WO2018036472A1 - 一种抗pd1单克隆抗体、其药物组合物及其用途 - Google Patents

一种抗pd1单克隆抗体、其药物组合物及其用途 Download PDF

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WO2018036472A1
WO2018036472A1 PCT/CN2017/098465 CN2017098465W WO2018036472A1 WO 2018036472 A1 WO2018036472 A1 WO 2018036472A1 CN 2017098465 W CN2017098465 W CN 2017098465W WO 2018036472 A1 WO2018036472 A1 WO 2018036472A1
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monoclonal antibody
antibody
antigen
binding fragment
seq
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PCT/CN2017/098465
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French (fr)
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李百勇
夏瑜
王忠民
张鹏
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中山康方生物医药有限公司
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Priority to CA3034849A priority patent/CA3034849A1/en
Priority to BR112019003775A priority patent/BR112019003775A2/pt
Application filed by 中山康方生物医药有限公司 filed Critical 中山康方生物医药有限公司
Priority to AU2017316255A priority patent/AU2017316255C1/en
Priority to KR1020197008464A priority patent/KR102469286B1/ko
Priority to EA201990570A priority patent/EA201990570A1/ru
Priority to US16/327,083 priority patent/US12076398B2/en
Priority to MX2019002268A priority patent/MX2019002268A/es
Priority to JP2019531512A priority patent/JP7082620B2/ja
Priority to EP17842895.9A priority patent/EP3505535B1/en
Publication of WO2018036472A1 publication Critical patent/WO2018036472A1/zh
Priority to IL264956A priority patent/IL264956B2/en
Priority to ZA2019/01781A priority patent/ZA201901781B/en
Priority to AU2024202527A priority patent/AU2024202527A1/en

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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70521—CD28, CD152
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7019—Ischaemia

Definitions

  • the invention belongs to the field of tumor treatment and molecular immunology, and relates to an anti-PD1 antibody, a pharmaceutical composition thereof and use thereof.
  • the invention relates to a monoclonal antibody against PD1.
  • the transmembrane receptor PD1 (programmed cell death 1, also known as PD-1) is a member of the CD28 gene family and is expressed in activated T cells, B cells and bone marrow cells.
  • PD1 receptors PDL1 and PDL2 belong to the B7 superfamily, in which PDL1 cells are expressed, including T cells, B cells, and endothelial cells and epithelial cells.
  • PDL2 is expressed only in antigen presenting cells such as dendritic cells and giant cells. Phagocytes.
  • T cells play a very important role in clearing viral infections, but T cell antiviral responses are often associated with immunopathology.
  • PD1 plays a very important role in the activation of negative regulatory T cells.
  • PD1-mediated negative regulation of T cells can reduce tissue damage caused by infection, but block or inhibit the negative regulation of PD1.
  • Leading to autoimmune diseases, for example, PD1 knockout mice are more effective in clearing pancreatic virus infection, but they cause more severe liver damage (Isai et al., 2003, J. Exp. Med. 198:39- 50).
  • tumors that highly express PD1 generally form cancers that are difficult to detect (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. USA 104: 3360-5).
  • An effective method for the implementation is to regulate the expression of PD1 by injecting antibodies in vivo.
  • PD1 antibodies became the hottest research in the global pharmaceutical industry after they unveiled their unprecedented clinical efficacy data at the annual meeting of the American Cancer Society (AACR) in 2012 and 2013 and the annual meeting of the American Society of Clinical Oncology (ASCO). New drug.
  • the present inventors After intensive research and creative labor, the present inventors used a mammalian cell expression system to express recombinant PD1 as an antigen to immunize mice, and fused the mouse spleen cells with myeloma cells to obtain hybridoma cells.
  • the inventors obtained a hybridoma cell line LT003 (Accession No. CCTCC NO: C2015105) by performing screening on a large number of samples.
  • the hybridoma cell line LT003 is capable of secreting a specific monoclonal antibody (designated 14C12) which specifically binds to PD1, and the monoclonal antibody is capable of blocking the binding of PD1 to PDL1 very efficiently.
  • the inventors creatively produced a humanized antibody against PD1 (designated 14C12H1L1).
  • the present inventors have also surprisingly found that the antibody 14C12, 14C12H1L1 of the present invention can efficiently bind to human T cells, and activate T cells to induce secretion of IFN- ⁇ and IL-2 by human lymphocytes.
  • the antibodies 14C12 and 14C12H1L1 of the present invention have potential for the preparation of a medicament for preventing cancers such as lung cancer, melanoma, renal tumor, ovarian cancer, leukemia, and anemia.
  • One aspect of the invention relates to a monoclonal antibody or antigen-binding fragment thereof, wherein
  • the heavy chain variable region ( VH ) of the monoclonal antibody comprises: a CDR having the amino acid sequence of SEQ ID NOs: 9-11;
  • the monoclonal antibody light chain variable region (V L) comprises: the amino acid sequence of SEQ ID NO: 12-14 of CDR.
  • the monoclonal antibody or antigen-binding fragment thereof wherein
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody is selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 6;
  • amino acid sequence of the light chain variable region of the monoclonal antibody is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8.
  • the monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody comprises:
  • VH as shown in SEQ ID NO: 2 and V L as shown in SEQ ID NO: 4;
  • variable regions of the light and heavy chains determine the binding of the antigen; the variable regions of each chain contain three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H) comprise HCDR1, HCDR2, HCDR3 , the CDR of the light chain (L) LCDR1, LCDR2, LCDR3 are included; which is named by Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Vol. 1-3, NIH Publication 91-3242, Bethesda Md).
  • CDRs complementarity determining regions
  • the antibodies 14C12, 14C12H1L1 of the invention have the same CDR:
  • amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:
  • HCDR1 GFAFSSYD (SEQ ID NO: 9),
  • HCDR2 ISGGGRYT (SEQ ID NO: 10),
  • HCDR3 ANRYGEAWFAY (SEQ ID NO: 11);
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCDR1 QDINTY (SEQ ID NO: 12),
  • LCDR2 RAN (SEQ ID NO: 13),
  • LCDR3 LQYDEFPLT (SEQ ID NO: 14).
  • the monoclonal antibody or antigen-binding fragment thereof wherein the monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab', F(ab') 2 , Fd, Fv a dAb, a complementarity determining region fragment, a single chain antibody (eg, scFv), a humanized antibody, a chimeric antibody, or a diabody.
  • the monoclonal antibody or antigen-binding fragment thereof wherein the monoclonal antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M,
  • the K D protein of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the PD1 protein; preferably, the K D is measured by a Fortebio molecular interaction instrument.
  • the monoclonal antibody or antigen-binding fragment thereof wherein the monoclonal antibody is less than about 100 nM, such as less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM EC 50 or less PD1 binding protein.
  • the EC 50 is measured by an indirect ELISA method.
  • the monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less PD1 K D binding protein.
  • the monoclonal antibody or antigen-binding fragment thereof wherein the monoclonal antibody comprises a non-CDR region, and the non-CDR region is from a species other than a murine, eg From human antibodies.
  • the monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody is a monoclonal antibody produced by the hybridoma cell line LT003, and the hybridoma cell line LT003 is preserved in China.
  • Typical Culture Collection (CCTCC) with accession number CCTCC NO: C2015105.
  • Another aspect of the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding an antibody heavy chain variable region, wherein
  • the heavy chain variable region of the antibody comprises: the CDR of the amino acid sequence of SEQ ID NOs: 9-11;
  • the heavy chain of the antibody has the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6;
  • nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5.
  • a further aspect of the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding a variable region of an antibody light chain, wherein
  • the antibody light chain variable region comprises a CDR having the amino acid sequence of SEQ ID NOs: 12-14;
  • the antibody light chain variable region has the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 8;
  • nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 7.
  • a further aspect of the invention relates to a vector comprising the isolated nucleic acid molecule of any of the invention.
  • a further aspect of the invention relates to a host cell comprising the isolated nucleic acid molecule of any of the invention, or the vector of the invention.
  • a further aspect of the invention relates to a method of producing a monoclonal antibody or antigen-binding fragment thereof according to any of the invention, which comprises culturing a host cell of the invention under suitable conditions, and from cell culture The step of recovering the monoclonal antibody or antigen-binding fragment thereof.
  • a further aspect of the invention relates to a hybridoma cell line LT003 deposited with the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO: C2015105.
  • a further aspect of the present invention relates to a conjugate comprising a monoclonal antibody or an antigen-binding fragment thereof, and a conjugated portion, wherein the monoclonal antibody is the monoclonal antibody of any one of the present invention or An antigen-binding fragment, the coupled moiety being a detectable label; in particular, the coupled moiety is a radioisotope, a fluorescent substance, a hair Light matter, colored matter or enzyme.
  • a further aspect of the invention relates to a kit comprising the monoclonal antibody or antigen-binding fragment thereof according to any of the invention, or comprising the conjugate of the invention;
  • the kit further comprises a second antibody that specifically recognizes the monoclonal antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance , luminescent substances, colored substances or enzymes.
  • a detectable label such as a radioisotope, a fluorescent substance , luminescent substances, colored substances or enzymes.
  • a further aspect of the invention relates to the use of a monoclonal antibody or antigen-binding fragment thereof according to any of the invention or a conjugate of the invention for the preparation of a kit for detecting PD1 in a sample The existence or level of it.
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof according to any of the invention or a conjugate according to the invention; optionally further comprising a pharmacy Acceptable carriers and/or excipients.
