WO2018133842A1 - 人程序性死亡受体pd-1的单克隆抗体及其片段 - Google Patents

人程序性死亡受体pd-1的单克隆抗体及其片段 Download PDF

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WO2018133842A1
WO2018133842A1 PCT/CN2018/073437 CN2018073437W WO2018133842A1 WO 2018133842 A1 WO2018133842 A1 WO 2018133842A1 CN 2018073437 W CN2018073437 W CN 2018073437W WO 2018133842 A1 WO2018133842 A1 WO 2018133842A1
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antibody
seq
antigen
binding fragment
amino acid
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French (fr)
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陈列平
罗利群
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大有华夏生物医药集团有限公司
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Definitions

  • the present invention relates to the field of biopharmaceuticals, and in particular to monoclonal antibodies directed against human PD-1 and their use in the treatment of diseases.
  • PD-1 Programmed Death Receptor 1
  • PD-L1 is an immunosuppressive receptor expressed on activated T cells and B cells. Blocking the interaction between PD-1 and one of its ligands, PD-L1, increases the immunity of tumor-specific CD8 + T cells, thus helping the immune system clear tumor cells.
  • Molecular and Biochemical Aspects of the PD-1 Checkpoint Pathway, Vassiliki A. Boussiotis, NEJM n engl j med 375; 18 nejm.org November 3, 2016 provides a detailed review of PD-1 and related studies.
  • PD-1 on tumor infiltrating lymphocytes
  • PD-L1 on tumor cells
  • Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma, and Pancreatic cancer and head and neck cancer.
  • Blocking the interaction of PD-1/PD-L1 leads to increased immunity of tumor-specific T cells and, therefore, helps to clear tumor cells by the immune system.
  • the PD-1/PD-L1 pathway is a well-established target for the development of antibody therapies for cancer therapy.
  • Anti-PD-1 antibodies can also be used for chronic viral infections. Recently, studies have shown that PD-1 is highly expressed in T cells from HIV-infected individuals.
  • the invention provides an isolated monoclonal antibody or antigen-binding fragment thereof directed against human PD-1 comprising: the heavy chain CDR1, CDR2 and CDR3 set forth in Table 2, and the light chain CDR1, CDR2 and CDR3.
  • the antibody or antigen-binding fragment thereof comprises: (i) the heavy chain amino acid sequence set forth in SEQ ID NO: 3 and the light chain amino acid sequence set forth in SEQ ID NO: 4; or (ii) no The heavy chain amino acid sequence of SEQ ID NO: 3 comprising the signal peptide portion and the light chain amino acid sequence of SEQ ID NO: 4 not comprising the signal peptide portion.
  • the first to the 19th position of the heavy chain amino acid sequence shown by SEQ ID NO: 3 is a signal peptide moiety
  • the first position of the light chain amino acid sequence shown by SEQ ID NO: 4 is -
  • the 20th position is the signal peptide moiety.
  • the signal peptide is added for the purpose of facilitating purification, etc., and is not an essential part of the antibody structure. It will be understood by those skilled in the art that in the antibody or antibody fragment of the present invention, the signal peptide is optional, that is, it may or may not contain a signal peptide.
  • the invention also provides a humanized isolated monoclonal antibody or antigen-binding fragment thereof directed against human PD-1 comprising:
  • the first to the 24th positions of the heavy chain amino acid sequence shown in SEQ ID NOS: 13, 14, 15 and 16 are signal peptide portions; SEQ ID NOs: 17, 18, 19 and 20
  • the first to the 20th position of the shown light chain amino acid sequence is a signal peptide moiety.
  • the antigen binding fragment is selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab') 2, and diabodies.
  • the invention provides an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof.
  • the heavy chain coding sequence of the polynucleotide is set forth in SEQ ID NO: 1
  • the light chain coding sequence is set forth in SEQ ID NO: 2.
  • the polynucleotide encodes a humanized antibody or antigen-binding fragment thereof, and the heavy chain coding sequence is selected from SEQ ID NOs: 5, 6, 7, and 8, and the light chain coding sequence is selected from the group consisting of SEQ ID NO : 9, 10, 11 and 12.
  • the invention provides an expression vector comprising the isolated polynucleotide, and a host cell comprising the expression vector.
  • the invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.
  • the invention provides a method of increasing the activity of an immune cell comprising contacting an immune cell with the antibody or antigen-binding fragment thereof.
  • the invention provides a method of treating a disease comprising administering a therapeutically effective amount of an antibody of the invention, or an antigen binding fragment thereof, to a subject in need of treatment.
  • the method comprises co-administering other drugs or therapies to the subject, including chemotherapy, radiation therapy, targeted therapy, gene therapy, cell therapy, stem cell therapy.
  • the invention provides the use of the antibody or antigen-binding fragment thereof for the preparation of a medicament for the treatment of a disease, wherein the disease is selected from the group consisting of cancer, infectious diseases, transplant rejection, autoimmune diseases and neurological diseases.
  • the invention provides a method of modulating cellular function comprising delivering a polynucleotide sequence encoding an antibody of the invention, or an antigen-binding fragment thereof, to said cell.
  • the method is for gene therapy, CAR-T therapy, or CRISPR therapy.
  • mice that have been knocked out of the PD-1 gene are used to produce an anti-human PD-1 mAb with higher affinity, better blocking effect, and higher cell killing activity.
  • the test results show that this method can obtain high affinity monoclonal antibodies. Its affinity is about 4-7 times higher than that of the currently marketed PD-1 antibody, and the interaction of blocking PD-L1/PD-1 in vitro is very effective. At the same time, the inhibition of tumor growth activity in vitro has also been improved.
  • Figure 1 shows the binding activity of PD-1 antibody to PD-1 protein.
  • Figure 2 shows the blocking effect of PD-1 antibody on PD-1 protein binding to PD-L1 protein.
  • Figure 3 shows the results of competitive binding assays for different antibodies.
  • Figure 4 shows the binding and blocking effects of recombinant DNA antibodies.
  • Figure 5 shows the binding activity of humanized antibodies to PD-1 protein.
  • Figure 6 shows the blocking effect of humanized antibodies.
  • Figure 7 shows a comparison of the affinity of different mutants of humanized maternal antibodies.
  • Figure 8 shows PD-1 protein binding of humanized PD-1 antibody and cynomolgus lymphocytes.
  • Figure 9 shows that humanized PD-1 antibody enhances effector cell killing of tumor cells in vitro.
  • Figure 10 shows that humanized PD-1 antibody blocks the induced expression of PD-1 in human lymphocytes.
  • Figure 11 shows a comparison of the kinetics and affinity of the self-developed PD-1 antibody to the marketed PD-1 antibody.
  • Figure 12 shows a comparison of inhibition of tumor growth in vitro.
  • Antibody refers to any form of antibody that exhibits the desired biological activity (eg, inhibits binding of a ligand to its receptor or by inhibiting ligand-induced receptor signaling).
  • antibody is used in its broadest sense and includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies).
  • Antibody fragments and “antigen-binding fragments” are used interchangeably herein to refer to antigen-binding fragments of antibodies and antibody analogs, which typically include at least a portion of the antigen-binding or variable region of a parental antibody ( For example one or more CDRs).
  • the antibody fragment retains at least some of the binding specificity of the parent antibody.
  • the antibody fragment retains at least 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv, single antibodies (technology from Genmab) Nanobodies (techniques from Domantis); domain antibodies (technology from Ablynx); and multispecific antibodies formed from antibody fragments.
  • Engineered antibody variants are reviewed in Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136.
  • a "Fab fragment” consists of a light chain and a heavy chain of CH1 and a variable region.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • Fc region contains two heavy chain fragments C H 1 and C H 2 domain of an antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic action of the CH3 domain.
  • Fab 'fragment contains one light chain and a portion comprising a V H domain and a C H 1 domain and a heavy chain region between a C H 2 domain and C H, whereby two Fab' fragments An interchain disulfide bond is formed between the two heavy chains to form an F(ab') 2 molecule.
  • F (ab ') 2 fragment contains two light chains and two heavy chain C H 1 portion comprises a C H 2 constant region and between the domains, thereby forming two inter-chain between the two heavy chains Sulfur bond.
  • the F(ab') 2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
  • the "Fv region” contains variable regions from both heavy and light chains, but lacks a constant region.
  • Single-chain Fv antibody refers to an antibody fragment comprises an antibody V H domain and V L, wherein these domains are present in a single polypeptide chain.
  • Fv polypeptide further comprises a polypeptide linker between the V H and V L, domains, linker which enables the scFv to form a desired structure for antigen binding. See International Patent Application WO 88/01649.
  • a “diabody” is a small antibody fragment that has two antigen binding sites.
  • the fragments comprise a heavy-chain variable domain (V H) (V H -V L or V L -V H) is connected to a light chain variable domain (V L) in the same polypeptide chain.
  • V H heavy-chain variable domain
  • V L light chain variable domain
  • chimeric antibody refers to an antibody and a fragment of the antibody that exhibits the desired biological activity, wherein a portion of the heavy and/or light chain corresponds to an antibody derived from a particular species or belonging to a particular antibody class or subclass.
