WO2015058573A1 - 拮抗抑制程序性死亡受体pd-1与其配体结合的单克隆抗体及其编码序列与用途 - Google Patents

拮抗抑制程序性死亡受体pd-1与其配体结合的单克隆抗体及其编码序列与用途 Download PDF

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WO2015058573A1
WO2015058573A1 PCT/CN2014/083780 CN2014083780W WO2015058573A1 WO 2015058573 A1 WO2015058573 A1 WO 2015058573A1 CN 2014083780 W CN2014083780 W CN 2014083780W WO 2015058573 A1 WO2015058573 A1 WO 2015058573A1
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antibody
seq
human
variable region
chain variable
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PCT/CN2014/083780
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English (en)
French (fr)
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罗师平
胡红群
宋晓琦
陈蕞
孙亚男
蔡明文
袁燕萍
周建鹰
罗昊
刘海云
周群敏
徐一清
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苏州思坦维生物技术有限责任公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention belongs to the field of biotechnology-monoclonal antibodies.
  • the present invention relates to an antagonistic inhibition of programmed death receptor PD-1 (programmed Death-1) A monoclonal antibody that binds to its ligand and its coding sequence and use.
  • programmed death receptor PD-1 programmed Death-1
  • T Lymphocytes can be roughly divided into two categories: functional helper cells (T helper cells) and cytotoxic T that directly participate in the recognition of target antigens and killer target cells. Cytotoxic T cells, CTL).
  • T helper cells functional helper cells
  • CTL Cytotoxic T cells
  • the activation and proliferation of these two types of T cells in vivo generally require the synergy of two signaling pathways: the first signaling pathway is a receptor protein (T-cell) that specifically recognizes antigens on T cells.
  • T-cell receptor protein
  • TcR tumors or antigen-presenting cells
  • APC antigen-presenting cells
  • the ligands on the cells or antigen presenting cells bind to and conduct, and the signaling pathway is non-specific for the antigen.
  • T Lymphocytes only activate and proliferate in the presence of both the first and second signaling pathways.
  • a representative factor that exerts a synergistic stimulatory effect (ie, positive or up-regulated immune function) in the second signaling pathway is CD28 and its ligands B7-1, B7-2; and exerts synergistic inhibition (ie, negative or down-regulated immune function)
  • Representative factors are CTLA-4 and its ligands B7-1, B7-2, and programmed death receptors (programmed Death-1) PD-1 and its ligands PD-L1 and PD-L2; these co-stimulation/synergy inhibitors are structurally similar and belong to members of the immunoglobulin superfamily (see review: Chen LP: Co-inhibitory molecules of the B7-CD28 family in the control of T cell Immunity, Nature Immunol 2004, 336-347).
  • Route 1 directly achieves the effect of up-regulating immune function by increasing the expression of co-stimulatory factors such as CD28;
  • Route 2 is by reducing or blocking CTLA-4 or
  • the expression of synergistic inhibitory factors such as PD-1/PD-L1 can remove or reduce the immunosuppression caused by tumor target cells, and indirectly achieve the effect of up-regulating immune function.
  • the PD-1 gene was first developed by Japanese scientist Tasuku founded and cloned by Honjo and colleagues in 1992, there is an IgV-like region in the extracellular region and 23% homology to CTLA-4 (Ishida, Y., Agata, Y., Shibahara, K. and Honjo, T. Induced expression of PD-1, a novel member of the Immunoglobulin gene superfamily, upon programmed cell death.
  • PD-1 is mainly expressed on activated T lymphocytes, B lymphocytes and monocytes (Yasutoshi Agata and other Internationl) Immunology 1996, 8: 675).
  • PD-L1 and PD-L2 are mainly expressed on target cells such as tumors or antigen-presenting cells (Thompson RH et al., Cancer Res). 2006; 66: 3881-3885).
  • target cells such as tumors or antigen-presenting cells.
  • the important fact that PD-1 is involved in the negative regulation of immune function in vivo was firstly developed by Tasuku. Honjo and colleagues observed in PD-1 knockout mice.
  • PD-1 knockout mice developed lupus-like glomerulonephritis and arthritis in the context of the C57BL/6 gene (Nishimura H,Development of lupus-like autoimmune diseases by disruption of the PD-1 Gene encoding an ITIM motif-carrying immunoreceptor.Immunity 1999, 11: 141); in the context of the Balb/c gene, high titers of anti-myocardial tissue antibodies are produced and thus cause severe autoimmune cardiomyopathy (Nishimura, H. et al. Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science 2001, 291: 319).
  • Immunosuppression or immune tolerance mediated by the expression of PD-L1 gene in normal tissue cells is an important mechanism for protecting normal tissues from attack and killing by surrounding autologous lymphocytes in vivo (Keir M.E. et al. Tissue expression of PD-L1 mediates peripheral T cell tolerance. J Exp Med 2006, 203: 883-895).
  • Tumor cells can transmit inhibition signals by increasing the expression of PD-L1 or PD-L2 protein and interacting with PD-1 receptors expressed on lymphocytes, thereby inhibiting or tolerating immunity in vivo.
  • BMS Bristol-Myers Squibb.
  • Merck The company and companies such as Roche/Genentech are stepping up efforts to develop new antagonistic antibody-based drugs for the purpose of blocking the binding of PD-1 to its ligands, and have applied for related patents.
  • a hybridoma and an antibody protein thereof which are obtained by screening a plurality of anti-human PD-1 antibodies obtained by immunizing a mouse with a DNA molecule containing the PD-1 gene, It encodes a nucleotide sequence, and the antibody is used for the purpose of treating tumors and infectious diseases by enhancing the immune function in the body.