  • a further aspect of the invention relates to a monoclonal antibody or antigen-binding fragment thereof according to any of the invention or a conjugate of the invention for the preparation of a prophylactic and/or therapeutic and/or adjuvant treatment and/or diagnosis of a tumor or anemia Use in a diseased drug; specifically, the tumor is selected from the group consisting of melanoma, renal tumor, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, non-small cell lung cancer, ovarian cancer, and leukemia.
  • the present inventors have found through animal experiments that 14C12H1L1 can effectively inhibit the growth of the right subcutaneously inoculated MC38 tumor cells in PD-1 HuGEMM mice, which shows that the administration of the antibody drug 14C12H1L1 can significantly inhibit the increase of tumor volume in PD-1 HuGEMM tumor-bearing mice, and It has been marketed with the same target drug Nivolumab mAb.
  • a further aspect of the invention relates to the use of a monoclonal antibody or antigen-binding fragment thereof according to any of the invention or a conjugate of the invention for the preparation of a medicament as follows:
  • the ligand of PD1 is PDL1 or PDL2, preferably PDL1.
  • a further aspect of the invention relates to an in vivo or in vitro method comprising the application of a cell or a need by a donor
  • the subject is an effective amount of the monoclonal antibody or antigen-binding fragment thereof of any one of the invention or the conjugate of the invention, the method being selected from the following:
  • the ligand of PD1 is PDL1 or PDL2, preferably PDL1.
  • the in vitro method is for non-therapeutic or diagnostic purposes.
  • Interferon gamma is mainly produced by natural killer cells (NK), natural killer T cells (NKT), and is produced by stimulation of specific antigens by CD4Th1 cells and CD8 cytotoxic T lymphocytes.
  • IFN ⁇ Interferon gamma
  • NK natural killer cells
  • NKT natural killer T cells
  • CD4Th1 cells CD8 cytotoxic T lymphocytes.
  • IFN ⁇ plays an important role in combating or inhibiting viruses, infection of certain bacteria and protozoal diseases.
  • IFN ⁇ activates macrophages, induces the expression of class II major histocompatibility complex (MHC), activates immune responses to control tumor development (Schoenborn JR, Wilson CB. Regulation of Interferon- ⁇ During Innate and Adaptive Immune Responses. Advances In Immunology 2007;96:41-101).
  • MHC major histocompatibility complex
  • the antibody anti-PD1 antibody can induce the secretion of IFN ⁇ to activate an immune response.
  • Interleukin 2 is produced by T cells, is a growth factor that regulates T cell subsets, is also an important factor regulating immune response, and can promote the proliferation of activated B cells, participate in antibody response, hematopoiesis and tumor surveillance.
  • Recombinant human IL-2 has been approved by the US FDA for the treatment of malignant tumors (including melanoma, kidney tumors, etc.) and is undergoing clinical research for the treatment of chronic viral infections (Chavez, AR, et al., Pharmacologic administration of interleukin- 2. Ann N Y Acad Sci, 2009.1182: p. 14-27).
  • the PD1 antibody of the present invention specifically abolishes the immunosuppression of PD1, activates T cells, induces IL-2 production, and has broad application prospects in gene therapy for diseases such as anti-tumor and parasitic diseases.
  • Monoclonal antibody or antigen-binding fragment or conjugate thereof for use in the prevention and/or treatment and/or adjuvant treatment and/or diagnosis of a tumor or anemia; in particular, the tumor It is selected from the group consisting of melanoma, renal tumor, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, non-small cell lung cancer, ovarian cancer and leukemia.
  • the ligand of PD1 is PDL1 or PDL2, preferably PDL1.
  • the monoclonal antibodies or antigen-binding fragments or conjugates thereof of the invention only block the binding of PD1 to PDL1.
  • a further aspect of the invention relates to a method of preventing and/or treating and/or adjunctively treating and/or diagnosing a tumor or anemia, comprising administering to a subject an effective amount of the monoclonal antibody of any of the invention Or a step of the antigen-binding fragment thereof or the monoclonal antibody conjugate of the present invention; specifically, the tumor is selected from the group consisting of melanoma, renal tumor, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer Non-small cell lung cancer, ovarian cancer and leukemia.
  • the amino acid sequence of the PD1 protein when referring to the amino acid sequence of the PD1 protein (Programmed cell death protein 1, NCBI GenBank: NP_005009.2), it includes the full length of the PD1 protein, or the extracellular fragment PD1ECD of PD1 or contains PD1ECD. Fragments; also include fusion proteins of PD1 ECD, such as fragments fused to a Fc protein fragment (mFc or hFc) of mouse or human IgG.
  • Fc protein fragment mFc or hFc
  • PD1 protein shall include all such sequences, as well as natural or artificial variants thereof. Also, when describing a sequence fragment of a PD1 protein, it includes not only sequence fragments but also corresponding sequence fragments in its natural or artificial variants.
  • the amino acid sequence of the PDL1 protein when referring to the amino acid sequence of the PDL1 protein (Programmed death-ligand 1, NCBI Genebank ID: NP_054862.1), it includes the full length of the PDL1 protein, or the extracellular fragment PDL1 ECD of PDL1 or contains PDL1 ECD. Fragments; also include fusion proteins of PDL1 ECD, such as fragments fused to a Fc protein fragment (mFc or hFc) of mouse or human IgG.
  • Fc protein fragment mFc or hFc
  • the term "PDL1 protein” should include All such sequences as well as natural or artificial variants thereof. Also, when a sequence fragment of a PDL1 protein is described, a PDL1 sequence fragment is included, and a corresponding sequence fragment in its natural or artificial variant is also included.
  • EC 50 refers to the term as used herein, half-maximal effective concentration (concentration for 50% of maximal effect ), the concentration refers to cause 50% of maximal effect.
  • antibody refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains (each pair having one "light” (L) chain and one "heavy” (H) chain. .
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids.
  • Each heavy chain is comprised of a heavy chain variable region (V H) and a heavy chain constant region (C H) composition.
  • the heavy chain constant region is comprised of three domains (C H 1, C H 2 and C H 3) components.
  • Each light chain is comprised of a light chain variable region (V L) and a light chain constant region (C L) components.
  • the light chain constant region is comprised of one domain, C L composition.
  • the constant region of the antibody mediates binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
  • V H regions may be subdivided into hypervariability regions (termed complementarity determining regions (CDR)), interspersed with regions are more conserved, termed framework regions (FR) of.
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4 from the amino terminus to the carboxy terminus arranged three four FR and CDR components.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J.
  • antibody is not limited by any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full length antibody that retains the ability to specifically bind to the same antigen to which the full length antibody binds, and/or compete with the full length antibody.
  • Specific binding to an antigen which is also referred to as an "antigen-binding portion.” See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Or producing an antigen-binding fragment of an antibody by enzymatic or chemical cleavage of an intact antibody.
  • the antigen-binding fragment includes Fab, Fab', F(ab') 2 , Fd, Fv, dAb and complementarity determining regions (CDRs) Fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.
  • CDRs complementarity determining regions
  • the antigen-binding fragments are diabodies, i.e., bivalent antibodies in which V H and V L, domains are expressed on a single polypeptide chain, but using a linker that is too short to not allow the same chain in two Pairing between domains forces the domain to pair with the complementary domain of another strand and create two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444 -6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)).
  • An antigen-binding fragment of an antibody can be obtained from a given antibody (eg, monoclonal antibody 14C12, 14C12H1L1 provided herein) using conventional techniques known to those skilled in the art (eg, recombinant DNA techniques or enzymatic or chemical cleavage methods) (eg, monoclonal antibody 14C12, 14C12H1L1 provided herein)
  • a given antibody eg, monoclonal antibody 14C12, 14C12H1L1 provided herein
  • conventional techniques known to those skilled in the art eg, recombinant DNA techniques or enzymatic or chemical cleavage methods
  • monoclonal antibody 14C12, 14C12H1L1 provided herein eg, monoclonal antibody 14C12, 14C12H1L1 provided herein
  • antibody As used herein, unless the context clearly dictates otherwise, when referring to the term “antibody”, it includes not only intact antibodies, but also antigen-binding fragments of antibodies.
  • mAb and “monoclonal antibody” refer to a fragment of an antibody or antibody from a population of highly homologous antibody molecules, ie, in addition to a natural mutation that may occur spontaneously, A group of identical antibody molecules.
  • Monoclonal antibodies are highly specific for a single epitope on the antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least two or more different antibodies, which typically recognize different epitopes on the antigen.
  • Monoclonal antibodies are typically obtained using hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see, for example, U.S. Patent 4,816,567).
  • humanized antibody means that all or part of the CDR regions of a human immunoglobulin (receptor antibody) are replaced by a CDR region of a non-human antibody (donor antibody).
  • An antibody or antibody fragment, wherein the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody having the desired specificity, affinity or reactivity.
  • some of the amino acid residues of the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of the corresponding non-human antibody or by amino acid residues of other antibodies to further refine or optimize the performance of the antibody.
  • the terms “isolated” or “isolated” refer to artificially obtained from a natural state. If a certain "separated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or that the substance has been isolated from the natural environment, or both. For example, a certain living animal has a naturally isolated polynucleotide or polypeptide that is not isolated, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called separation. of.
  • separation of the term “separated” or “separated” does not exclude mixing There are artificial or synthetic substances, and there are no other impurities that do not affect the activity of the substance.
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC).
  • Phage such as lambda phage or M13 phage and animal virus.
  • Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, such as a fungal cell such as a yeast cell or an Aspergillus.
  • a prokaryotic cell such as Escherichia coli or Bacillus subtilis
  • a fungal cell such as a yeast cell or an Aspergillus.