  • the sequence is identical or homologous, while the remainder of the strand is identical or homologous to the sequence corresponding to an antibody from another species or belonging to another antibody class or subclass (see, for example, U.S. Patent No. 4,816,567 and Morrison et al. 1984, Proc. Natl. Acad. Sci. USA 81: 6851-6855).
  • a "humanized" form of a non-human (eg, murine) antibody is a chimeric antibody that contains minimally derived non-human immunoglobulin sequences.
  • a humanized antibody comprises at least one and usually almost all of two variable domains, wherein all or nearly all hypervariable loops correspond to hypervariable loops of non-human immunoglobulins, all or almost all FR regions The FR region of the human immunoglobulin sequence.
  • the humanized antibody also optionally comprises at least a portion of an immunoglobulin (typically human immunoglobulin) constant region (Fc).
  • an immunoglobulin typically human immunoglobulin constant region (Fc).
  • an “isolated” antibody is one that has been identified and separated from its natural environmental components.
  • the antibody is purified to the extent that: (1) more than 95% by weight of antibody, most preferably more than 99% by weight, as determined by the Lowry method; (2) sufficient to obtain by using a rotary cup sequencer The extent of at least 15 residues of the N-terminal or internal amino acid sequence; or (3) is determined to be homogenous by SDS-PAGE staining with Coomassie blue or preferably silver under reducing or non-reducing conditions.
  • nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule, typically associated with it in the natural source of the antibody nucleic acid. Thus, an isolated nucleic acid molecule differs from a nucleic acid molecule that is present in its natural cells.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., in addition to the possible natural mutations that may be present in minor amounts, the individual antibodies comprising the population are identical. Monoclonal antibodies are highly specific and can target individual antigenic sites. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include a plurality of different antibodies directed against a plurality of different determinants (epitopes), each monoclonal antibody is directed only to a single determinant on the antigen.
  • Monoclonal antibodies denotes the property of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring preparation of the antibody by any particular method.
  • Monoclonal antibodies herein specifically include “chimeric" antibodies.
  • Immune cell includes cells that have a hematopoietic origin and function in an immune response.
  • Immune cells include: lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • a sequence “variant” refers to a sequence that differs from the sequence shown at one or more amino acid residues but retains substantially its biological activity. For example, a variant may retain at least 50%, preferably at least 70%, or even 90% of the biological activity of the original sequence.
  • references to an animal, a human, a cell or a subject by "administering” and “treating” means contacting the exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, human, cell or subject.
  • administering and “treating” means contacting the exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, human, cell or subject.
  • administering and “treatment” may refer to, for example, methods of treatment, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods.
  • administering” and “treatment” also mean in vitro and ex vivo treatment of cells, for example, by reagents, diagnostics, therapies, combinations, or by other cells.
  • terapéuticaally effective amount refers to an amount of active compound that is sufficient to cause a biological or medical response desired by a clinician in a subject.
  • the "therapeutically effective amount” of an antibody of the invention can be determined by one skilled in the art based on factors such as the route of administration, the subject's weight, age, condition, and the like. For example, a typical total weekly dose can be at least 0.05 [mu]g/kg body weight, typically at least 1 [mu]g/kg body weight.
  • the medicament provided by the present invention can be formulated into a clinically acceptable dosage form such as a powder or an injection.
  • the pharmaceutical composition of the present invention can be administered to a subject using any suitable route, for example, by oral, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or by inhalation, transdermal, and the like. Route administration.
  • the pharmaceutical preparation of the present invention comprises the PD-1 antibody of the present invention or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition of the present invention may further comprise other agents including, but not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic drugs or antimetabolites, targeted tumor drugs, immunostimulants or immunomodulators or with cytotoxic agents, cells. Growth inhibitor or other toxic drug conjugated antibody.
  • the pharmaceutical composition can also be administered with other forms of treatment, such as surgery, chemotherapy, and radiation.
  • An antibody or antigen-binding fragment of the invention that specifically binds to human PD-1 can be used to increase, augment, stimulate or upregulate an immune response.
  • the antibodies and antibody fragments of the invention are particularly useful for treating a subject suffering from a disease that can be treated by increasing a T cell mediated immune response.
  • Preferred subjects include human patients in need of an immune response.
  • the antibodies or antigen-binding fragments of the invention can be used to treat cancer (i.e., inhibit the growth or survival of tumor cells).
  • Preferred cancers which can be inhibited by the antibodies of the invention include cancers which are generally responsive to immunotherapy, but also include cancers which have not been associated with immunotherapy to date.
  • Non-limiting examples of preferred cancers for treatment include melanoma (e.g., malignant metastatic melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., prostate cancer that is difficult to control), pancreatic adenocarcinoma , breast cancer, colon cancer, lung cancer (eg non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymph Tumors and other malignant tumors.
  • the invention encompasses refractory or recurrent cancers which can be inhibited by the antibodies of the invention.
  • the antibody of the present invention or antigen-binding fragment thereof can be used alone or in combination with other anti-tumor drugs or immunogenic agents (for example, attenuated cancer cells, tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), antigen presenting cells)
  • immunogenic agents for example, attenuated cancer cells, tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), antigen presenting cells
  • dendritic cells stimulated with tumor-derived antigens or nucleic acids, immunostimulatory cytokines (eg, IL-2, IFNa2, GM-CSF_), and genes transfected with immunostimulatory cytokines (such as, but not limited to, GM-CSF) Dyed cells; standard cancer treatments (eg chemotherapy, radiotherapy or surgery); or other antibodies (including but not limited to antibodies against VEGF, EGFR, Her2/neu, VEGF receptors, other growth factor receptors, CD20, CD40, CTLA
  • the antibodies or antigen-binding fragments thereof of the invention may also be used to prevent or treat infections and infectious diseases.
  • the antibody or antigen-binding fragment thereof can be used alone or in combination with a vaccine to stimulate an immune response against pathogens, toxins, and autoantigens.
  • Antibodies or antigen-binding fragments thereof can be used to stimulate an immune response to a human infected virus such as, but not limited to, human immunodeficiency virus, hepatitis A, hepatitis B, hepatitis C virus, Epstein-Barr virus, human cytomegalovirus, Human papillomavirus, herpes virus.
  • Antibodies or antigen-binding fragments thereof can be used to stimulate an immune response to infections caused by bacterial or fungal parasites and other pathogens.
  • the antibodies of the invention or antigen-binding fragments thereof are also useful for preventing or treating transplant rejection, autoimmune diseases, and neurological diseases.
  • autoimmune disease refers to any disease caused by an autoimmune response, ie, an immune response against a substance in the body of a subject.
  • autoimmunity can affect any organ or tissue of a subject, such as the brain, skin, kidney, lung, liver, heart or thyroid of the subject, the clinical manifestations of the disease depend on the affected site.
  • Typical autoimmune diseases include, but are not limited to, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, Still's disease, juvenile arthritis, lupus, diabetes, myasthenia gravis, Hashimoto's thyroiditis, Aode thyroiditis, Graves' disease, rheumatoid arthritis syndrome, multiple sclerosis, infectious neuronitis, acute disseminated encephalomyelitis, Addison's disease, visual ocular palsy-muscle array Sputum syndrome, ankylosing spondylitis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hepatitis, celiac disease, Goodpasch syndrome, idiopathic thrombocytopenic purpura, optic neuritis, scleroderma , primary biliary cirrhosis, Lytle syndrome, high arteritis, temporal arteritis, warm autoimmune hemolytic anemia, Wegener's granulomatos
  • Neurological disease refers to a disease or condition that affects the central nervous system and/or has a cause in the central nervous system.
  • exemplary neurological diseases include, but are not limited to, Parkinson's disease, Alzheimer's disease, prion disease, Huntington's disease, Rett syndrome. Wait.
  • the anti-PD-1 antibodies or antigen-binding fragments thereof of the invention may be used in combination with other drugs or therapies, for example, in combination with one or more other therapeutic agents, such as cytotoxic agents, radiotoxic agents or immunosuppressive agents.
  • the antibody can be linked to the agent (as an immune complex) or can be administered separately from the therapeutic agent, for example, the antibody can be administered before, after or simultaneously with administration of the therapeutic agent.
  • the antibody of the present invention can be used in combination with chemotherapy, radiotherapy, targeted therapy, gene therapy, cell therapy, stem cell therapy and the like, especially for the treatment of cancer.
  • the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention or a gene encoding the same can also be introduced into a cell to regulate cell function or activity, or to treat a disease.
  • the method includes in vitro and in vivo applications. This method is particularly suitable for gene therapy, CAR-T therapy or CRISPR therapy.
  • Non-therapeutic applications of the antibodies and antibody fragments of the invention include: for flow cytometry analysis, immunohistochemistry and in vitro functional assays, Western blotting and ELISA, and the like.
  • the antibodies of the invention can also be used as affinity purification reagents.
  • the antibodies can also be used in diagnostic assays, for example, to detect expression of PD-1 in a particular cell, tissue or serum.
  • antibodies are typically labeled (directly or indirectly) with a detectable moiety.
  • markers can be utilized, which are generally classified into the following classes: biotin, fluorescent dyes, radionucleotides, enzymes, iodine, and biosynthetic markers.
  • the antibodies of the invention can be used in any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.
  • Antibodies can also be used in in vivo diagnostic assays.
