fisher Scientific, A29129) were supplemented to the cells. The shaker flask was gently shaken during the addition process, and the cells were transferred to a shaker for 4 days of culture. On the 5th day after transfection, ExpiCHOTM Feed (Thermo Fisher Scientific, A29129) equivalent to 16% of the cell volume was supplemented to the transfected cells. The shaker flask was gently shaken during the addition process. On the 12th day after transfection, the culture medium was taken, centrifuged for 10 mins, and the supernatant was harvested.

[0093] 2.2 Purification of Humanized Antibody

[0094] The cell culture supernatant collected in Example 2.1 of high-speed centrifugation was filtered through a 0.45 + 0.22 μm filter membrane, and the first-step purification was carried out by affinity chromatography. The chromatography medium was protein A packing Mbaselect Sure (GE, 17543803) that interacts with Fc, and the equilibration buffer was PBS (2.5 g / L Na2HPO4·12H2O, 0.408 g / L NaH2PO4, 8.76 g / L NaCl, pH 7.2). After equilibration for 4 column volumes, the cell supernatant was loaded and bound, and the flow rate was controlled so that the retention time of the sample on the column was ≥5 min. After the loading was completed, the column was washed with PBS (pH 7.2) until the A280 UV absorption dropped to the baseline. Then, it was washed with 20 mM PB + 1 M NaCl (pH 6.0) to remove impurities for 2 column volumes. The column was washed with PBS (pH 7.2) again until the A280 UV absorption and conductivity reached the baseline. Finally, the chromatography column was washed with an elution buffer of 20 mM citric acid (pH 3.4), and the elution peak was collected according to the A280 UV absorption peak. The collected elution sample was neutralized to neutral with 1 M Tris-HCl (pH 9.0).

[0095] The above neutralized elution sample was ultrafiltered and concentrated and then subjected to size exclusion chromatography. The buffer was 20 mM citric acid (pH 5.5), the chromatography column was GE HiLoad Superdex 26 / 600 200 pg (GE, 28989336), the flow rate was controlled at 2.6 ml / min, the loading volume was less than 10 ml, and the target protein peaks were combined according to the A280 UV absorption. The collected protein, as Figure 1 shown, was identified by SEC-HPLC to have a purity greater than 95% and could be used for subsequent studies.

[0096] Example 3 Determination of KD of the antibody binding to human and cynomolgus monkey PD-L1 recombinant proteins

[0097] The binding affinity of the PD-L1 antibody for human and cynomolgus monkey PD-L1-His proteins was determined using a Biacore T200 (GE Healthcare). Anti-human IgG Fc (Genway, Cat. GWB-20A705) was immobilized on a CM5 chip (GE Healthcare, Cat. BR-1005-30) at 25 °C. Anti-human Fc (Genway, Cat. GWB-20A705) was diluted to 20 μg / mL with Acetate pH 5.0 (GE Healthcare, BR-1003-51). Immobilization was performed using the Amine method in the Immobilization method. Alternatively, a commercial Protein A (GE Healthcare, Cat. 29127556) chip was used for detection. The affinity of the antibody for the antigen was determined by the multi-cycle kinetics method at 25 °C. In each cycle, the antibody to be tested was first captured on the immobilized CM5 chip, then recombinant human PD-L1-His (Novoprotein, Cat. 315) and cynomolgus monkey PD-L1-His protein (Sino Biological, Cat. 90251-C08H) were injected, and finally, it was regenerated with Glycine pH 1.5 (Shanghai Reagent, Cat. 62011516). The mobile phase was HBS-EP+ Buffer (GE Healthcare, Cat. BR-1006-69), the flow rate was 30 μL / min, and the binding time was 300 seconds. The regeneration flow rate was 30 μL / min, and the time was 30 seconds. The Biacore T200 Evaluation Software (version 3.0) was used to analyze the experimental data with a 1:1 binding model, fit the equilibrium dissociation constant KD of the antibody-antigen, and determine the association rate constant ka and the dissociation rate constant kd.

[0098] As can be seen from the results, the tested PD-L1 humanized antibodies all showed an affinity of nM or higher for the binding of human PD-L1 recombinant protein and cynomolgus monkey PD-L1 recombinant protein, as shown in Table 2 below.