  • S2 Drosophila cells or insect cells such as Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • an antibody that specifically binds to an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Affinity (K D ) of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the antigen.
  • K D refers to a particular antibody - antigen interaction dissociation equilibrium constant, which is used to describe the binding affinity between antibody and antigen.
  • the antibody e.g., monoclonal antibody 14C12, 14C12H1L1 of the invention
  • the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10
  • An M or smaller dissociation equilibrium constant (K D ) binds to an antigen (eg, a PD1 protein).
  • K D can be determined using methods known to those skilled in the art, for example using molecular interactions Fortebio analyzer.
  • amino acids are generally represented by single letter and three letter abbreviations as are known in the art.
  • alanine can be represented by A or Ala.
  • hybridomas and “hybridoma cell lines” are used interchangeably and when reference is made to the terms In the case of “hybridomas” and “hybridoma cell lines”, they also include subclones and progeny cells of hybridomas. For example, when referring to the hybridoma cell line LT003, it also refers to the subcloned and progeny cells of the hybridoma cell line LT003.
  • pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancement. Agent.
  • pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, a desired effect.
  • an effective amount to prevent a disease eg, a tumor
  • an effective amount to prevent, prevent, or delay the onset of a disease eg, a tumor
  • treating an effective amount of the disease means sufficient to cure or at least partially arrest a patient already suffering from the disease.
  • the amount of disease and its complications Determination of such an effective amount is well within the capabilities of those skilled in the art.
  • the amount effective for therapeutic use will depend on the severity of the condition to be treated, the overall condition of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments for simultaneous administration. and many more.
  • the monoclonal antibody of the present invention can bind specifically to PD1 well, and can effectively block the binding of PD1 to PDL1, specifically relieve PD1 from immunosuppression and activate T lymphocytes.
  • PD1 antibody 14C12H1L1 induced the secretion of IFN- ⁇ and IL-2 significantly stronger than the control antibody 5C4 (5C4: PD1antibody, from Medarex: Alan J. Korman, et al., Human monoclonal antibodies to programmed death 1 ( PD1) and methods for treating cancer using anti-PD1 antibodies alone or in combination with other immunotherapeutics, United States Patent, Patent No. US 8008449B2).
  • the anti-tumor effect of the monoclonal antibody of the present invention is comparable to that of the target drug Nivolumab.
  • the antibodies of the present invention have potential for the preparation or use as a medicament for the prevention and treatment of non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma, leukemia or anemia.
  • Figure 1 Results of SDS-PAGE detection of monoclonal humanized antibody 14C12H1L1. 4 lanes from left to right The sample and its loading amount are as follows: 1. Non-reducing protein electrophoresis loading buffer sample antibody, 1 ⁇ g; 2. Reduced protein electrophoresis loading buffer sample antibody, 1 ⁇ g; Marker, 5 ⁇ l; 3. BSA, 1 ⁇ g .
  • Figure 5 Indirect ELISA detects binding of antibodies 14C12H1L1 and 5C4 to PD1.
  • Figure 7 antibody binding EC 50 14C12H1L1 PD1 protein to a target cell surface 293T-PD1.
  • Figure 8 Binding activity of antibody 14C12H1L1 to T cell surface antigen PD1.
  • Figure 9 Effect of antibody 14C12H1L1 on IFN- ⁇ secretion by mixed lymphocytes.
  • Figure 10 Effect of antibody 14C12H1L1 on IL-2 secretion by mixed lymphocytes.
  • Figure 11 Effect of antibody 14C12H1L1 on secretion of cytokine IL-2 induced by co-culture of mixed PBMC, MDA-MB-231 and Raji cells.
  • Figure 12 Effect of antibody 14C12H1L1 on tumor growth of PD-1 HuGEMM mouse MC38 tumor model.
  • Hybridoma cell line LT003 which was deposited with the China Center for Type Culture Collection (CCTCC) on June 16, 2015, with the accession number CCTCC NO: C2015105, and the deposit address is Wuhan University, Wuhan, China, 430072.
  • the T cells used were from Zhongshan Kangfang Biomedical Co., Ltd.; the BALB/C mice used were purchased from the Guangdong Medical Laboratory Animal Center.
  • the PD-1 HuGEMM mice used were from Nanjing Yinhe Biomedical Co., Ltd.; the MC38 cells used were from Shanghai Fudan IBS Cell Resource Center; the same target drug Nivolumab was used.
  • Monoclonal antibody is from Bristol-Myers Squibb.
  • Example 1 Obtainment of hybridoma cell line LT003 and preparation of monoclonal antibody 14C12
  • PD1-mFc Programmed cell death protein 1, NCBI GenBank ID: NP_005009.2
  • spleen cells and mouse myeloma were obtained from immunized BALB/C mice (purchased from Guangdong Medical Laboratory Animal Center). Fusion of cells into hybridoma cells, refer to currently established methods (eg, Stewart, SJ, "Monoclonal Antibody Production", in Basic Methods in antibody Production and Characterization, Eds. GC Howard and DR Bethell, Boca Raton: CRC Press, 2000 ).
  • the PD1-mFc was used as an antigen-coated ELISA plate, and subjected to indirect ELISA screening to obtain a hybridoma cell secreting a novel antibody which specifically binds to PD1.
  • a hybridoma cell line capable of secreting a monoclonal antibody that competes with PD1 binding to the ligand PDL1-hFc fusion protein (PDL1: Programmed death-ligand 1, NCBI Genebank ID: NP_054862.1) was screened by competitive ELISA and obtained by limiting dilution method.
  • a stable hybridoma cell line was obtained and a LT003 stable cell line was obtained by limiting dilution (the monoclonal antibody secreted by the antibody was named 14C12).
  • Hybridoma cell line LT003 (PD1-14C12), which was deposited with the China Center for Type Culture Collection (CCTCC) on June 16, 2015, with the accession number CCTCC NO: C2015105, deposited at Wuhan University, Wuhan, China. :430072.
  • the LT003 cell line of the present invention was cultured in an IMDM medium containing 10% of low IgG fetal bovine serum, and after 7 days, the cell culture supernatant was collected and purified to obtain antibody 14C12.
  • the mRNA was extracted from the hybridoma cell line LT003 prepared in Example 1 according to the method of culturing the cell bacterial total RNA extraction kit (Tiangen, Cat. No. DP430).
  • the cDNA was synthesized by the III First-Strand Synthesis System for RT-PCR kit and subjected to PCR amplification.
  • the PCR amplification product was directly subjected to TA cloning, and the specific procedure was carried out by referring to the pEASY-T1 Cloning Kit (Transgen CT101) kit instructions.
  • the heavy chain cDNA sequence of 14C12 (Re) (the variable region sequence thereof is shown in SEQ ID NO: 1) and the cDNA sequence of the light chain (the variable region sequence thereof are shown in SEQ ID NO: 3) were cloned into pUC57simple, respectively. (provided by Kingsray) vector (enzyme cleavage site: XbaI & BamHI), respectively obtained pUC57simple-14C12H and pUC57simple-14C12L plasmid.
  • the plasmids pUC57simple-14C12H and pUC57simple-14C12L were digested (HindIII & EcoRI), and the heavy chain light chains recovered by electrophoresis were subcloned into pcDNA3.1 vector, and the recombinant plasmid was co-transfected into 293F cells.
  • the culture solution was vacuum filtered through a high-speed centrifugation filter, and then applied to a HiTrap MabSelectSuRe column.
  • the protein was eluted in one step with Elution Buffer, and the target sample was recovered and replaced with HiTrap Desalting to obtain PBS.
  • the recombinant antibody 14C12(Re) was verified to have a binding activity comparable to that of antibody 14C12 by ELISA binding activity, and can be used for the next human antibody design.
  • Example 3 Design of heavy and light chain sequences of humanized antibody 14C12H1L1
  • variable region sequence of the antibody 14C12H1L1 was obtained by computer simulation of the antibody model and the mutation was designed according to the model (the heavy chain constant region was Ig gamma-1 chain C region, ACCESSION: P01857, and the light chain constant region was Ig kappa chain C region, ACCESSION: P01834). ), the variable region sequence is as follows:
  • the heavy chain cDNA of 14C12H1L1 (the variable region sequence of which is SEQ ID NO: 5, the heavy chain constant region is Ig gamma-1 chain C region, ACCESSION: P01857) and the cDNA of the light chain (the variable region sequence thereof is SEQ ID NO :7, the light chain constant region is Ig kappa chain C region, ACCESSION: P01834) was cloned into pUC57simple (provided by Kingsray) vector (enzyme cleavage site: XbaI & BamHI), and pUC57simple-14C12H1 and pUC57simple-14C12L1 plasmids were obtained, respectively. And subcloned into pcDNA3.1 vector (enzyme cleavage site: XbaI & BamHI), and the recombinant plasmid was co-transfected into 293F cells.
  • the culture solution was vacuum filtered through a high-speed centrifugation filter, and then applied to a HiTrap MabSelectSuRe column.
  • the protein was eluted in one step with Elution Buffer, and the target sample was recovered and replaced with HiTrap Desalting to obtain PBS.
  • the reduced protein sample target protein was approximately 24.5 kD and 49 KD, and the non-reduced protein sample target protein was approximately 147 kD.
  • the kinetic parameters of binding of antibody 14C12 and humanized 14C12H1L1 to antigen PD1 were determined using a Fortebio molecular interaction instrument.
  • the PD1-mFc protein was digested with TEV protease and purified by column to obtain PD1 antigen.