  • an antibody is labeled with a radionuclide (eg, 111 In, 99 Tc, 4 C, 3 1I, 125 I, 3 H, 32 P, 35 S, or 18 F) to localize the antigen or cells expressing the antibody.
  • a radionuclide eg, 111 In, 99 Tc, 4 C, 3 1I, 125 I, 3 H, 32 P, 35 S, or 18 F
  • T lymphocytes were isolated from human peripheral blood mononuclear cells (PBMC) using MACS magnetic beads (Miltenyi Biotec), RNA (RNeasy mini kit, QIAGEN) was extracted and first-strand cDNA (SuperScript First-Strand Synthesis System, Invitrogen,) was obtained.
  • the full length sequence of the human PD-1 molecule was obtained by a PCR reaction according to the human PD-1 gene DNA sequence (NCBI Reference Sequence: NM_005018.2).
  • the human PD-1 fusion protein inserts the extracellular partial sequence of PD-1 into an expression plasmid mIgV (HDong et al Nat Med. 1999; 5: 1365-1369) containing the mouse immunoglobulin G2aCH2-CH3 domain. .
  • the plasmid DNA was transfected into 293T cells with PEI reagent (Sigma-Aldrich) by transient expression method to generate human PD-1 mouse Ig fusion protein (hPD-1mIg), and the cell secreted supernatant was subjected to IgA purification column (HiTrap Protein A HP, GE healthcare) purified protein and confirmed by SDS-PAGE gel electrophoresis.
  • PEI reagent Sigma-Aldrich
  • IgA purification column HiTrap Protein A HP, GE healthcare
  • a human PD-1 protein stably expressing cell line To detect and identify PD-1 antibodies, we constructed a human PD-1 protein stably expressing cell line.
  • the full-length PD-1 sequence obtained above was inserted into the pcDNA3.1(-) expression plasmid, and transfected into Chinese hamster ovary fibroblasts (CHO) by lipofactamin 2000 transfection reagent (Thermo Fisher Scientific), and stably expressed by G418. Positive cell line (CHO/hPD1).
  • mice Female Balb/c mice, 6-8 weeks old, were immunized with hPD-1mIg fusion protein and Freund's complete adjuvant (CFA, Sigma) mixed emulsion subcutaneously, 80-100 ⁇ g protein per mouse. After 3 weeks, the same fusion protein and the incomplete adjuvant (IFA, Sigma) emulsion were used for booster immunization, and the immunization was performed 3 times. Serum titers were determined about 2 weeks after immunization of the mice, and serum titers were determined by flow cytometry using CHO/hPD1 cells. The secondary antibody was fluorescently labeled with goat anti-mouse IgG (eBioscience). When the serum titer was sufficiently high, 80 ⁇ g of hPD-1mIg fusion protein was dissolved in PBS, boosted once by intraperitoneal injection, and spleen was taken out for fusion 5 days later.
  • CFA complete adjuvant
  • Hybridoma cell production was performed by spleen cells of immunized mice and mouse myeloma cell line (SP2/0-Ag14) fused by polyethylene glycol (PEG1500, Roche) fusion agent, and the fused cells were subjected to HAT (Sigma). Aldrich) selection culture, using the high-throughput transfection and screening system established by our laboratory (S Yao et al. Immunity. 2011; 34(5): 729-40.) to identify the culture supernatant, screening for positive monoclonal The antibody was confirmed by flow cytometry to be able to bind to PD-1 positively expressing cells. The selected positive cloned cell lines were subcloned after 5 times of limiting dilution to ensure a positive clone from a single source.
  • Blocking effect of PD-1 antibody on PD-1 receptor protein and its ligand PD-L1 binding expression of PD-L1 ligand protein on tumor cells and its binding to PD-1 receptor expressed by lymphocytes Inhibition of immune cell function leads to tumor evasion of host immune system surveillance, and blocking the interaction between PD-1 and PD-L1 is an important mechanism of anti-tumor immunotherapy of PD-1 antibody.
  • To detect the blocking activity of the antibody we mixed 100 ng of hPD-1hIg fusion protein with different doses of 100 ng, 200 ng, 400 ng and 800 ng of different PD-1 antibodies, and set up a control group of mIgG and PBS buffer.
  • Antibody competition binding assay To clarify whether these PD-1 antibodies recognize the same or different epitopes, we performed antibody competition binding experiments. First, 10 ⁇ g of different PD-1 antibodies were incubated with CHO/hPD-1 cells for 30 minutes at 4 ° C, and after centrifugation with 1% fetal calf serum in PBS buffer, 0.1 ⁇ g of Biotin-labeled Ab4 or Ab5, incubate at 4 ° C for 20 minutes, wash and add fluorescently labeled streptavidin (SA; eBioscience), incubate at 4 ° C for 20 minutes, wash and analyze by flow cytometry. The results show that Ab4 and Ab5 compete with each other for binding to the same epitope, which has partial competitive binding to Ab3 and no competitive binding to Ab1 and Ab2 (Fig. 3).
  • the downstream primer was subjected to PCR cycle using SMART first-strand cDNA as a template.
  • the PCR products were then ligated to the T vector (Zero Blunt TOPO PCR Cloning Kit, Invitrogen K2875-J10), respectively.
  • the clones were selected for sequence determination and analysis, and the light chain and heavy chain variable region sequences of the antibody were obtained.
  • the primers were designed according to the heavy chain variable region sequence and the mouse IgG1 sequence, and the full-length sequence of the heavy chain was obtained by PCR, and the VBASE2 pair was used.
  • the sequence was analyzed to determine the amino acid sequence of the light and heavy chains of the antibody and the corresponding CDR regions (see Table 2). See Table 1 for the DNA coding sequence.
  • the full-length sequences of the light and heavy chains of the antibody were inserted into the pcDNA3.1(-) expression plasmid, and the protein product was obtained by transient transfection of 293T cells. The protein product was confirmed by purification and identification.
  • the original antibody was able to bind to the PD-1 protein (Fig. 4.A) and was able to block the binding of the PD-1 protein to PD-L1 (Fig. 4.B).
  • the antibody sequences were humanized.
  • the Fc portion of the antibody heavy chain was replaced with the human IgG4 sequence, and the CPSCP of the IgG hinge region 4 was replaced with the IgG1 CPPCP (S228P), and the antibody light chain was replaced with the adult kappa chain to obtain the human-matrix chimeric parent.
  • Antibody (Chimeric Ab) Humanized heavy chain variable region and light chain variable region sequences were humanized after confirming the functional activity of the maternal antibody, and the resulting three humanized heavy chain variants were obtained (Table 3, Sequences 6–8, Table 4).
  • Sequences 14–16), 3 humanized light chain variants (Table 3, Sequence 10–12, Table 4, Sequences 18–20) were combined with each other to obtain 9 different variant sequences (Variants 1 to Variants 9).
  • One maternal and nine variant sequences were cloned into a high expression vector, and the antibody supernatant was obtained by transient transfection of 293 cells. The purified antibody was confirmed by SDS-PAGE gel electrophoresis and sequencing verification. The DNA and amino acid sequences of the antibodies are shown in Tables 3 and 4.
  • Variants 3 has a heavy chain amino acid sequence of SEQ ID NO: 6, a light chain amino acid sequence of SEQ ID NO: 12;
  • Variants 4 has a heavy chain amino acid sequence of SEQ ID NO: 7, and a light chain amino acid sequence of SEQ ID NO: 10;
  • Variants 5 has a heavy chain amino acid sequence of SEQ ID NO: 7, a light chain amino acid sequence of SEQ ID NO: 11;
  • Variants 6 has a heavy chain amino acid sequence of SEQ ID NO: 7, and a light chain amino acid sequence of SEQ ID NO: 12;
  • Variants 7 has a heavy chain amino acid sequence of SEQ ID NO: 8, a light chain amino acid sequence of SEQ ID NO: 10;
  • Variants 8 has a heavy chain amino acid sequence
  • Binding activity of humanized antibody to PD-1 protein The chimeric parental and mutant antibodies were tested and compared, and CHO/hPD-1 cells were suspended in PBS containing 1% calf serum and dispensed. In the flow tube, different doses of maternal and mutant antibodies were added, incubated at 4 ° C for 30 minutes, and washed with 1% calf serum in PBS buffer and then fluorescently labeled goat anti-human IgG secondary antibody (eBioscience). After incubation at 4 ° C for 20 minutes, the cells were analyzed by flow cytometry. The results showed that all humanized mutants could bind to PD-1 protein. Compared with the chimeric parental antibody, some of the mutant antibodies had higher binding ability and some were relatively lower (Fig. 5).
  • Blockade of PD-1 receptor protein and its ligand PD-L1 by humanized antibody 400ng, 300ng, 200ng, 100ng and 50ng different PD-1 antibodies were mixed with 100ng hPD-1mIg fusion protein, respectively.
  • the hIgG and PBS buffer control group were incubated at 4 ° C for 30 minutes, and then treated with PD-L1 positive expression cells (CHO/PD-L1) for 30 minutes, washed with 1% calf serum in PBS buffer and then added.
  • Fluorescently labeled goat anti-mouse IgG- incubated at 4 ° C for 20 minutes, washed and detected by flow cytometry.
  • the results showed that all humanized mutant antibodies blocked the binding of PD-1 to PD-L1 protein in a dose-dependent manner, and some mutants blocked the parent antibody more strongly (Fig. 6).