[0099] Table 2. Results of Biacore binding affinity KD determination of humanized PD-L1 antibodies

[0100] Antibody number Human PD-L1 (M) Cynomolgus monkey PD-L1 (M) 156-BM12 2.04E-09 1.17E-09

[0101] Example 4 Determination of the IC50 of antibody blocking the interaction between PD-L1 and PD-1

[0102] The IC50 of the anti-PD-L1 antibody blocking the binding of PD-L1 protein to PD-1 protein was determined by a competitive ELISA method. Human recombinant PD-L1 protein (Sino Biological, Cat.10084-H05H) was diluted with carbonate buffer and added to a 96-well ELISA plate at a final concentration of 1 μg / ml. After blocking with PBS solution containing 3% BSA, gradient-diluted anti-PD-L1 antibody (40 nM - 0.02 nM) and human PD-1-His recombinant protein (Sino Biological, Cat.10377-H08H) were added for co-incubation. Then, HRP-labeled anti-His tag antibody (MBL, Cat.D291-7) was added, and TMB (Thermo, Cat.34029) was used for color development. After termination with 1 M sulfuric acid, the OD value was read (dual wavelength 450 nm - 630 nm). By correlating the antibody concentration with the OD value, the competitive binding curve of the test antibody could be plotted, and the IC50 value could be calculated. Figure 2 The competitive binding curve of the anti-PD-L1 antibody to human recombinant PD-L1 protein is shown. The results indicate that the tested 156-BM12 antibody can effectively block the interaction between human PD-L1 protein and human PD-1 protein, with an IC50 of 1.489 nM, and the positive control Avelumab (Pfizer, lot: AU020322) has an IC50 of 1.263 nM.

[0103] Example 5 FACS assay for the EC50 of the PD-L1 antibody binding to cell surface PD-L1

[0104] CHO-PD-L1 cells with high cell surface expression of PD-L1 (Nanjing Yongshan Biotechnology Co., Ltd., 10 5 cells / well) were co-incubated with gradient concentrations of the antibody to be tested (antibody concentration: 10000 ng / ml - 0.1 ng / ml) at 4°C for 30 min. After incubation, 1:250 diluted anti-human IgG PE fluorescent antibody (eBioscience, Cat.12-4998-8) was added and co-incubated at 4°C for 30 min. The fluorescent antibody specifically binds to the Fc segment of the antibody to be tested, and the ability of the antibody to be tested to bind to the PD-L1 protein highly expressed on the cell surface was analyzed by detecting the level of PE fluorescence intensity through FACS. Figure 3 The results showed that the EC50 of the 156-BM12 antibody was 104.9 ng / ml, which was similar to that of the positive control Avelumab (EC50 was ~72 ng / ml) in this experiment. This detection quantitatively confirmed the ability of the 156-BM12 antibody to bind to the PD-L1 target on the cell surface in a dose-dependent manner. MFI fold = MFI value of the experimental group / MFI value of the control group without drug.

[0105] Example 6 PD-1 / PD-L1-NFAT Reporter Gene Assay for Inhibitory Effect of Anti-PD-L1 Antibody on PD-1:PD-L1 Binding and Signaling

[0106] The antagonistic effects of PD-L1 antibodies on PD-1 / PD-L1 protein interaction and its signaling pathway were compared using Jurkat cell line stably transfected with PD-1 (GenScript, Cat. 00612) and CHO cell line stably transfected with PD-L1 (GenScript, Cat. M00613). When the inhibitory signal pathway is blocked, the expression of the NFAT-controlled luciferase reporter gene is enhanced and the luciferase signal value increases. The blocking effect of the antibody on PD-L1 is reflected by the intensity of the luciferase reading (relative light units, RLU).

[0107] The CHO cell line stably transfected with PD-L1 was seeded on a 96-well white bottom plate at 40,000 cells per well, 100 μl per well, and returned to the incubator overnight. The next day, the plate was taken out, the medium was aspirated, and the cell line stably transfected with PD-1 and the PD-L1 antibody to be tested were added for co-incubation. The addition amount of PD-1 cells was 16,000 cells per well, and the antibody was serially diluted. There were 3 replicates for each dose, the incubation volume was 100 μl per well, and the incubation duration was 6 hours. When the incubation was completed, the plate was taken out, and the luciferase detection reagent was added in an equal volume (100 μl) for reading. The EC50 values of each antibody were obtained by performing a 4-parameter analysis using Graphpad on the detected values to generate a regression curve. Figure 4 It was shown that the EC50 value of the tested 156-BM12 antibody (340.8 ng / ml) was similar to that of the positive control antibody Avelumab (291 ng / ml). This assay quantitatively confirmed the dose-dependent inhibitory ability of the 156-BM12 antibody on the inhibition of T cell activity caused by the cell surface PD-1:PD-L1 interaction, thereby enhancing the activity of the reporter gene in Jurkat cells in a dose-dependent manner.