  • the antigen PD1 (antigen concentration: 1 ⁇ g/ml) was biotinylated and fixed on the surface of the SA sensor. After equilibration in PBST, it was bound to antibodies 14C12, 14C12H1L1 and 5C4, respectively, and the antibody was diluted three times from 200 nM with PBST. Dissociated in PBST.
  • the kinetic parameters of the antibodies 14C12, 14C12H1L1 and 5C4 are shown in Table 1, and the kinetic characteristic parameter detection results are shown in Fig. 2, Fig. 3 and Fig. 4, respectively.
  • the results showed that 14C12 and 14C12H1L1 had good affinity with antigen, and the affinity was stronger than that of the control antibody 5C4.
  • Example 6 Detection of binding activity of antibody to antigen PD1 by indirect ELISA
  • the binding activity of 14C12H1L1 and 5C4 to PD1 was determined by indirect ELISA. The method is as follows:
  • Fig. 5 The results of binding of the detection antibodies 14C12H1L1 and 5C4 to the antigen PD1 are shown in Fig. 5. As can be seen from Figure 5, both antibodies 14C12H1L1 and 5C4 were able to bind PD1 efficiently, and their binding efficiency was dose-dependent. The fluorescence intensity of each dose is shown in Table 2. 14C12H1L1 by antibody binding and 5C4 fluorescence quantitative analysis, simulation curves 14C12H1L1 5C4 antibody binding efficiency and EC 50 respectively 0.032nM and 0.043nM.
  • Example 7 Competitive ELISA method to detect the binding of antibodies to PDL1 to bind antigen PD1
  • the competitive antibody ELISA method was used to determine the binding of the humanized antibody 14C12H1L1 and 5C4 to PDL1 to bind the antigen PD1.
  • the method is as follows:
  • the cells were coated with PD1-mFc at 4 ° C overnight, and blocked with 1% BSA for 2 h.
  • Different concentrations of antibodies 14C12H1L1 and 5C4 were mixed with PDL1-mFc for 10 min (see Table 3 for dilution), and incubated at 37 ° C for 30 min.
  • the corresponding anti-human and anti-mouse enzyme secondary antibodies were incubated at 37 ° C for 30 min.
  • the absorbance at 450 nm was measured on a microplate reader.
  • Fig. 6 The results of binding of the detection antibodies 14C12H1L1 and 5C4 to the antigen PD1 are shown in Fig. 6.
  • antibody 14C12H1L1 was able to compete with PDL1 for binding to PD1 protein, and its binding efficiency was dose-dependent.
  • the fluorescence intensity of each dose is shown in Table 3.
  • 14C12H1L1 by antibody binding and 5C4 fluorescence quantitative analysis, simulation curve 14C12H1L1 antibodies 5C4 and binding efficiency of EC 50 were: 1.322nM and 1.199nM.
  • Example 8 Flow cytometry method for detecting binding activity of antibody to cell surface antigen PD1
  • Host cell 293T expressing the PD1 antigen was first constructed, and then the host cell was labeled with the humanized antibody 14C12H1L1 (see Example 4) prepared in the present invention. Flow cytometry analysis was then used to verify the antigen-specific binding ability of antibody 14C12H1L1 to the native conformation of the cell surface.
  • Construction of host cell 293T expressing PD1 antigen The vector pLenti6.3-PD1 (vector pLenti6.3 purchased from Invitrogen) containing PD1 was transfected into 293T cells according to the lipofectamin transfection kit (purchased from Invitrogen). A clonal population 293T-PD1 stably expressing PD1 was obtained.
  • Antibody labeling and flow cytometry detection Host cell 293T-PD1 expressing PD1 antigen obtained by the above steps of the conventional trypsin digestion method, and the number of cells per collection tube was 2 ⁇ 10 5 , using PBS (1% BSA) PD1 antibody dilutions at 50 nM, 10 nM, 5 nM, 1 nM, 0.1 nM, and 0.01 nM were prepared and incubated with 293T cells expressing PD1 for 2 hours on ice, and 100 ⁇ L of FITC goat anti-human IgG (1:500) ice was added to each tube. After incubating for 1 hour, the cells were washed 3 times with PBS, and then resuspended in 300 ⁇ L of PBS, and the fluorescent signal was detected by flow cytometry using a FITC channel.
  • PBS 1% BSA
  • the results of binding of the humanized antibody 14C12H1L1 to 293T-PD1 cells are shown in FIG.
  • the 14C12H1L1 antibody efficiently binds to the target PD1 protein on the surface of the host cell 293T-PD1, and its binding efficiency is dose-dependent.
  • the fluorescence intensity of each dose is shown in Table 4. 14C12H1L1 antibody binding by fluorescence quantitative analysis, simulation curve of antibody binding efficiency 14C12H1L1 EC 50 of 1.89nM.
  • Table 4 Fluorescence intensity analysis of 14C12H1L1 binding to PD1 host cell 293T-PD1 surface antigen by flow cytometry
  • Example 9 Flow cytometry method for detecting binding activity of antibodies to T cell surface antigen PD1
  • PBMC peripheral blood mononuclear cells
  • PHA Stai Disease Control Biotechnology Co., Ltd., 50 ⁇ l/ml
  • the binding results of the humanized antibody 14C12H1L1 to T cells are shown in FIG. As can be seen from Figure 8, the 14C12H1L1 antibody efficiently binds to the target PD1 protein on the surface of T cells, and its binding efficiency is dose dependent.
  • Example 10 Mixed lymphocyte reaction: secretion of cytokine IFN- ⁇ , IL-2
  • PBMC peripheral blood mononuclear cells
  • Ficoll-Paque Plus GE Healthcare LOT No.: 171440-02
  • IL-4 Peprotech K2513, 1000 U/ml
  • GM-CSF Peprotech H1513, 1000 U/ml
  • DC cells were obtained by inducing TNF- ⁇ (Peprotech G1513, 200 U/ml) for 3 days.
  • T cells were isolated from PBMC, and the obtained DC cells were mixed with T cells in a ratio of 1:10, and different ratios of antibodies 14C12H1L1, 5C4 and hIgG (hIgG as isotype control) were added for 5-6 days, and ELISA was used.
  • the kit was used to detect the secretion of IFN- ⁇ (purchased from Daktronics) and IL-2 (purchased from Daktronics).
  • 14C12H1L1 antibody can effectively induce the secretion of IFN- ⁇ and IL-2 by mixed lymphocytes, and its secretion The amount was dose dependent with antibody 14C12H1L1. The effect of antibody 14C12H1L1 on the secretion of IFN- ⁇ and IL-2 was stronger than that of the control antibody 5C4.
  • the isolated PBMC (method as in Example 10) was stimulated with PHA (Shanghai CDC Biotech Co., Ltd., 50 ⁇ l/mL) for 3 days, and then stimulated mature PBMC (5 ⁇ 10 4 cells/well) was added to the 96-well plate.
  • Raji cells (Shanghai Academy of Sciences) (5 ⁇ 10 4 cells/well) and MDA-MB-231 cells (ATCC) (1 ⁇ 10 4 cells/well), 100 nM 14C12H1L1 or control antibody 5C4 or hIgG (hIgG is isotype control) Mix and homogenize.
  • the secretion amount of IL-2 (purchased from Daktronics Co., Ltd.) was measured using an ELISA kit, and the specific procedure was carried out in accordance with the kit instructions.
  • FIG. 11 The results of secretion detection of IL-2 after mixed culture of cells are shown in FIG. As can be seen from Fig. 11, the 14C12H1L1 antibody can effectively induce the secretion of IL-2 by PBMC, and the induction effect of antibody 14C12H1L1 on IL-2 secretion is significantly stronger than that of the control antibody 5C4.
  • Example 12 Effect of antibody 14C12H1L1 on tumor growth of PD-1 HuGEMM mouse MC38 tumor model ring
  • MC38 tumor cells (1 ⁇ 10 6 /only) were subcutaneously inoculated into the right side of PD-1 HuGEMM mice (human PD-1 transgenic mice). When the average tumor volume reached approximately 118 mm 3 , mice were randomly divided according to tumor volume. Four experimental groups were administered intraperitoneally, with 8 mice in each group. The specific grouping and dosage are as follows:
  • Nivolumab group (administered dose 8 mg/kg).
  • the above four groups were administered twice a week for a total of 5 times. Tumor size was measured twice weekly after group administration.
  • the tumor volume of Nivolumab group, 14C12H1L1 high dose group and 14C12H1L1 low dose group was statistically significantly lower than that of Isotype control group (P ⁇ 0.01, ⁇ 0.01, ⁇ 0.05).
  • the 14C12H1L1 high-dose group (8 mg/kg) showed a statistically significant anti-tumor effect in the PD-1 HuGEMM mouse MC38 tumor model and was comparable to the target-marketed drug Nivolumab (8 mg/kg).