  • Humanized PD-1 antibody cross-links with lymphocyte PD-1 protein of cynomolgus monkey For future preclinical toxicological testing, we tested whether PD-1 antibody binds to monkey PD-1 protein.
  • the peripheral mononuclear cells of the cynomolgus monkey were taken, and the lymphocytes were separated from the lymphocyte separation solution in 10% fetal calf serum RPMI 1640 culture medium, and added to a 24-well culture plate coated with 1 ⁇ g/ml anti-human CD3 antibody or added.
  • 10 ng/ml anti-hIFN ⁇ and 50 ⁇ L hIL2, 5% CO2 were added to the culture medium, and cultured at 37 ° C for 2 days.
  • the harvested cells were washed once with PBS, PD-1 antibody was added, and incubation was carried out for 30 minutes at 4 ° C. After washing, fluorescently labeled anti-human IgG was added, and the mixture was incubated at 4 ° C for 20 minutes, washed and detected by flow cytometry. The results showed that the humanized PD-1 antibody was able to bind to the cynomolgus lymphocyte PD-1 protein (Fig. 8).
  • the allo CTL effector cells and the 624Mel tumor cells are mixed in a certain ratio, and then the PD-1 antibody or the control protein is added for 5 days, and the cells are stained with 0.5% crystal violet, and then the OD (450 nm) value is read by a microplate reader, according to Tumor cell survival was calculated for killing activity.
  • Humanized PD-1 antibody inhibits the expression of PD-1 in human lymphocytes: Tumor cells expressing PD-L1 can induce peripheral lymphocytes to express PD-1, which leads to inhibition of lymphocyte activity and can not kill tumor cells. The in vitro model mimicked this situation and examined whether the expression of PD-1 in human lymphocytes can be inhibited by the addition of PD-1 antibody to restore normal lymphocyte function.
  • the self-developed PD-1 antibody and three listed antibodies or control proteins were co-cultured for 5 days, and the cells were stained with 0.5% crystal violet.
  • the OD (450 nm) value was read by a microplate reader, and the killing activity was calculated according to the survival of the tumor cells.
  • the results showed that the self-developed PD-1 antibody and the three marketed antibodies can enhance the function of the effector cells to kill tumor cells in vitro, and the self-developed antibody.
  • Monoclonal antibody parental heavy chain hIgG4-S228P (SEQ ID NO: 5)
  • Humanized heavy chain variant 1 (SEQ ID NO: 6)
  • Humanized heavy chain variant 2 (SEQ ID NO: 7)
  • Humanized heavy chain variant 3 (SEQ ID NO: 8)
  • Monoclonal antibody maternal kappa light chain (SEQ ID NO: 9)
  • Humanized light chain variant 1 (SEQ ID NO: 10)
  • Humanized heavy chain variant 1 (SEQ ID NO: 14)
  • Humanized heavy chain variant 2 (SEQ ID NO: 15)
  • Humanized light chain variant 1 (SEQ ID NO: 18)

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Abstract

本发明提供了针对人PD-1的分离的单克隆抗体或其抗原结合片段，包含表2中所列的重链CDR1、CDR2和CDR3以及表2中所列的轻链CDR1、CDR2和CDR3。

Description

人程序性死亡受体PD-1的单克隆抗体及其片段 技术领域
本发明涉及生物制药领域，具体而言，涉及针对人PD-1的单克隆抗体及其在疾病治疗中的应用。
背景技术
程序性死亡受体1(PD-1)是在激活的T细胞和B细胞上表达的免疫抑制性受体。阻断PD-1与其配体之一PD-L1间的相互作用提高肿瘤特异性CD8 +T细胞的免疫性，因此有助于免疫系统清除肿瘤细胞。Molecular and Biochemical Aspects of the PD-1 Checkpoint Pathway,Vassiliki A.Boussiotis,NEJM n engl j med 375；18 nejm.org November 3,2016中详细综述了PD-1及相关研究。
在文献中已确定了PD-1在癌症中的作用。业已在人的很多原发性肿瘤中发现PD-1(在肿瘤浸润淋巴细胞上)和/或PD-L1(在肿瘤细胞上)。这样的组织包括肺癌、肝癌、卵巢癌、宫颈癌、皮肤癌、结肠癌、神经胶质瘤、膀胱癌、乳腺癌、肾癌、食道癌、胃癌、口腔鳞状细胞癌、尿道上皮细胞癌和胰腺癌以及头颈肿瘤。
阻断PD-1/PD-L1的相互作用可导致肿瘤特异性T细胞的免疫性提高，因此，有助于由免疫系统清除肿瘤细胞。
PCT国际申请PCT/US2008/007463披露了一种针对人程序性死亡受体PD-1的抗体。
PD-1/PD-L1途径是开发癌症治疗的抗体疗法的被充分证实的靶标。抗PD-1抗体还可用于慢性病毒性感染。最近，有研究显示PD-1在来自HIV感染的个体的T细胞中高度表达。
发明内容
本发明提供了针对人PD-1的分离的单克隆抗体或其抗原结合片段，包含：表2中所列的重链CDR1、CDR2和CDR3，以及表2中所列的轻链CDR1、CDR2和CDR3。
在一个实施方案中，所述抗体或其抗原结合片段包含：(i)SEQ ID NO:3所示的重链氨基酸序列和SEQ ID NO:4所示的轻链氨基酸序列；或(ii)不包含信号肽部分的SEQ ID NO:3所示的重链氨基酸序列和不包含信号肽部分的SEQ ID NO:4所示的轻链氨基酸序列。
其中，SEQ ID NO:3所示的重链氨基酸序列的第1位-第19位(即，MNFGLSLIFLVLVLKGVLC)是信号肽部分；SEQ ID NO:4所示的轻链氨基酸序列的第1位-第20位(即，MEKDTLLLWVLLLWVPGSTG)是信号肽部分。
信号肽是出于方便纯化等目的而加入的，并非抗体结构中必不可少的部分。本领域技术人员可以理解，在本发明的抗体或抗体片段中，信号肽是任选的，也就是说，可以包含信号肽，也可以不包含信号肽。