[0108] Example 7 ELISA Detection of IFN-γ Secreted by T Cells in Mixed Lymphocyte Reaction

[0109] The activity of PD-L1 monoclonal antibody in enhancing T cells was measured by mixed lymphocyte reaction (MLR). CD14 was isolated from peripheral blood mononuclear cells (PBMC) of a healthy human donor 1 +Monocytes were induced to differentiate into dendritic cells (DCs) in vitro using recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF, Peprotech, Cat.300-03) and recombinant human interleukin 4 (rhIL-4, Peprotech, Cat.200-04). On the 6th day of culture, lipopolysaccharide (LPS, Sigma, Cat: L4516) was added to stimulate the maturation of DCs. On the 7th day, the DC cells from donor 1 were mixed and co-cultured with CD4 + T cells enriched from the PBMCs of healthy donor 2. The ratio of DC:CD4 + T cells was 1:10. The test antibody, negative control antibody anti-Hel, and positive control antibody Avelumab (antibody concentration: 12.5 nM - 0.125 nM) were added, and the co-culture was carried out for 4 days. After 4 days, the cell culture supernatant was collected, and the content of IFN-γ in the supernatant was detected by ELISA. As Figure 5 shown, compared with the anti-Hel monoclonal antibody negative control group, 156-BM12 and the positive control antibody Avelumab could significantly enhance the ability of CD4 + T cells to secrete IFN-γ in the MLR experiment, and as the concentration of the PD-L1 antibody drug decreased, the activity of increasing IFN-γ secretion also decreased. This result indicates that 156-BM12 can enhance the function of T cells and has a dose-dependent relationship. (T-test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.)

[0110] Example 8 Evaluation of the Efficacy of HuPBMC Humanized Mice in Vivo

[0111] A375 cells (Beina Biotechnology, BNCC100266) were inoculated subcutaneously into the right side of female 5-6-week-old NPG mice (Beijing Vitalstar Biotechnology Co., Ltd.) at a concentration of 5×10 6 cells / 0.1 mL. After the inoculation of A375 cells, Hu PBMC cells (ALLCELLs, PB005F-C) were injected into the mice via the tail vein at a concentration of 5×10 6 cells / 0.2 mL. When the tumor grew to approximately 100 mm 3 in volume, 24 mice were randomly selected and divided into 3 groups of 8 mice each according to the tumor volume. The three groups were: Vehicle (PBS), Tecentriq (30 mg / kg; lot NO. HK65567, Roche), and 156-BM12 (30 mg / kg). The administration route for all groups was intraperitoneal injection, and the drug was administered 3 times a week for 5 consecutive times. The experiment ended 2 days after the last administration. During the administration and observation period, the body weight and tumor volume of the mice were measured 3 times a week, and the measured values were recorded, and the tumor volume was calculated (long diameter × short diameter2 / 2) and tumor growth inhibition rate TGI TV (%) = (1 - (Tn / T0) / (Vn / V0)) × 100%, where Tn represents the average tumor volume at the experimental endpoint of the treatment group; T0 represents the average tumor volume at the starting point of the treatment group; Vn represents the average tumor volume at the endpoint of the vehicle group; V0 represents the tumor volume at the starting point of the vehicle group. On the 11th day of grouped drug administration, compared with the control group, the positive drug Tecentriq group and the PD-L1 antibody 156-BM12 group had significant and similar inhibitory effects on tumor volume, and there were statistical differences (P < 0.05). For details, see Figure 6 Table 3.

[0112] Table 3. Effects of test substances on the tumor volume of A375 cell-transplanted HuPBMC NPG mice

[0113]

[0114] Note: a: Mean ± standard error;

[0115] b: The tumor volume of the drug administration group was statistically compared with that of the Vehicle control group on the 11th day of grouped drug administration. Two-way ANOVA analysis, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0116] The experimental animals had good activity and feeding status during drug administration, and their body weights showed an overall upward trend, indicating that 156-BM12 had high safety. For details, see Figure 7 Table 4.