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Abstract

一种抗PD1单克隆抗体或其抗原结合片段、其药物组合物及其用途。其中，所述单克隆抗体的重链可变区包含：氨基酸序列为SEQ ID NO:9－11的CDR；和/或所述单克隆抗体的轻链可变区包含：氨基酸序列为SEQ ID NO:12－14的CDR。所述单克隆抗体能够很好地特异性与PD1结合，特异地解除PD1对机体免疫抑制，激活T淋巴细胞。

Description

一种抗PD1单克隆抗体、其药物组合物及其用途 技术领域
本发明属于肿瘤治疗和分子免疫学领域，涉及一种抗PD1抗体、其药物组合物及其用途。具体地，本发明涉及一种抗PD1的单克隆抗体。
背景技术
跨膜受体PD1(programmed cell death 1，程序性细胞死亡因子1，亦简称为PD-1)是CD28基因家族成员之一，在活化的T细胞，B细胞以及骨髓系细胞都有表达。PD1的受体PDL1和PDL2均属于B7超家族，其中PDL1多种细胞都有表达，包括T细胞，B细胞以及内皮细胞和上皮细胞，PDL2则仅表达于抗原呈递细胞如树突状细胞和巨噬细胞。
T细胞对清除病毒感染起着非常重要的作用，但T细胞抗病毒反应通常与免疫病理相关。PD1在负调节T细胞的活化过程中起着非常重要的作用，PD1介导的对T细胞负调节作用可减少促进感染过程中所引起的组织损伤，但是阻断或抑制PD1的负调节作用可导致自身免疫性疾病，例如，PD1基因敲除小鼠能更有效的清除胰腺病毒感染，但是却导致了更严重的肝脏损伤(Isai et al.,2003,J.Exp.Med.198:39-50)。另外，高表达PD1的肿瘤一般形成很难被检测到的癌症(Hamanishi et al.,2007,Proc.Natl.Acad.Sci.USA 104:3360-5)。一种实施有效的方法是通过体内注射抗体对PD1的表达进行调控。
由于PD1抗体的广谱抗肿瘤前景和惊人的药效，业界普遍认为针对PD1通路的抗体将导致治疗多种肿瘤治疗的突破性的进展：用于治疗非小细胞性肺癌，肾细胞癌，卵巢癌，黑色素瘤(Homet M.B.,Parisi G.,et al.,Anti-PD1Therapy in Melanoma.Semin Oncol.2015Jun；42(3):466-473)，白血病以及贫血病(Held SA,Heine A,et al.,Advances in immunotherapy of chronic myeloid leukemia CML.Curr Cancer Drug Targets.2013Sep；13(7):768-74)。
PD1抗体在2012年和2013年的美国癌症协会(AACR)年会以及美国临床肿瘤协会(ASCO)年会上揭晓了其前所未有的临床药效的数据后，即成为全球制药行业最炙手可热的在研新药。
目前，尚需要开发新的具有更好的结合效率的抗PD1抗体，以有效地阻断PD1与PDL1的结合。
发明内容
本发明人经过深入的研究和创造性的劳动，利用哺乳动物细胞表达系统表达出重组的PD1作为抗原免疫小鼠，经小鼠脾脏细胞与骨髓瘤细胞融合获得杂交瘤细胞。发明人通过进行对大量样本的筛选，得到了杂交瘤细胞株LT003(保藏编号为CCTCC NO:C2015105)。
本发明人惊奇地发现，杂交瘤细胞株LT003能够分泌产生与PD1特异性结合的特异性单克隆抗体(命名为14C12)，并且该单克隆抗体能够十分有效地阻断PD1与PDL1的结合。
进一步地，本发明人创造性地制得了抗PD1的人源化抗体(命名为14C12H1L1)。
本发明人还惊奇地发现，本发明的抗体14C12、14C12H1L1能有效地结合人T细胞，并且激活T细胞，诱导人淋巴细胞分泌IFN-γ和IL-2。本发明的抗体14C12、14C12H1L1具有用于制备防治肺癌、黑色素瘤、肾肿瘤、卵巢癌、白血病等癌症以及贫血病的药物的潜力。
由此提供了下述发明：
本发明的一个方面涉及单克隆抗体或其抗原结合片段，其中，
所述单克隆抗体的重链可变区(VH)包含：氨基酸序列为SEQ ID NO:9－11的CDR；
和/或
所述单克隆抗体的轻链可变区(VL)包含：氨基酸序列为SEQ ID NO:12－14的CDR。
在本发明的一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中，
所述单克隆抗体的重链可变区的氨基酸序列选自SEQ ID NO:2和SEQ ID NO:6；
和/或
所述单克隆抗体的轻链可变区的氨基酸序列选自SEQ ID NO:4和SEQ ID NO:8。
在本发明的一个实施方案中，所述的单克隆抗体或其抗原结合片段，其中，所述单克隆抗体包括：
(1)如SEQ ID NO:2所示的VH和如SEQ ID NO:4所示的VL；
(2)如SEQ ID NO:6所示的VH和如SEQ ID NO:8所示的VL。
轻链和重链的可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)(重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR 包含LCDR1、LCDR2、LCDR3；其由Kabat等人命名，见Sequences of Proteins of Immunological Interest,Fifth Edition(1991),第1-3卷,NIH Publication 91-3242,Bethesda Md)。
通过本领域技术人员所熟知的技术手段，例如通过VBASE2数据库分析上面的(1)-(2)项中的单克隆抗体序列的CDR区的氨基酸序列：
本发明的抗体14C12、14C12H1L1具有相同的CDR：
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1:GFAFSSYD(SEQ ID NO:9)，
HCDR2:ISGGGRYT(SEQ ID NO:10)，
HCDR3:ANRYGEAWFAY(SEQ ID NO:11)；
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1:QDINTY(SEQ ID NO:12)，
LCDR2:RAN(SEQ ID NO:13)，
LCDR3:LQYDEFPLT(SEQ ID NO:14)。
在本发明的一些实施方式中，所述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选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、人源化抗体、嵌合抗体或双抗体。
在本发明的一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中，所述的单克隆抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的KD结合PD1蛋白；优选地，所述KD通过Fortebio分子相互作用仪测得。
在本发明的一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中，所述的单克隆抗体以小于大约100nM，例如小于大约10nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM或更小的EC50结合PD1蛋白。具体地，所述EC50通过间接ELISA方法测得。
在本发明的一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中，所述的单克隆抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的KD结合PD1蛋白。
在本发明的一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中，所述的单克隆抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
在本发明的一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中所述单克隆抗体是由杂交瘤细胞株LT003产生的单克隆抗体，所述杂交瘤细胞株LT003保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2015105。
本发明的另一方面涉及一种分离的核酸分子，其包含能够编码抗体重链可变区的核酸序列，其中，
所述抗体的重链可变区包含：氨基酸序列为SEQ ID NO:9－11的CDR；
具体地，所述抗体的重链具有SEQ ID NO:2或SEQ ID NO:6所示的氨基酸序列；
更具体地，所述核酸分子具有SEQ ID NO:1或SEQ ID NO:5所示的核苷酸序列。
本发明的再一方面涉及一种分离的核酸分子，其包含能够编码抗体轻链可变区的核酸序列，其中，
所述抗体轻链可变区包含氨基酸序列为SEQ ID NO:12－14的CDR；
具体地，所述抗体轻链可变区具有SEQ ID NO:4或SEQ ID NO:8所示的氨基酸序列；
更具体地，所述核酸分子具有SEQ ID NO:3或SEQ ID NO:7所示的核苷酸序列。
本发明的再一方面涉及一种载体，其包本发明中任一项所述的分离的核酸分子。
本发明的再一方面涉及一种宿主细胞，其包含本发明中任一项所述的分离的核酸分子，或者本发明的载体。
本发明的再一方面涉及一种制备本发明中任一项所述的单克隆抗体或其抗原结合片段的方法，其包括在合适的条件下培养本发明的宿主细胞，以及从细胞培养物中回收所述单克隆抗体或其抗原结合片段的步骤。
本发明的再一方面涉及杂交瘤细胞株LT003，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2015105。
本发明的再一方面涉及一种偶联物，其包括单克隆抗体或其抗原结合片段以及偶联部分，其中，所述单克隆抗体为本发明中任一项所述的单克隆抗体或其抗原结合片段，所述偶联部分为可检测的标记；具体地，所述偶联部分为放射性同位素、荧光物质、发 光物质、有色物质或酶。
本发明的再一方面涉及一种试剂盒，其包括本发明中任一项所述的单克隆抗体或其抗原结合片段，或者包括本发明的偶联物；
具体地，所述试剂盒还包括第二抗体，其特异性识别所述单克隆抗体或其抗原结合片段；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物在制备试剂盒中的用途，所述试剂盒用于检测PD1在样品中的存在或其水平。
本发明的再一方面涉及一种药物组合物，其包含本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明所述的偶联物；可选地，其还包括药学上可接受的载体和/或赋形剂。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物在制备预防和/或治疗和/或辅助治疗和/或诊断肿瘤或者贫血病的药物中的用途；具体地，所述肿瘤选自黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌、肝癌、非小细胞性肺癌、卵巢癌和白血病。
本发明人通过动物实验发现，14C12H1L1能够有效地抑制PD-1HuGEMM小鼠右侧皮下接种MC38肿瘤细胞的生长，表现为给予抗体药14C12H1L1可显著抑制PD-1HuGEMM荷瘤小鼠肿瘤体积的增加，与已上市同靶点药物Nivolumab单抗药效相当。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物在制备如下药物中的用途：
阻断PD1与PD1的配体结合的药物，
调节(例如下调)PD1活性或水平的药物，