本发明还提供了针对人PD-1的人源化的分离的单克隆抗体或其抗原结合片段，其包含：
(1)选自SEQ ID NO:13、14、15和16的重链氨基酸序列或不包含信号肽部分的SEQ ID NO:13、14、15和16的重链氨基酸序列；以及
(2)选自SEQ ID NO:17、18、19和20的轻链氨基酸序列或不包含信号肽部分的SEQ ID NO:17、18、19和20的轻链氨基酸序列。
其中，SEQ ID NO:13、14、15和16所示的重链氨基酸序列的第1位-第24位(即，MDPKGSLSWRILLFLSLAFELSYG)是信号肽部分；SEQ ID NO:17、18、19和20所示的轻链氨基酸序列的第1位-第20位(即，METDTLLLWVLLLWVPGSTG)是信号肽部分。
在一些实施方案中，所述抗原结合片段选自：Fab、Fab’、Fab’-SH、Fv、scFv、F(ab’) 2和双抗体。
本发明提供了编码所述抗体或其抗原结合片段的分离的多核苷酸。
在一些实施方案中，所述多核苷酸的重链编码序列如SEQ ID NO:1所示，轻链编码序列如SEQ ID NO:2所示。
在一些实施方案中，所述多核苷酸编码人源化抗体或其抗原结合片段，并且重链编码序列选自SEQ ID NO:5、6、7和8，轻链编码序列选自SEQ ID NO:9、10、11和12。
本发明提供了包含所述分离的多核苷酸的表达载体，以及包含所述表达载体的宿主细胞。
本发明提供了药物组合物，包含所述抗体或其抗原结合片段以及可药用载体。
本发明提供了提高免疫细胞活性的方法，包括使免疫细胞与所述抗体或其抗原结合片段接触。
本发明提供了治疗疾病的方法，所述方法包括将治疗有效量的本发明的抗体或其抗原结合片段给予需要治疗的受试者。
在一些实施方案中，其中所述疾病选自癌症、感染性疾病、移植排斥、自身免疫性疾病和神经疾病。在一些实施方案中，所述方法包括对受试者联合应用其他药物或疗法，包括化疗、放疗、靶向治疗、基因治疗、细胞治疗、干细胞治疗。
本发明提供了所述抗体或其抗原结合片段在制备用于治疗疾病的药物中的用途，其中所述疾病选自：癌症、感染性疾病、移植排斥、自身免疫性疾病和神经疾病。
本发明提供了调节细胞功能的方法，包括将编码本发明的抗体或其抗原结合片段的多核苷酸序列递送至所述细胞中。
在一些实施方案中，所述方法用于基因治疗、CAR-T治疗或CRISPR治疗。
在一些实施方案中，利用敲除了PD-1基因的小鼠来产生抗人PD-1单抗，亲和力更高，阻断效果更好，细胞杀伤活力更高。试验结果表明这种方法能够得到高亲和力的单克隆抗体。其亲和力比目前上市的PD-1抗体亲和力高出大约4-7倍，体外阻断PD-L1/PD-1的相互作用非常有效。同时体外抑制肿瘤生长活性也有所提高。
附图说明
图1显示了PD-1抗体与PD-1蛋白的结合活性。
图2显示了PD-1抗体对PD-1蛋白与PD-L1蛋白结合的阻断效应。
图3显示了不同抗体竞争结合实验结果。
图4显示了DNA重组抗体的结合和阻断作用。
图5显示了人源化抗体的与PD-1蛋白的结合活性
图6显示了人源化抗体的阻断作用。
图7显示了人源化母本抗体不同突变体亲和力比较。
图8显示了人源化PD-1抗体和食蟹猴淋巴细胞PD-1蛋白结合。
图9显示了人源化PD-1抗体体外增强效应细胞杀伤肿瘤细胞。
图10显示了人源化PD-1抗体阻断人淋巴细胞的PD-1的诱导表达。
图11显示了自研PD-1抗体与上市PD-1抗体动力学和亲和力比较。
图12显示了体外抑制肿瘤生长作用比较。
具体实施方式
“抗体”是指表现出所需生物学活性(例如抑制配体与其受体的结合或通过抑制配体诱导的受体信号转导)的抗体的任何形式。因此，“抗体”以其最广泛的意义来使用，并明确包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体和多特异性抗体(例如双特异性抗体)。
“抗体片段”和“抗原结合片段”在本文中可互换使用，意指抗体的抗原结合片段及抗体类似物，其通常包括至少部分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个CDR)。抗体片段保留母体抗体的至少某些结合特异性。优选地，抗体片段保留至少50％、70％、80％、90％、95％或100％或更多的母体抗体对靶标的结合亲和力。抗体片段实例包括但不限于：Fab、Fab′、F(ab′) 2和Fv片段；双抗体；线性抗体 (linear antibody)；单链抗体分子，例如sc-Fv、单抗体(技术来自Genmab)；纳米抗体(技术来自Domantis)；结构域抗体(技术来自Ablynx)；和由抗体片段形成的多特异性抗体。工程改造的抗体变体综述于Holliger和Hudson(2005)Nat.Biotechnol.23：1126-1136中。
“Fab片段”由一条轻链和一条重链的C H1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。
“Fc”区含有包含抗体的C H1和C H2结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。
“Fab′片段”含有一条轻链和包含V H结构域和C H1结构域以及C H1和C H2结构域之间区域的一条重链的部分，由此可在两个Fab′片段的两条重链之间形成链间二硫键以形成F(ab′) 2分子。
“F(ab′) 2片段”含有两条轻链和两条包含C H1和C H2结构域之间的恒定区的部分的重链，由此在两条重链间形成链间二硫键。因此，F(ab′) 2片段由通过两条重链间的二硫键保持在一起的两个Fab′片段组成。
“Fv区”包含来自重链和轻链二者的可变区，但缺少恒定区。
“单链Fv抗体”(或“scFv抗体”)是指包含抗体的V H和V L结构域的抗体片段，其中这些结构域存在于单个多肽链中。一般而言，Fv多肽另外在V H和V L结构域之间包含多肽接头，该接头使得scFv能形成用于抗原结合的所需结构。参见国际专利申请WO 88/01649。
“双抗体”为具有两个抗原结合位点的小抗体片段。所述片段包含在相同的多肽链中与轻链可变结构域(V L)连接的重链可变结构域(V H)(V H-V L或V L-V H)。通过使用短至不能在同一链的两个结构域之间配对的接头，迫使所述结构域与另一条链的互补结构域配对并形成两个抗原结合位点。
术语“嵌合”抗体指这样的抗体以及表现出所需生物学活性的所述抗体的片段：其中部分重链和/或轻链与来源于特定物种或属于特定抗体类别或亚类的抗体相应的序列等同或同源，同时所述链的其余部分与来源于另一物种或属于另一抗体类别或亚类的抗体相应的序列等同或同源(参见例如美国专利第4,816,567号和Morrison等，1984、Proc.Natl.Acad.Sci.USA 81：6851-6855)。
非人类(例如鼠)抗体的“人源化”形式为含有最小限度的来源于非人类免疫球蛋白序列的嵌合抗体。一般而言，人源化抗体包含至少一个且通常为两个可变结构域的几乎全部，其中全部或几乎全部超变环对应于非人类免疫球蛋白的超变环，全部或几乎全部FR区为人免疫球蛋白序列的FR区。人源化抗体还任选包含至少部分免疫球蛋白(通常为人免疫球蛋白)恒定区(Fc)。更详细的资料参见Jones et al，Nature 321：522-525(1986)；Riechmann et al，Nature 332：323-329(1988)。
“分离的”抗体为业已被鉴定并与其天然环境组分相分离的抗体。在一些实施方案中，将所述抗体纯化到以下程度：(1)超过95％重量的抗体，最优选超过99％重量，其由Lowry法测定；(2)足以通过用旋杯式测序仪获得N-末端或内部氨基酸序列的至少15个残基的程度；或(3)通过在还原或非还原条件下用考马斯蓝或优选银染色的SDS-PAGE确定为同质。
“分离的”核酸分子为被鉴定并与至少一种污染性核酸分子(通常在抗体核酸的天然来源中与其缔合)分离的核酸分子。因此，分离的核酸分子与在其天然细胞中存在的核酸分子有区别。
本文所用术语“单克隆抗体”是指从基本上同种抗体群中获得的抗体，即除了可能少量存在的可能的天然突变体外，构成所述群的各个抗体是一致的。单克隆抗体具有高度特异性，可针对单个的抗原位点。此外，与通常包括针对多个不同的决定簇(表位)的多种不同抗体的常规(多克隆)抗体制备物相反，每种单克隆抗体仅针对抗原上的单个决定簇。修饰语“单克隆”表示从基本上同种抗体群获得的抗体的特性，不能理解为需要通过任何特定方法来制备所述抗体。本文单克隆抗体明确包括“嵌合”抗体。
关于抗体及其应用的更多介绍参见中国专利申请CN201510367410.4，在此通过援引将其全部内容并入本文。
本文所用术语“免疫细胞”包括具有造血的起源并在免疫应答中起作用的细胞。免疫细胞包括：淋巴细胞，例如B细胞和T细胞；天然杀伤细胞；髓样细胞，例如单核细胞、巨噬细胞、嗜曙红细胞、肥大细胞、嗜碱细胞和粒细胞。
本文所用序列“变体”是指在一个或多个氨基酸残基处不同于所示的序列但基本保留其生物学活性的序列。例如，变体可以保留原序列至少50％，优选至少70％，甚至90％的生物学活性。
本文所用术语“约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，“约”或“基本上包含”可意味着不超过20％的范围，优选不超过10％，更优选不超过5％。除非另外说明，否则当具体值在本文中出现时，“约”的含义应该假定为在该具体值的可接受误差范围内。