[0117] Table 4. Effects of test substances on the body weights of A375 cell-transplanted HuPBMC NPG mice

[0118]

[0119] Note: a: Mean ± standard error;

[0120] The final results showed that the humanized PD-L1 antibody 156-BM12 had a significant inhibitory effect on the growth of A375 subcutaneous tumor xenografts and showed high safety. The TGI levels of the two were comparable to those of the positive control antibody Tecentriq. SEQUENCE LISTING <110> Shandong Simcere Biopharmaceutical Co., Ltd. <120> Antibody against human programmed death ligand-1 (PD-L1) and its application <130> simcere03 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 1 Glu Val Gln Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Asn Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Asp Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Ser Val Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110 Ser Ser <210> 2 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 2 Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Val 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Leu Thr Phe Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 3 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Asn Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser <210> 4 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 4 Glu Ile Val Leu Thr Gln Ser Pro Ala Leu Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Asn Tyr Val 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Leu Ile Tyr 35 40 45 Leu Thr Phe Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105

Claims

1. An isolated antibody or antigen-binding fragment that specifically binds to human programmed death ligand-1 (PD-L1), characterized in that, The sequences of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment are as shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

2. The antibody or antigen-binding fragment according to claim 1, wherein The antibody or antigen-binding fragment is a humanized antibody or a fragment thereof.

3. The antibody or antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody comprises the sequence of any one of the constant regions of human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

4. The antibody or antigen-binding fragment according to claim 3, wherein The antibody comprises the sequence of the constant region of human or murine antibodies IgG1, IgG2, IgG3 or IgG4.

5. The antibody or antigen-binding fragment according to claim 1 or 2, characterized in that, The antigen-binding fragment is selected from one or more of F(ab’)2, Fab’, Fab, Fv, scFv.

6. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody, antigen-binding fragment, or any combination thereof as claimed in any one of claims 1-5.

7. An expression vector comprising the isolated nucleic acid molecule as claimed in claim 6.

8. An isolated host cell comprising the isolated nucleic acid molecule as claimed in claim 6, or the expression vector as claimed in claim 7.

9. The host cell according to claim 8, characterized in that, The host cell is a eukaryotic cell or a prokaryotic cell.

10. The host cell according to claim 8, characterized in that, The host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli and / or Bacillus subtilis.

11. The host cell according to claim 8, characterized in that, The host cell is selected from Chinese hamster ovary cells (CHO).

12. A method for preparing the antibody or antigen-binding fragment according to any one of claims 1-5, characterized in that, Culturing the host cell as claimed in any one of claims 8-11 under appropriate conditions, and isolating the antibody or antigen-binding fragment.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody or antigen-binding fragment as claimed in any one of claims 1-5, the isolated nucleic acid molecule as claimed in claim 6, the expression vector as claimed in claim 7, the host cell as claimed in any one of claims 8-11, or the product prepared by the method as claimed in claim 12.

14. The pharmaceutical composition according to claim 13, characterized in that, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier or adjuvant.

15. The pharmaceutical composition according to claim 13 or 14, characterized in that, The pharmaceutical composition further comprises an additional anti-tumor agent.

16. Use of the antibody or antigen-binding fragment as claimed in any one of claims 1-5, the isolated nucleic acid molecule as claimed in claim 6, the expression vector as claimed in claim 7, the host cell as claimed in any one of claims 8-11, the product prepared by the method as claimed in claim 12, or the pharmaceutical composition as claimed in any one of claims 13-15 in the preparation of a medicament for treating tumors, wherein the tumors are selected from melanoma, glioma, breast cancer, lung cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, testicular cancer, salivary gland cancer, thyroid cancer, thymic cancer, epithelial cancer, colon cancer, rectal cancer, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body cancer and osteosarcoma.

17. A kit comprising the antibody or its antigen-binding fragment as claimed in any one of claims 1-5, the isolated nucleic acid molecule as claimed in claim 6, the expression vector as claimed in claim 7, the host cell as claimed in any one of claims 8-11, the product prepared by the method as claimed in claim 12, or the pharmaceutical composition as claimed in any one of claims 13-15.

18. The kit according to claim 17, characterized in that, The kit further comprises instructions for use.

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