解除PD1对机体免疫抑制的药物，或者
提高T淋巴细胞中IFN-γ和/或IL-2表达的药物；
具体地，所述PD1的配体为PDL1或PDL2，优选为PDL1。
本发明的再一方面涉及一种在体内或体外方法，包括施加细胞或给予者有需求的受 试者以有效量的本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物的步骤，所述方法选自如下：
阻断PD1与PD1的配体结合的方法，
调节(例如下调)PD1活性或水平的方法，
解除PD1对机体免疫抑制的方法，或者
提高T淋巴细胞中IFN-γ和/或IL-2表达的方法；
具体地，所述PD1的配体为PDL1或PDL2，优选为PDL1。
在本发明的一个具体的实施方案中，所述体外方法是非治疗目的的或诊断目的的。
干扰素γ(IFNγ)，主要由自然杀伤细胞(NK)，自然杀伤T细胞(NKT)先天产生，和由CD4Th1细胞和CD8细胞毒性T淋巴细胞这些效应T细胞经由特定抗原刺激后产生。IFNγ作为一种重要的先天性和获得性免疫细胞因子在对抗或抑制病毒，某些细菌和原虫性疾病感染起着重要作用。同时，IFNγ能激活巨噬细胞，诱导Ⅱ类主要组织相容性复合体(MHC)的表达激活免疫反应控制肿瘤的发展(Schoenborn JR,Wilson CB.Regulation of Interferon-γDuring Innate and Adaptive Immune Responses.Advances in Immunology 2007；96:41-101)。本发明的体外实验中，抗体anti-PD1抗体能诱导IFNγ的分泌激活免疫反应。
白细胞介素2(IL-2)由T细胞产生，是调节T细胞亚群的生长因子，也是调控免疫应答的重要因子，并可促进活化B细胞增殖，参与抗体反应、造血和肿瘤监视。重组的人IL-2已经被美国FDA批准用于治疗恶性肿瘤(包括黑色素瘤、肾肿瘤等)，同时正在进行治疗慢性病毒感染的临床研究(Chavez,A.R.,et al.,Pharmacologic administration of interleukin-2.Ann N Y Acad Sci,2009.1182:p.14-27)。体外实验中，本发明的PD1抗体可特异地解除PD1对机体免疫抑制，激活T细胞，诱导IL-2产生，在抗肿瘤及寄生虫等疾病的基因治疗中有广泛应用前景。
根据本发明任一项所述的单克隆抗体或其抗原结合片段或者偶联物，其用于预防和/或治疗和/或辅助治疗和/或诊断肿瘤或者贫血病；具体地，所述肿瘤选自黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌、肝癌、非小细胞性肺癌、卵巢癌和白血病。
根据本发明任一项所述的单克隆抗体或其抗原结合片段或者偶联物，其用于：
阻断PD1与PD1的配体结合，
调节(例如下调)PD1活性或水平，
解除PD1对机体免疫抑制，或者
提高T淋巴细胞中IFN-γ和/或IL-2表达；
具体地，所述PD1的配体为PDL1或PDL2，优选为PDL1。
在本发明的一个具体的实施方案中，本发明的单克隆抗体或其抗原结合片段或者偶联物仅阻断PD1与PDL1的结合。
本发明的再一方面涉及一种预防和/或治疗和/或辅助治疗和/或诊断肿瘤或者贫血病的方法，包括给予受试者有效量的本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明所述的单克隆抗体偶联物的步骤；具体地，所述肿瘤选自黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌、肝癌、非小细胞性肺癌、卵巢癌和白血病。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，当提及PD1蛋白(Programmed cell death protein 1,NCBI GenBank:NP_005009.2)的氨基酸序列时，其包括PD1蛋白的全长，或者PD1的胞外片段PD1ECD或者包含PD1ECD的片段；还包括PD1ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在PD1蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“PD1蛋白”应包括所有此类序列，及其天然或人工的变体。并且，当描述PD1蛋白的序列片段时，其不仅包括序列片段，还包括其天然或人工变体中的相应序列片段。
如本文中所使用的，当提及PDL1蛋白(Programmed death-ligand 1,NCBI Genebank ID:NP_054862.1)的氨基酸序列时，其包括PDL1蛋白的全长，或者PDL1的胞外片段PDL1ECD或者包含PDL1ECD的片段；还包括PDL1ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在PDL1蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“PDL1蛋白”应包括 所有此类序列以及其天然或人工的变体。并且，当描述PDL1蛋白的序列片段时，包括PDL1序列片段，还包括其天然或人工变体中的相应序列片段。
如本文中所使用的，术语EC50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”是指，是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab'、F(ab')2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
在一些情况下，抗体的抗原结合片段是双抗体，即，双价抗体，其中VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的单克隆抗体14C12、14C12H1L1)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片断，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(Nature,256:495,1975)，但也可采用重组DNA技术获得(如参见U.S.P 4,816,567)。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.,Nature,321:522 525(1986)；Reichmann et al.,Nature,332:323 329(1988)；Presta,Curr.Op.Struct.Biol.,2:593 596(1992)；和Clark,Immunol.Today 21:397 402(2000)。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混 有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的亲和力(KD)结合该抗原。
如本文中所使用的，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体(例如，本发明的单克隆抗体14C12、14C12H1L1)以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的解离平衡常数(KD)结合抗原(例如，PD1蛋白)。可以使用本领域技术人员知悉的方法测定KD，例如使用Fortebio分子相互作用仪测定。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用；术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“杂交瘤”和“杂交瘤细胞株”可互换使用，并且当提及术语 “杂交瘤”和“杂交瘤细胞株”时，其还包括杂交瘤的亚克隆和后代细胞。例如，当提及杂交瘤细胞株LT003时，其还指杂交瘤细胞株LT003的亚克隆和后代细胞。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
发明的有益效果
本发明的单克隆抗体特别是14C12H1L1能够很好地特异性与PD1结合，并且能够十分有效地阻断PD1与PDL1的结合，特异地解除PD1对机体免疫抑制，激活T淋巴细胞。其中，PD1抗体14C12H1L1对IFN-γ和IL-2分泌的诱导效果明显强于对照抗体5C4(5C4：PD1antibody，来源于Medarex公司：Alan J.Korman,et al.,Human monoclonal antibodies to programmed death 1(PD1)and methods for treating cancer using anti-PD1antibodies alone or in combination with other immunotherapeutics,United States Patent,Patent No.US 8008449B2)。本发明的单克隆抗体特别是14C12H1L1的抗肿瘤作用与同靶点上市药物Nivolumab效果相当。本发明的抗体具有用于制备或者作为防治非小细胞性肺癌、肾细胞癌、卵巢癌、黑色素瘤、白血病或者贫血病的药物的潜力。
附图说明
图1：单克隆人源化抗体14C12H1L1的SDS-PAGE检测结果。从左至右的4个泳道 的样品及其上样量依次为：1.非还原型蛋白电泳上样缓冲液样品抗体，1μg；2.还原型蛋白电泳上样缓冲液样品抗体，1μg；Marker，5μl；3.BSA，1μg。
图2：抗体14C12的动力学特征参数检测结果。
图3：抗体14C12H1L1的动力学特征参数检测结果。
图4：抗体5C4的动力学特征参数检测结果。
图5：间接ELISA检测抗体14C12H1L1和5C4与PD1的结合。
图6：竞争ELISA检测抗体14C12H1L1和5C4与PDL1竞争结合PD1。
图7：抗体14C12H1L1与293T-PD1细胞表面的靶标PD1蛋白结合EC50。
图8：抗体14C12H1L1与T细胞表面抗原PD1的结合活性。
图9：抗体14C12H1L1对混合淋巴细胞的IFN-γ分泌的影响。
图10：抗体14C12H1L1对混合淋巴细胞的IL-2分泌的影响。
图11：抗体14C12H1L1对混合PBMC、MDA-MB-231和Raji细胞共培养所诱导的细胞因子IL-2的分泌的影响。
图12：抗体14C12H1L1对PD-1HuGEMM小鼠MC38肿瘤模型的肿瘤生长的影响。
杂交瘤细胞株LT003，其于2015年6月16日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2015105，保藏地址为中国.武汉.武汉大学，邮编：430072。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市场购买获得的常规产品。
在下面的实施例中，使用的T细胞来自中山康方生物医药有限公司；使用的BALB/C小鼠购自广东省医学实验动物中心。使用的PD-1HuGEMM小鼠来源于南京银河生物医药有限公司；使用的MC38细胞来源于上海复旦IBS细胞资源中心；使用的同靶点上市药物Nivolumab
Figure PCTCN2017098465-appb-000001
单抗来源于百时美施贵宝公司。
实施例1：杂交瘤细胞株LT003的获得以及单克隆抗体14C12的制备
1.杂交瘤细胞株LT003的建立