当用“给予”和“治疗”提及动物、人、细胞或受试者时，是指将外源性药物、治疗剂、诊断剂或组合物与动物、人、细胞或受试者接触。“给予”和“治疗”可指例如治疗方法、药动学方法、诊断方法、研究方法和实验方法。“给予”和“治疗”还意味着例如通过试剂、诊断剂、疗法、联合用药或通过其他细胞对细胞进行体外和离体治疗。
本文所使用的术语“治疗有效量”指的是足以在受试者体内引起临床医师所期望的生物学或医学反应的活性化合物的量。本发明抗体的“治疗有效量”可由本领域技术人 员根据给药途径、受试者的体重、年龄、病情等因素而确定。例如，典型的周总剂量可以为至少0.05μg/kg体重，典型地为至少1μg/kg体重。
本发明所提供的药物可以制成粉末、注射剂等临床可接受的剂型。可以使用任何适当的途径给受试者施用本发明的药物组合物，例如可通过口服、静脉内输注、肌肉内注射、皮下注射、腹膜下、直肠、舌下，或经吸入、透皮等途径给药。
本发明的药物制剂包含本发明PD-1抗体或其抗原结合片段以及可药用载体。为了制备药物组合物或无菌组合物，让抗体或其片段与可药用载体(或赋形剂)混合。
本发明药物组合物还可含有其它药剂，包括但不限于细胞毒剂、细胞生长抑制剂、抗血管形成药物或抗代谢药物、靶向肿瘤药物、免疫刺激剂或免疫调节剂或与细胞毒剂、细胞生长抑制剂或其它毒性药物缀合的抗体。也可与其它治疗形式(例如手术、化疗及放射)一起施用所述药物组合物。
与人PD-1特异性结合的本发明抗体或抗原结合片段可用于增加、提高、刺激或上调免疫应答。本发明抗体及抗体片段尤其适用于治疗罹患可通过提高T细胞介导的免疫应答来治疗的疾病的受治疗者。优选的受治疗者包括需要提高免疫应答的人患者。
本发明抗体或抗原结合片段可用于治疗癌症(即抑制肿瘤细胞的生长或存活)。可用本发明抗体抑制其生长的优选的癌症包括通常对免疫疗法有反应的癌症，但还包括迄今与免疫疗法尚无关联的癌症。用于治疗的优选癌症的非限制性实例包括黑素瘤(例如恶性转移性黑素瘤)、肾癌(例如透明细胞癌)、前列腺癌(例如激素难控制的前列腺腺癌)、胰腺腺癌、乳腺癌、结肠癌、肺癌(例如非小细胞肺癌)、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、宫颈癌、甲状腺癌、胶质母细胞瘤、神经胶质瘤、白血病、淋巴瘤和其它恶性肿瘤。另外，本发明包括可用本发明抗体抑制其生长的难治性或复发性癌。
本发明抗体或其抗原结合片段可单独使用或与以下其它物质联合使用：抗肿瘤药或免疫原剂(例如减弱的癌细胞、肿瘤抗原(包括重组蛋白质、肽和糖类分子)、抗原呈递细胞例如用来源于肿瘤的抗原或核酸刺激的树突细胞、免疫刺激细胞因子(例如IL-2、IFNa2、GM-CSF_)和用编码免疫刺激细胞因子(例如但不限于GM-CSF)的基因转染的细胞；标准癌症治疗(例如化疗、放疗或手术)；或其它抗体(包括但不限于针对以下物质的抗体：VEGF、EGFR、Her2/neu、VEGF受体、其它生长因子受体、CD20、CD40、CTLA-4、OX-40、4-IBB和ICOS)。
本发明抗体或其抗原结合片段还可用于防止或治疗感染和感染性疾病。抗体或其抗原结合片段可单独使用，或与疫苗联合使用，以刺激针对病原体、毒素和自体抗原的免疫应答。抗体或其抗原结合片段可用于刺激对感染人的病毒的免疫应答，这些病毒例如但不限于人免疫缺陷病毒、甲、乙、丙型肝炎病毒、爱泼斯坦-巴尔病毒、人巨细胞病毒、人乳头瘤病毒、疱疹病毒。抗体或其抗原结合片段可用于刺激对细菌或真菌寄生虫及其它病原体引起的感染的免疫应答。
本发明抗体或其抗原结合片段还可用于防止或治疗移植排斥、自身免疫性疾病和神经疾病。
本文中使用的术语“自身免疫性疾病”是指由自身免疫反应所引起的任何疾病，即，免疫应答针对受试者身体内的物质。
应当注意的是由于自身免疫性可以影响受试者的任何器官或组织，例如受试者的大脑、皮肤、肾脏、肺、肝脏、心脏或甲状腺，疾病的临床表现取决于受影响的部位。
典型的自身免疫性疾病包括但不限于类风湿性关节炎、银屑病性关节炎、骨关节炎、斯蒂尔病、青少年关节炎、狼疮、糖尿病、重症肌无力症、桥本甲状腺炎、奥德甲状腺炎、格雷夫斯病、类风湿性关节炎综合征、多发性硬化症、传染性神经元炎、急性播散性脑脊髓炎、阿狄森病、视性眼阵挛-肌阵挛综合征、强直性脊椎炎、抗磷脂抗体综合征、再生障碍性贫血、自身免疫性肝炎、乳糜泻、古德帕斯彻综合征、特发性血小板减少性紫癜、视神经炎、硬皮病、原发性胆汁性肝硬化、莱特尔综合征、高安动脉炎、颞动脉炎、温型自身免疫性溶血性贫血、韦格纳肉芽肿病、银屑病、全身脱毛、贝赫切特病、慢性疲劳、家族性自主神经功能异常、子宫内膜异位、间质性膀胱炎、神经肌强直、硬皮病和外阴痛等。
本文中使用的术语"神经疾病"是指影响中枢神经系统和/或具有在中枢神经系统中的病因的疾病或病症。示例性的神经疾病的具体例子包括但不限于：帕金森病、阿尔茨海默病(Alzheimer’s disease)、朊病毒病(prion disease)、亨廷顿病(Huntington's disease)、雷特综合征(Rett syndrome)等。
本发明抗PD-1抗体或其抗原结合片段可以与其他药物或疗法联合应用，例如与一种或多种其它治疗剂(例如细胞毒剂、放射性毒性剂或免疫抑制剂)联合应用。所述抗体可与所述药剂连接(作为免疫复合体)，或可与治疗剂分开给予，例如可在给予治疗剂之前、之后或同时给予抗体。此外，本发明抗体可与化疗、放疗、靶向治疗、基因治疗、细胞治疗、干细胞治疗等联合应用，尤其是用于癌症的治疗。
本发明抗PD-1抗体或其抗原结合片段或其编码基因还可以被导入细胞中，以调节细胞功能或活性，或用于治疗疾病。该方法包括体外应用和体内应用。该方法特别适合用于基因治疗、CAR-T治疗或CRISPR治疗。
本发明抗体及抗体片段的非治疗性应用包括：用于流式细胞术分析、免疫组织化学和体外功能分析、蛋白质印迹和ELISA等。本发明抗体还可用作亲和纯化试剂。
所述抗体还可用于诊断测定，例如用于检测PD-1在特定的细胞、组织或血清中的表达。为了诊断应用，通常用可检测的部分标记(直接或间接)抗体。可利用众多标记物，其通常分为以下类别：生物素、荧光染料、放射性核苷酸、酶、碘和生物合成标记物。
本发明抗体可用于任何已知的测定法中，例如竞争性结合测定法，直接和间接夹心测定法和免疫沉淀测定法。抗体还可用于体内诊断测定。例如用放射性核素(例如 111In、 99Tc、 4C、 31I、 125I、 3H、 32P、 35S或 18F)标记抗体，以便定位抗原或表达抗体的细胞。
通过参考以下实施例将更充分地理解本发明。然而，这些实施例不应该理解为限制本发明范围。
实施例1 产生和筛选PD-1抗体
1.产生人PD-1融合蛋白
从人外周血单个核细胞(PBMC)利用MACS磁珠(MiltenyiBiotec)分离出T淋巴细胞，提取RNA(RNeasy mini kit，QIAGEN)并获得第一链cDNA(SuperScript First-Strand Synthesis System，Invitrogen,)并根据人PD-1基因DNA序列(NCBI Reference Sequence:NM_005018.2)通过PCR反应获得人PD-1分子全长序列。人PD-1融合蛋白是将PD-1分子胞外部分序列插入到一个含有小鼠免疫球蛋白G2aCH2-CH3结构域的表达质粒mIgV(HDong et al Nat Med.1999；5:1365-1369)中。利用瞬时表达方法将质粒DNA用PEI试剂(Sigma-Aldrich)转染293T细胞产生人PD-1小鼠Ig融合蛋白(hPD-1mIg)，细胞分泌上清液经IgA纯化柱(HiTrap Protein A HP，GE healthcare)纯化蛋白并经SDS-PAGE凝胶电泳鉴定确认。
2.构建PD-1稳定表达细胞株
为检测和鉴定PD-1抗体，我们构建了人PD-1蛋白稳定表达细胞株。将上述得到的PD-1全长序列插入到pcDNA3.1(-)表达质粒，利用lipofactamin 2000转染试剂(Thermo Fisher Scientific)转染中国仓鼠卵巢成纤维细胞(CHO)，经G418筛选得到稳定表达的阳性细胞株(CHO/hPD1)。
3.免疫小鼠
取6-8周龄大小的雌性Balb/c小鼠，用hPD-1mIg融合蛋白与福氏完全佐剂(CFA,Sigma)混合乳化液皮下多点接种免疫，每只小鼠80-100μg蛋白，3周后用相同融合蛋白与不完全佐剂(IFA,Sigma)乳化液进行加强免疫，共免疫3次。每次在小鼠免疫后2周左右取血清测定抗体效价，血清效价用CHO/hPD1细胞通过流式细胞仪测定。二抗用荧光标记的羊抗鼠IgG(eBioscience)。当血清效价达到足够高时，用80μg hPD-1mIg融合蛋白溶于PBS中，腹腔注射加强免疫一次，5天后取出脾脏进行融合。
4.制备和筛选杂交瘤
杂交瘤细胞的产生通过免疫小鼠的脾细胞与小鼠骨髓瘤细胞系(SP2/0-Ag14)在聚乙二醇(PEG1500，Roche)融合剂作用下进行融合，融合的细胞经过HAT(Sigma Aldrich)选择培养，利用我们实验室建立的高通量转染和筛选系统(S Yao et al.Immunity.2011； 34(5):729-40.)鉴定培养上清，筛选得到的阳性单克隆抗体再经过流式细胞仪检测确认能够和PD-1阳性表达细胞结合。挑选出的阳性克隆细胞株经过5遍以上有限稀释法进行亚克隆选择，确保是单一来源的阳性克隆。
实施例2 抗PD-1单抗的特性分析