用PD1-mFc(PD1:Programmed cell death protein 1,NCBI GenBank ID:NP_005009.2)融合蛋白作为抗原，取免疫BALB/C小鼠(购自广东医学实验动物中心)的脾细胞与小鼠骨髓瘤细胞融合成杂交瘤细胞，参考目前已确立的方法(例如，Stewart,S.J.,“Monoclonal Antibody Production”,in Basic Methods in antibody Production and Characterization,Eds.G.C.Howard and D.R.Bethell,Boca Raton:CRC Press,2000)。
用PD1-mFc作为抗原包被酶标板，进行间接ELISA法筛选，得到分泌与PD1特异性结合的新的抗体的杂交瘤细胞。
通过竞争ELISA筛选出能够分泌与配体PDL1-hFc融合蛋白(PDL1：Programmed death-ligand 1,NCBI Genebank ID:NP_054862.1)竞争结合PD1的单克隆抗体的杂交瘤细胞株，经过有限稀释法得到稳定的杂交瘤细胞株，并经过有限稀释法得到LT003稳定细胞株(其分泌的单克隆抗体命名为14C12)。
杂交瘤细胞株LT003(PD1-14C12)，其于2015年6月16日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2015105，保藏地址为中国.武汉.武汉大学，邮编：430072。
2.单克隆抗体14C12的制备
用含10％的低IgG胎牛血清的IMDM培养基对本发明LT003细胞株进行培养，7天后收集细胞培养上清进行纯化制备获得抗体14C12。
实施例2：单克隆抗体14C12的轻链和重链序列的获得
按照培养细胞细菌总RNA提取试剂盒(Tiangen，货号DP430)的方法，从实施例1制得的杂交瘤细胞株LT003中提取mRNA。
按照Invitrogen 
Figure PCTCN2017098465-appb-000002
III First-Strand Synthesis System for RT-PCR试剂盒说明书合成cDNA，并进行PCR扩增。
PCR扩增产物直接进行TA克隆，具体操作参考pEASY-T1Cloning Kit(Transgen CT101)试剂盒说明书进行。
将TA克隆的产物直接进行测序，测序结果如下：
重链可变区的DNA测序结果：(354bp)
Figure PCTCN2017098465-appb-000003
Figure PCTCN2017098465-appb-000004
其编码的蛋白序列：(118aa)
Figure PCTCN2017098465-appb-000005
轻链可变区的DNA测序结果：(318bp)
Figure PCTCN2017098465-appb-000006
其编码的蛋白序列：(106aa)
Figure PCTCN2017098465-appb-000007
2.重组抗体14C12(Re)的制备
将14C12(Re)的重链cDNA序列(其可变区序列如SEQ ID NO:1所示)和轻链的cDNA序列(其可变区序列如SEQ ID NO:3所示)分别克隆到pUC57simple(金斯瑞公司提供)载体中(酶切位点：XbaI&BamHI)，分别获得pUC57simple-14C12H和 pUC57simple-14C12L质粒。
分别将质粒pUC57simple-14C12H和pUC57simple-14C12L进行酶切(HindIII&EcoRI)，电泳回收得到的重链轻链分别亚克隆到pcDNA3.1载体中，提取重组质粒共转染293F细胞。
细胞培养7天后，将培养液通过高速离心、微孔滤膜抽真空过滤后，上样至HiTrap MabSelectSuRe柱，用Elution Buffer一步洗脱蛋白，回收目标样品并用HiTrap Desalting换液至PBS后进行纯化获得重组抗体14C12(Re)。
重组抗体14C12(Re)经ELISA结合活性验证具有与抗体14C12相当的结合活性，可以用于进行下一步抗体人源化设计。
实施例3：人源化抗体14C12H1L1的重链和轻链序列的设计
1.人源化抗体14C12H1L1的轻链和重链序列的设计
根据PD1蛋白的三维晶体结构(Shinohara T,et al.,Structure and chromosomal localization of the human PD1gene(PDCD1).Genomics 1995,23(3):704–6)以及实施例2获得的抗体14C12的序列，通过计算机模拟抗体模型，根据模型设计突变，得到抗体14C12H1L1的可变区序列(重链恒定区为Ig gamma-1chain C region，ACCESSION:P01857，轻链恒定区为Ig kappa chain C region，ACCESSION:P01834)，可变区序列如下：
重链可变区的DNA序列：(354bp)
Figure PCTCN2017098465-appb-000008
其编码的蛋白序列：(118aa)
Figure PCTCN2017098465-appb-000009
Figure PCTCN2017098465-appb-000010
轻链可变区的DNA序列：(321bp)
Figure PCTCN2017098465-appb-000011
其编码的蛋白序列：(107aa)
Figure PCTCN2017098465-appb-000012
实施例4：人源化抗体14C12H1L1的制备和SDS-PAGE电泳检测
将14C12H1L1的重链cDNA(其可变区序列为SEQ ID NO:5，重链恒定区为Ig gamma-1chain C region，ACCESSION:P01857)和轻链的cDNA(其可变区序列为SEQ ID NO:7，轻链恒定区为Ig kappa chain C region，ACCESSION:P01834)分别克隆到pUC57simple(金斯瑞公司提供)载体中(酶切位点：XbaI&BamHI)，分别获得pUC57simple-14C12H1和pUC57simple-14C12L1质粒，并分别亚克隆到pcDNA3.1载体中(酶切位点：XbaI&BamHI)，提取重组质粒共转染293F细胞。
细胞培养7天后，将培养液通过高速离心、微孔滤膜抽真空过滤后，上样至HiTrap MabSelectSuRe柱，用Elution Buffer一步洗脱蛋白，回收目标样品并用HiTrap Desalting换液至PBS后进行纯化获得重组抗体14C12H1L1。进行SDS-PAGE电泳检测。
结果如图1所示，还原型蛋白样品目标蛋白大约在24.5kD和49KD处，非还原型蛋白样品目标蛋白大约在147kD处。
实施例5：抗体的动力学参数测定
使用Fortebio分子相互作用仪测定抗体14C12及人源化14C12H1L1与抗原PD1结合的动力学参数。
1.用TEV蛋白酶酶切PD1-mFc蛋白，并过柱纯化获得PD1抗原。
2.抗原PD1(抗原浓度为1μg/ml)经生物素标记后固定于SA传感器表面，于PBST中平衡后，分别与抗体14C12、14C12H1L1和5C4结合，抗体用PBST从200nM往下三倍稀释，于PBST中解离。
抗体14C12、14C12H1L1和5C4的动力学参数见表1，动力学特征参数检测结果分别如图2、图3和图4所示。结果表明，14C12和14C12H1L1与抗原均有较好的亲和力，且亲和力均强于对照抗体5C4。
表1：抗体14C12动力学参数
Figure PCTCN2017098465-appb-000013
KD为亲和力常数；kon为抗原抗体结合速率；kdis为抗原抗体解离速率；KD＝kdis/kon。
实施例6：间接ELISA方法检测抗体与抗原PD1的结合活性
采用间接ELISA方法分别测定14C12H1L1和5C4分别与PD1的结合活性。方法具体如下：
酶标板中加入PD1-mFc孵育，4℃过夜，用1％的BSA 37℃封闭2h后，分别加入抗体，37℃孵育30min，加入HRP标记羊抗人IgG(H+L)二抗(Jackson，109-035-088)，用TMB(Neogen，308177)进行显色反应5min，并在酶标仪中检测450nm波长吸光度。
检测抗体14C12H1L1和5C4与抗原PD1结合结果如图5所示。由图5可见，抗体14C12H1L1和5C4均能有效地结合PD1蛋白，并且其结合效率呈剂量依赖关系，各剂量的荧光强度见表2。通过对结合的抗体14C12H1L1和5C4进行荧光定量分析，曲线模拟14C12H1L1和5C4抗体的结合效率EC50分别为0.032nM和0.043nM。
表2：抗体14C12H1L1和5C4分别与PD1结合的荧光强度
Figure PCTCN2017098465-appb-000014
实施例7：竞争ELISA方法检测抗体与PDL1竞争结合抗原PD1的活性
采用竞争ELISA方法分别测定人源化抗体14C12H1L1以及5C4与PDL1的竞争结合抗原PD1。方法具体如下：
用PD1-mFc包被酶标板4℃过夜，1％BSA封闭2h后，分别将不同浓度的抗体14C12H1L1和5C4分别与PDL1-mFc混合10min(稀释浓度见表3)，37℃孵育30min后加入相应的抗人和抗鼠酶标二抗37℃孵育30min。在酶标仪上检测450nm的吸光值。
检测抗体14C12H1L1和5C4与抗原PD1结合结果如图6所示。由图6可见，抗体14C12H1L1能有效地与PDL1竞争结合PD1蛋白，并且其结合效率呈剂量依赖关系，各剂量的荧光强度见表3。通过对结合的抗体14C12H1L1和5C4进行荧光定量分析，曲线模拟抗体14C12H1L1和5C4的结合效率EC50分别为：1.322nM和1.199nM。
表3：抗体14C12H1L1和5C4与PDL1竞争结合PD1
Figure PCTCN2017098465-appb-000015
Figure PCTCN2017098465-appb-000016
实施例8：流式细胞仪方法检测抗体与细胞表面抗原PD1的结合活性
首先构建表达PD1抗原的宿主细胞293T，然后用本发明中制备的人源化抗体14C12H1L1(见实施例4)对改宿主细胞进行标记。然后采用流式细胞术分析验证抗体14C12H1L1对细胞表面天然构象的抗原特异性的结合能力。
1.表达PD1抗原的宿主细胞293T的构建
具体步骤如下：
表达PD1抗原的宿主细胞293T的构建：按照lipofectamin转染试剂盒(购自Invitrogen公司)方法将包含PD1的载体pLenti6.3-PD1(载体pLenti6.3购自Invitrogen公司)转染293T细胞，经筛选获得稳定表达PD1的克隆群体293T-PD1。
2.抗体对293T-PD1细胞表面抗原的结合
抗体标记和流式细胞仪检测：采用常规胰酶消化方法上述步骤获得的表达PD1抗原的宿主细胞293T-PD1，并使每个收集管细胞数为2×105，用PBS(1％BSA)配制浓度分别为50nM，10nM，5nM，1nM，0.1nM，0.01nM的PD1抗体稀释液，冰上与表达PD1的293T细胞孵育2小时，每管加入100μL FITC羊抗人IgG(1：500)冰上孵育1小时用PBS洗3次后加入300μL PBS重悬细胞，在流式细胞仪上用FITC通道检测荧光信号。
人源化抗体14C12H1L1与293T-PD1细胞的结合结果如图7所示。由图7可见，14C12H1L1抗体能有效地结合宿主细胞293T-PD1表面的靶标PD1蛋白，并且其结合效率呈剂量依赖关系，各剂量的荧光强度见表4。通过对结合的14C12H1L1抗体进行荧光定量分析，曲线模拟14C12H1L1抗体的结合效率EC50为1.89nM。
表4：流式细胞仪检测14C12H1L1结合PD1宿主细胞293T-PD1表面抗原的荧光强度分析
浓度(nM) 0.01 0.1 1 5 10 50
荧光强度 8.32 20.31 174.62 579.41 686.49 669.54
实施例9：流式细胞仪方法检测抗体与T细胞表面抗原PD1的结合活性
采用Ficoll-Paque Plus(GE Healthcare LOT No.:171440-02)分离PBMC，从PBMC中分离得到CD4+细胞，用PHA(上海疾控生物科技有限公司，50μl/ml)刺激三天后用PBS洗一次，加入不同浓度的抗体，冰上孵育1.5h。孵育完成后，用PBS洗涤一次，加入FITC-标记抗人二抗IgG(Jackson immunoresearch lot.102155)。冰上避光孵育1h后，用PBS洗涤一次并用流式细胞仪检测。
人源化抗体14C12H1L1与T细胞的结合结果如图8所示。由图8可见，14C12H1L1抗体能有效地结合T细胞表面的靶标PD1蛋白，并且其结合效率呈剂量依赖关系。
实施例10：混合淋巴细胞反应：细胞因子IFN-γ，IL-2的分泌
采用Ficoll-Paque Plus(GE Healthcare LOT No.:171440-02)分离PBMC，将分离出来的PBMC加入IL-4(Peprotech K2513，1000U/ml)和GM-CSF(Peprotech H1513，1000U/ml)诱导6天后，加入TNF-α(Peprotech G1513，200U/ml)诱导3天获得DC细胞。
PBMC中分离得到T细胞，将获得的DC细胞与T细胞按1:10的比例混合培养，同时加入不同比例的抗体14C12H1L1、5C4和hIgG(hIgG做为同型对照)培养5-6天后，采用ELISA试剂盒检测IFN-γ(购自达科为公司)和IL-2(购自达科为公司)的分泌量。
DC细胞和T细胞混合培养后IFN-γ和IL-2的分泌检测结果分别如图9、图10所示：14C12H1L1抗体能有效地诱导混合淋巴细胞分泌IFN-γ和IL-2，并且其分泌量与抗体14C12H1L1呈剂量依赖关系。抗体14C12H1L1对IFN-γ和IL-2分泌量的诱导效果均强于对照抗体5C4。
实施例11：诱导IL-2分泌
将分离得到的PBMC(方法同实施例10)用PHA(上海疾控生物科技有限公司，50μl/mL)刺激3天后，96孔板中加入刺激成熟的PBMC(5×104cells/孔)，Raji细胞(上海中科院)(5×104cells/孔)和MDA-MB-231细胞(ATCC)(1×104cells/孔)，加入100nM 14C12H1L1或对照抗体5C4或hIgG(hIgG为同型对照)混合均匀共培养。3天 后，采用ELISA试剂盒检测IL-2(购自达科为公司)的分泌量，具体操作按照试剂盒说明书进行。
细胞混合培养后的IL-2的分泌检测结果如图11所示。由图11可见，14C12H1L1抗体能有效地诱导PBMC分泌IL-2，抗体14C12H1L1对IL-2分泌的诱导效果显著强于对照抗体5C4。
实例12：抗体14C12H1L1对PD-1HuGEMM小鼠MC38肿瘤模型的肿瘤生长的影 响
在PD-1HuGEMM小鼠(人PD-1转基因小鼠)右侧皮下接种MC38肿瘤细胞(1×106/只)，当平均肿瘤体积达到约118mm3时，根据肿瘤体积将小鼠随机分入4个实验组并进行腹腔给药，每组小鼠均为8只。具体分组和给药剂量如下：
Isotype Control组(给药剂量8mg/kg)，
14C12H1L1高剂量组(给药剂量8mg/kg)，
14C12H1L1低剂量组(给药剂量0.8mg/kg)，
Nivolumab组(给药剂量8mg/kg)。
以上4组均按照每周给药2次，共给药5次。分组给药后，每周测量肿瘤大小两次。
结果如图12所示。
结果显示：
Nivolumab组、14C12H1L1高剂量组、14C12H1L1低剂量组的肿瘤体积在统计学上均显著小于Isotype control组(P<0.01、<0.01、<0.05)。14C12H1L1高剂量组(8mg/kg)在PD-1HuGEMM小鼠MC38肿瘤模型中显示有统计学显著意义的抗肿瘤作用，并且与同靶点上市药物Nivolumab(8mg/kg)药效相当。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (22)

  1. 单克隆抗体或其抗原结合片段，其中，
    所述单克隆抗体的重链可变区包含：氨基酸序列为SEQ ID NO:9－11的CDR；
    和/或
    所述单克隆抗体的轻链可变区包含：氨基酸序列为SEQ ID NO:12－14的CDR。
  2. 根据权利要求1所述的单克隆抗体或其抗原结合片段，其中，
    所述单克隆抗体的重链可变区的氨基酸序列选自SEQ ID NO:2和SEQ ID NO:6；
    和/或
    所述单克隆抗体的轻链可变区的氨基酸序列选自SEQ ID NO:4和SEQ ID NO:8。
  3. 根据权利要求1至2中任一权利要求所述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选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、人源化抗体、嵌合抗体或双抗体。
  4. 根据权利要求1至2中任一权利要求所述的单克隆抗体或其抗原结合片段，其中，所述的单克隆抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的KD结合PD1蛋白；优选地，所述KD通过Fortebio分子相互作用仪测得。
  5. 根据权利要求1至2中任一权利要求所述的单克隆抗体或其抗原结合片段，其中，
    所述的单克隆抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
  6. 根据权利要求1至2中任一权利要求所述的单克隆抗体或其抗原结合片段，其中所述单克隆抗体是由杂交瘤细胞株LT003产生的单克隆抗体，所述杂交瘤细胞株LT003保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2015105。
  7. 分离的核酸分子，其包含能够编码抗体重链可变区的核酸序列，其中，
    所述抗体的重链可变区包含：氨基酸序列为SEQ ID NO:9－11的CDR；
    优选地，所述抗体的重链具有SEQ ID NO:2或SEQ ID NO:6所示的氨基酸序列；
    更优选地，所述核酸分子具有SEQ ID NO:1或SEQ ID NO:5所示的核苷酸序列。
  8. 分离的核酸分子，其包含能够编码抗体轻链可变区的核酸序列，其中，
    所述抗体轻链可变区包含氨基酸序列为SEQ ID NO:12－14的CDR；
    优选地，所述抗体轻链可变区具有SEQ ID NO:4或SEQ ID NO:8所示的氨基酸序列；
    更优选地，所述核酸分子具有SEQ ID NO:3或SEQ ID NO:7所示的核苷酸序列。
  9. 一种载体，其包含权利要求7和/或8所述的分离的核酸分子。
  10. 一种宿主细胞，其包含权利要求7和/或8所述的分离的核酸分子，或者权利要求9所述的载体。
  11. 制备权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段的方法，其包括在合适的条件下培养权利要求10的宿主细胞，以及从细胞培养物中回收所述单克隆抗体或其抗原结合片段的步骤。
  12. 杂交瘤细胞株LT003，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2015105。
  13. 偶联物，其包括单克隆抗体或其抗原结合片段以及偶联部分，其中，所述单克隆抗体为权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段，所述偶联部分为可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。
  14. 试剂盒，其包括权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段，或者包括权利要求13所述的偶联物；
    优选地，所述试剂盒还包括第二抗体，其特异性识别所述单克隆抗体或其抗原结合 片段；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。
  15. 权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求13所述的偶联物在制备试剂盒中的用途，所述试剂盒用于检测PD1在样品中的存在或其水平。
  16. 一种药物组合物，其包含权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求13所述的偶联物；可选地，其还包括药学上可接受的载体和/或赋形剂。
  17. 权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求13所述的偶联物在制备预防和/或治疗肿瘤或者贫血病的药物中的用途，或者用于制备诊断肿瘤或者贫血病的药物中的用途；优选地，所述肿瘤选自黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌、肝癌、非小细胞性肺癌、卵巢癌和白血病。
  18. 权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求13所述的偶联物在制备如下药物中的用途：
    阻断PD1与PD1的配体结合的药物，
    调节(例如下调)PD1活性或水平的药物，
    解除PD1对机体免疫抑制的药物，或者
    提高T淋巴细胞中IFN-γ和/或IL-2表达的药物；
    优选地，所述PD1的配体为PDL1或PDL2，更优选为PDL1。
  19. 一种在体内或体外方法，包括给予细胞或者有需求的受试者以有效量的权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求13所述的偶联物的步骤，所述方法选自如下：
    阻断PD1与PD1的配体结合的方法，
    调节(例如下调)PD1活性或水平的方法，
    解除PD1对机体免疫抑制的方法，或者
    提高T淋巴细胞中IFN-γ和/或IL-2表达的方法；
    优选地，所述PD1的配体为PDL1或PDL2，更优选为PDL1。
  20. 根据权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求13所述的偶联物，其用于预防和/或治疗肿瘤或者贫血病，或者用于诊断肿瘤或者贫血病；优选地，所述肿瘤选自黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌、肝癌、非小细胞性肺癌、卵巢癌和白血病。
  21. 根据权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求13所述的偶联物，其用于：
    阻断PD1与PD1的配体结合，
    调节(例如下调)PD1活性或水平，
    解除PD1对机体免疫抑制，或者
    提高T淋巴细胞中IFN-γ和/或IL-2表达；
    优选地，所述PD1的配体为PDL1或PDL2，更优选为PDL1。
  22. 一种预防和/或治疗肿瘤或者贫血病的方法，或者一种诊断肿瘤或者贫血病的方法，包括给予有需求的受试者以有效量的权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求13所述的偶联物的步骤；优选地，所述肿瘤选自黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌、肝癌、非小细胞性肺癌、卵巢癌和白血病。
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