1.单抗同种型(isotype)的鉴定
得到的几株单克隆抗体用小鼠IgG同种型检测试剂盒(Mouse Immunoglobulin Isotyping Kit,BD Biosciences)进行测定，5个单克隆抗体重链均为IgG1，轻链均为κ链。
2.单抗特异性结合的评估
1)种属交叉反应的鉴定显示几株PD-1抗体克隆只和人PD-1蛋白结合，与小鼠PD-1无交叉结合。
2)PD-1抗体与PD-1蛋白的结合反应：将我们实验室得到的5个PD-1抗体用流式细胞仪检测和比较和CHO/hPD-1细胞结合活性，结果显示这些抗体与PD-1蛋白都有较强的结合活性(图1)。
3)PD-1抗体对PD-1受体蛋白和其配体PD-L1结合的阻断作用：肿瘤细胞上表达PD-L1配体蛋白与其在淋巴细胞表达的PD-1受体结合会诱导产生免疫细胞功能抑制而导致肿瘤逃避宿主免疫系统监视作用，阻断PD-1与PD-L1之间的相互作用是PD-1抗体抗肿瘤免疫治疗的一个重要作用机制。为检测抗体的阻断活性，我们将100ng hPD-1hIg融合蛋白分别与100ng、200ng、400ng和800ng不同剂量的不同PD-1抗体混合，并设mIgG和PBS缓冲液对照组。4℃孵育30分钟，然后与PD-L1阳性表达细胞(CHO/PD-L1)在4℃作用30分钟，用含1％小牛血清PBS缓冲液离心洗一遍，加入萤光标记的羊抗人IgG，4℃孵育20分钟，洗后用流式细胞仪检测。结果显示2个PD-1抗体没有阻断活性，其余3个抗体都能够以剂量依赖性阻断PD-1与PD-L1蛋白间的结合(图2)。
4)抗体竞争结合实验：为弄清这些PD-1抗体是否识别同一个或不同抗原表位，我们做了抗体竞争结合实验。首先分别用10μg不同PD-1抗体与CHO/hPD-1细胞在4℃孵育30分钟，用含1％小牛血清PBS缓冲液离心洗一遍后加入0.1μg的生物素(Biotin)标记的Ab4或Ab5，4℃孵育20分钟，洗后加入荧光标记的链霉亲和素(Streptavidin,SA；eBioscience),4℃孵育20分钟，洗后用流式细胞仪进行分析。结果显示Ab4和Ab5相互竞争结合同一抗原表位，这两个抗体与Ab3有部分竞争结合，与Ab1和Ab2不存在竞争结合(图3)。
实施例3 抗PD-1抗体的序列测定和人源化
为了将PD-1抗体能够用于临床肿瘤治疗，我们根据上述抗体活性的测试结果选择了Ab4进行序列测定和人源化。
1.单克隆抗体序列测定
首先利用RNA纯化试剂盒(RAeasy Mini Kit，Qiagen 74104，QIAshredder，Qiagen 79654)从杂交瘤细胞提取和纯化总RNA。然后用试剂盒(SMARter RACE cDNA Amplification Kit,Clontech)并按公司试剂操作手册将RNA反转录合成第一链cDNA链。利用5’RACE技术，用试剂盒提供的通用引物UPM(universal primer)作为上游引物，和根据小鼠IgG1重链可变区和轻链κ链基因序列设计的基因特异引物GSP(genespecific primer)作为下游引物，以SMART第一链cDNA为模板进行PCR循环。然后将PCR产物分别连接到T载体(Zero Blunt TOPO PCR Cloning Kit，Invitrogen K2875-J10)。挑选克隆进行序列测定和分析，分别得到抗体的轻链和重链可变区序列，再根据重链可变区序列和小鼠IgG1序列设计引物，通过PCR获得重链全长序列，利用VBASE2对序列进行分析，确定了该抗体轻、重链的氨基酸序列以及相应的CDR区域(见表2)。其DNA编码序列参见表1。
2.单克隆抗体DNA重组序列和抗体表达
为确保克隆抗体序列的正确性，分别把抗体的轻、重链全长序列插入到pcDNA3.1(-)表达质粒中，并通过瞬转293T细胞得到蛋白产物，经纯化和鉴定确认蛋白产物与原抗体一样能够和PD-1蛋白结合(图4.A)并能够阻断PD-1蛋白与PD-L1的结合(图4.B)。
3.抗体序列人源化
为了使抗体能够应用临床病人治疗，对抗体序列进行了人源化改造。首先将抗体重链的Fc部分换为人IgG4序列，并将IgG铰链区4的CPSCP换为IgG1的CPPCP(S228P)，同时把抗体轻链换成人的κ链，得到人鼠嵌合的母本抗体(Chimeric Ab)。在确认母本抗体的功能活性后对重链可变区和轻链可变区序列进行人源化改造，将所得到的3个人源化重链变异体(表3序列6–8，表4序列14–16)、3个人源化轻链变异体(表3序列10–12，表4序列18–20)互相组合，共得到9个不同的变异体序列(Variants 1至Variants 9)。将1个母本和9个变异体序列分别克隆到高表达载体中，用瞬时转染293细胞得到抗体上清，纯化后的抗体经SDS-PAGE胶蛋白电泳和测序验证确认正确。抗体的DNA和氨基酸序列见表3、表4。其中，Variants 1的重链氨基酸序列为SEQ ID NO：6，轻链氨基酸序列为SEQ ID NO：10；Variants 2的重链氨基酸序列为SEQ ID NO：6，轻链氨基酸序列为SEQ ID NO：11；Variants 3的重链氨基酸序列为SEQ ID NO：6，轻链氨基酸序列为SEQ ID NO：12；Variants 4的重链氨基酸序列为SEQ ID NO：7，轻链氨基酸序列为SEQ ID NO:10；Variants 5的重链氨基酸序列为SEQ ID NO：7，轻链氨基酸序列为SEQ ID NO：11；Variants 6的重链氨基酸序列为SEQ ID NO：7，轻链氨基酸序列为SEQ ID NO：12；Variants 7的重链氨基酸序列为SEQ ID NO：8，轻链氨基酸序列为SEQ ID NO：10；Variants 8的重链氨基酸序列为SEQ ID NO：8，轻链氨基酸序列为SEQ ID NO：11；Variants 9的重链氨基酸序列为SEQ ID NO：8，轻链氨基酸序列为SEQ ID NO：12。
实施例4 人源化抗体的特性和功能
1.人源化抗体的与PD-1蛋白的结合活性：对嵌合母本和突变抗体进行测试和比较，取CHO/hPD-1细胞悬于含1％小牛血清的PBS并分装于流式管中，分别加入不同剂量的母本和突变体抗体，4℃孵育30分钟，用含1％小牛血清PBS缓冲液离心洗一遍后加入荧光标记的羊抗人IgG二抗(eBioscience)，4℃孵育20分钟洗后用流式细胞仪进行分析。结果显示所有人源化突变体均可与PD-1蛋白结合，与嵌合母本抗体比较，部分突变抗体的结合能力更高，有的相对要低一些(图5)。
2.人源化抗体对PD-1受体蛋白和其配体PD-L1结合的阻断作用：用400ng、300ng、200ng、100ng和50ng不同PD-1抗体分别与100ng hPD-1mIg融合蛋白混合，设hIgG和PBS缓冲液对照组，4℃孵育30分钟，然后与PD-L1阳性表达细胞(CHO/PD-L1)作用30分钟，用含1％小牛血清PBS缓冲液离心洗一遍后加入荧光标记的羊抗鼠IgG-，4℃孵育20分钟，洗后用流式细胞仪检测。结果显示所有人源化突变体抗体都能够阻断PD-1与PD-L1蛋白间的结合并呈剂量依赖性，部分突变体的阻断作用高于母本抗体(图6)。
3.人源化抗体亲和力；利用Biacore仪器(Biacore T100，GE Healthcare Life Sciences)对母本抗体和突变体抗体进行了亲和力的分析比较。将蛋白抗原hPD-1mIg偶联在CM5芯片表面，检测抗体作为分析物(流动相)，检测相互作用。结果显示突变体抗体与母本抗体比较，结合速率(Ka)差异不大，解离速率(Kd)有2个突变体较嵌合抗体略慢，亲和力(KD)母本抗体为9.89E-11M，具有较强的亲和力，有2个突变体KD值与嵌合抗体接近(图7)。
实施例5 人源化抗体其它生物功能特性
1.人源化PD-1抗体与食蟹猴的淋巴细胞PD-1蛋白交叉结合：为今后临床前的毒理测试需要，我们测试PD-1抗体能否结合猴子PD-1蛋白。取食蟹猴的外周单个核细胞，用淋巴细胞分离液分离出淋巴细胞悬于10％小牛血清RPMI 1640培养液里，加入包被1μg/ml抗人CD3抗体的24孔培养板中或加入没有包被抗体的24孔培养板中，培养液中加入10ng/ml抗hIFNγ和50imL hIL2，5％CO2，37℃条件培养2天。收获细胞用PBS洗1遍，加入PD-1抗体，4℃孵育30分钟，洗后再加入荧光标记的抗人IgG，4℃孵育20分钟，洗后用流式细胞仪检测。结果显示人源化PD-1抗体能够和食蟹猴淋巴细胞PD-1蛋白结合(图8)。
2.体外抗肿瘤作用：我们根据PD-1抗肿瘤作用的机理设计了一个体外模型。首先利用从人外周血淋巴细胞(PBMC)分离出的CD8+淋巴细胞，与经过放射线照射的转染hB7-1的人黑色素瘤细胞(624Mel/B7-1)共培养，产生能够杀伤624Mel瘤细胞的同种异体细胞毒性淋巴细胞(alloCD8+CTL)。然后将allo CTL效应细胞和624Mel瘤细胞按一定比例混合后，加入PD-1抗体或对照蛋白培养5天，用0.5％结晶紫染色细胞后，用酶标仪读取OD(450nm)值，根据瘤细胞存活计算杀伤活性，结果显示部分人源化的PD-1突变体 抗体体外能够增强效应细胞杀伤肿瘤细胞的功能(图9，E：T＝效应细胞(Effect cells)：靶细胞(target cells))。
3.人源化PD-1抗体抑制人淋巴细胞PD-1的表达：肿瘤细胞表达PD-L1可诱导周边的淋巴细胞表达PD-1而导致淋巴细胞活性受抑制而不能杀伤肿瘤细胞，我们利用体外模型模拟这种情况，检测在加入PD-1抗体后能否抑制人淋巴细胞PD-1的表达从而恢复淋巴细胞的正常功能。我们将转染PD-L1的人黑色素瘤细胞(624Mel/hPD-L1)与alloCD8+CTL按比例混合加入24孔培养板，并在培养液中分别加入10μg/ml不同PD-1抗体和对照蛋白，培养4天后，用荧光标记的PD-1和CD8抗体染色细胞后用流式细胞仪检测淋巴细胞上PD-1的表达情况。结果显示培养液和hIgG对照组的CD8+淋巴细胞表达PD-1表达阳性，而加入母本抗体和突变抗体的各组CD8+淋巴细胞PD-1蛋白均为阴性(图10)，说明加入PD-1抗体后能抑制淋巴细胞表面的PD-1表达。
实施例6 人源化PD-1抗体与商业化抗体功能比较
我们选择了目前已获批在临床治疗肿瘤病人的2个厂家的商业化PD-1抗体和我们的抗体做基本特性的比较，包括Keytruda(penbrolizumab)(Merck&CO.,INC),100mg/4ml,Lot MO23694Exp 2017JUL13；Opdivo(nivolumab)(Bristol-Myers Squibb),100mg/10ml,Lot AAK5824Exp 02.2018；Opdivo(nivolumab)(Bristol-Myers Squibb),40mg/4ml,Lot AAK2713Exp10.2017。
6.1抗体动力学和亲和力比较：
抗体动力学和亲和力比较我们使用Biacore T200仪器(GE Healthcare Life Sciences)，在GE公司技术工程师操作下，检测了自研的PD-1抗体FMU100(即，Variant 3)与3个上市抗体对PD-1mFc蛋白的动力学和亲和力的差异。将PD1-mFc蛋白分别偶联在CM5芯片的4通道(33RU)，自研和商品抗体作为分析物(流动相)，检测相互作用。经摸索后，将PD1抗体均稀释到50nM作为最高浓度；1.5625，3.125，6.25，12.5，25，50nM进行浓度梯度进样，使用Glycine pH1.7作为再生条件。测试结果显示结合速率Ka差异不大，自研抗体较商品抗体稍低；解离速率Kd，自研抗体较商品抗体慢10倍；亲和力KD，自研抗体较商品抗体高4-7倍(图11)。
6.2体外抑制肿瘤生长作用比较
如前所述，将alloCD8+CTL效应细胞和624Mel瘤细胞按一定比例混合后，加入自研PD-1抗体和3个上市抗体或对照蛋白共培养5天，用0.5％结晶紫染色细胞后，用酶标仪读取OD(450nm)值，根据瘤细胞存活计算杀伤活性，结果显示自研的PD-1抗体和3个上市抗体体外都能够增强效应细胞杀伤肿瘤细胞的功能，自研的抗体增强作用高于2个上市抗体(图12，E：T＝效应细胞(Effect cells):靶细胞(target cells))。
表1.PD-1单克隆抗体DNA序列(对应于Ab4)
重链序列(SEQ ID NO:1)
Figure PCTCN2018073437-appb-000001
轻链序列(SEQ ID NO:2)
Figure PCTCN2018073437-appb-000002
表2.PD-1单克隆抗体的氨基酸序列(对应于Ab4)
重链序列(SEQ ID NO:3)
Figure PCTCN2018073437-appb-000003
轻链序列(SEQ ID NO:4)
Figure PCTCN2018073437-appb-000004
表3.PD-1单克隆抗体母本和人源化变异体DNA序列
单克隆抗体母本重链hIgG4-S228P(SEQ ID NO:5)
Figure PCTCN2018073437-appb-000005
人源化重链变异体1(SEQ ID NO:6)
Figure PCTCN2018073437-appb-000006
人源化重链变异体2(SEQ ID NO:7)
Figure PCTCN2018073437-appb-000007
Figure PCTCN2018073437-appb-000008
人源化重链变异体3(SEQ ID NO:8)
Figure PCTCN2018073437-appb-000009
单克隆抗体母本kappa轻链(SEQ ID NO:9)
Figure PCTCN2018073437-appb-000010
人源化轻链变异体1(SEQ ID NO:10)
Figure PCTCN2018073437-appb-000011
人源化轻链变异体2(SEQ ID NO:11)
Figure PCTCN2018073437-appb-000012
Figure PCTCN2018073437-appb-000013
人源化轻链变异体3(SEQ ID NO:12)
Figure PCTCN2018073437-appb-000014
表4.PD-1单克隆抗体母本和人源化变异体氨基酸序列
单克隆抗体母本重链hIgG4-S228P(SEQ ID NO:13)
Figure PCTCN2018073437-appb-000015
人源化重链变异体1(SEQ ID NO:14)
Figure PCTCN2018073437-appb-000016
人源化重链变异体2(SEQ ID NO:15)
Figure PCTCN2018073437-appb-000017
人源化重链变异体3(SEQ ID NO:16)
Figure PCTCN2018073437-appb-000018
单克隆抗体母本kappa轻链(SEQ ID NO:17)
Figure PCTCN2018073437-appb-000019
人源化轻链变异体1(SEQ ID NO:18)
Figure PCTCN2018073437-appb-000020
人源化轻链变异体2(SEQ ID NO:19)
Figure PCTCN2018073437-appb-000021
人源化轻链变异体3(SEQ ID NO:20)
Figure PCTCN2018073437-appb-000022

Claims (21)

  1. 针对人PD-1的分离的单克隆抗体或其抗原结合片段，包含：表2中所列的重链CDR1、CDR2和CDR3，以及表2中所列的轻链CDR1、CDR2和CDR3。
  2. 权利要求1的单克隆抗体或其抗原结合片段，包含：(i)SEQ ID NO:3所示的重链氨基酸序列和SEQ ID NO:4所示的轻链氨基酸序列；或(ii)不包含信号肽部分的SEQ ID NO:3所示的重链氨基酸序列和不包含信号肽部分的SEQ ID NO:4所示的轻链氨基酸序列。
  3. 权利要求1的单克隆抗体或其抗原结合片段，其中所述单克隆抗体或其抗原结合片段被人源化，并且包含：
    (1)选自SEQ ID NO:13、14、15和16的重链氨基酸序列或不包含信号肽部分的SEQ ID NO:13、14、15和16的重链氨基酸序列；以及
    (2)选自SEQ ID NO:17、18、19和20的轻链氨基酸序列或不包含信号肽部分的SEQ ID NO:17、18、19和20的轻链氨基酸序列。
  4. 权利要求1的单克隆抗体或其抗原结合片段，包含：SEQ ID NO:13所示的重链氨基酸序列和SEQ ID NO:17所示的轻链氨基酸序列。
  5. 权利要求1-4中任一项的单克隆抗体或其抗原结合片段，其中所述抗原结合片段选自：Fab、Fab’、Fab’-SH、Fv、scFv、F(ab’)2和双抗体。
  6. 编码权利要求1-5中任一项的抗体或其抗原结合片段的分离的多核苷酸。
  7. 权利要求6的分离的多核苷酸，其中，重链编码序列如SEQ ID NO:1所示，轻链编码序列如SEQ ID NO:2所示。
  8. 权利要求6的分离的多核苷酸，该多核苷酸编码人源化抗体或其抗原结合片段，并且其中：
    重链编码序列选自SEQ ID NO:5、6、7和8，
    轻链编码序列选自SEQ ID NO:9、10、11和12。
  9. 包含权利要求6-8中任一项的分离的多核苷酸的表达载体。
  10. 包含权利要求9的表达载体的宿主细胞。
  11. 药物组合物，包含权利要求1-5中任一项的抗体或其抗原结合片段以及可药用载体。
  12. 提高免疫细胞活性的方法，包括使所述免疫细胞与权利要求1-5中任一项的抗体或其抗原结合片段接触。
  13. 治疗疾病的方法，所述方法包括将治疗有效量的权利要求1-5中任一项的抗体或其抗原结合片段给予需要治疗的受试者。
  14. 权利要求13的方法，其中所述疾病选自：癌症、感染性疾病、移植排斥、自身免疫性疾病和神经疾病。
  15. 权利要求13或14的方法，包括对所述受试者联合应用其他药物或疗法。
  16. 权利要求15的方法，其中所述其他疗法选自：化疗、放疗、靶向治疗、基因治疗、细胞治疗、干细胞治疗。
  17. 权利要求1-5中任一项的抗体或其抗原结合片段在制备用于治疗疾病的药物中的用途，其中所述疾病选自：癌症、感染性疾病、移植排斥、自身免疫性疾病和神经疾病。
  18. 权利要求17的应用，其中所述药物与其他药物或疗法进行联合应用。
  19. 权利要求18的应用，其中所述其他疗法选自：化疗、放疗、靶向治疗、基因治疗、细胞治疗、干细胞治疗。
  20. 调节细胞功能的方法，包括将编码权利要求1-5中任一项的抗体或其抗原结合片段的多核苷酸序列递送至所述细胞中。
  21. 权利要求20的方法，其中所述方法用于基因治疗、CAR-T治疗或CRISPR治疗。
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