  • anti-PD-1 or anti-PD-L1 antibody drugs are mainly used to treat lung cancer Melanoma, Many advanced tumors such as kidney cancer, among which early clinical trials (stage I) showed that anti-PD-1 or anti-PD-L1 antibody drugs not only have the objective effect of inhibiting tumor growth and prolonging the survival of patients, but also their safety after long-term use. Also in acceptable range (Brahmer JR et al, JCO 2010, 28: 3167-3175; Topalian S, etc., NEJM 2012, 366: 2443-2454; Brahmer JR Etc., NEJM 2012, 366: 2455-2465; Hamid O et al, NEJM 2013, 369: 134-144).
  • One of the technical problems to be solved by the present invention provides an antagonistic inhibition of programmed death receptor PD-1 (programmed Death-1)
  • PD-1 programmed death receptor 1
  • Derivatives of the murine monoclonal antibody include human-mouse chimeric antibodies, humanized antibodies, Antibody Fab fragments, single chain antibodies, and the like.
  • a second technical problem to be solved by the present invention is to provide a DNA molecule or gene encoding the above antibody.
  • the third technical problem to be solved by the present invention is to provide a method for preparing the above antibody.
  • the fourth technical problem to be solved by the present invention is to provide the use of the antibody, comprising using the above antibody as a detection reagent for quantitatively and qualitatively analyzing PD-1 protein in a biological sample.
  • the recombinant human PD-1 extracellular protein expressed by mammals is selected as an immunizing antigen in the present invention, and obtained by using traditional mouse hybridoma technology and recombinant genetic engineering method. Multiple new monoclonal antibodies against PD-1.
  • the hybridoma strain and the secreted antibody thereof are provided; another aspect of the present invention discloses a heavy chain variable region and a light chain variable region amino acid sequence of the antibody and a DNA nucleotide sequence encoding the same;
  • the invention also provides a human-mouse chimeric antibody of the antibody and a derivative thereof; the invention also provides an expression vector for expressing and preparing the human-mouse chimeric antibody and the derivative thereof , engineering cells and methods.
  • an aspect of the present invention provides an antagonistic inhibition of PD-1 (programmed Death-1) a murine monoclonal antibody that binds to its ligand, characterized in that its light chain variable region has the amino acid sequence shown in SEQ ID NO.: 1, and the heavy chain variable region has the SEQ ID NO.: The amino acid sequence shown in 2.
  • the murine monoclonal antibody is derived from a mouse hybridoma cell line code-named AB7, which was on October 17, 2013. It is deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee (Accession No. CGMCC No. 8351. Deposit Location: Beijing, China).
  • the present invention provides a DNA molecule encoding the above-described murine monoclonal antibody, wherein the light chain variable region has the nucleotide sequence shown in SEQ ID NO.: 3, and the heavy chain variable region has SEQ ID NO.: nucleotide sequence of 4.
  • Another aspect of the invention provides a derivative derived from the above-described murine monoclonal antibody, such as a human-mouse chimeric antibody, a humanized antibody, Antibody Fab fragment, Single-chain antibodies and the like.
  • human-mouse chimeric antibodies or humanized antibodies have long-term half-life (up to 20 days) and low immunogenicity in humans, and are convenient for long-term or repeated times. Used in the body.
  • the heavy chain variable region and the light chain variable region gene of the murine monoclonal antibody were ligated to the human antibody heavy chain constant region and the light chain constant region gene, respectively, to obtain a human-mouse chimeric antibody cAB7.
  • the human-mouse chimeric antibody or derivative thereof is characterized by
  • the light chain antigen complementarity determining region has the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7;
  • the heavy chain antigen complementarity determining region has SEQ ID NO: 8, the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10.
  • the present invention also provides for encoding the above - A DNA molecule or gene of a murine chimeric antibody or a derivative thereof, wherein the light chain variable region has the nucleotide sequence of SEQ ID NO: 3, and the heavy chain variable region has SEQ ID NO: 4 Nucleotide sequence.
  • a third aspect of the present invention provides an expression vector comprising the DNA encoding the above human-mouse chimeric antibody or a derivative thereof A molecular sequence and an expression control sequence operably linked to the sequence.
  • the fourth aspect of the invention provides A recombinant host cell which is transformed from the above expression vector.
  • the recombinant host cell or a progeny cell thereof expresses the above human-mouse chimeric antibody or a derivative thereof.
  • a fifth aspect of the invention provides a person for preparing said person - A method of a murine chimeric antibody or derivative thereof, the method comprising the steps of:
  • an expression vector comprising the DNA of claim 4 a molecular sequence and an expression control sequence operably linked to the sequence;
  • step b) transforming the host cell with the expression vector of step a);
  • the antibody is obtained by isolation and purification from host cell culture medium by affinity chromatography.
  • a sixth aspect of the present invention provides a detection reagent or kit for quantitatively or qualitatively detecting PD-1 protein in a sample for analysis.
  • a detection reagent or kit for quantitatively or qualitatively detecting PD-1 protein in a sample for analysis Including the above-mentioned AB7 murine monoclonal antibody or human-mouse chimeric antibody, preferably, it uses an unlabeled AB7 antibody as a primary antibody, and is chemically labeled (eg, fluorescein, enzyme, A second antibody such as biotin or the like, such as goat anti-mouse IgG, is used in combination as a kit for quantitative and qualitative detection of PD-1 protein in various biological samples.
  • Quantitative and qualitative assays employed are well known in the art and include, for example, ELISA, immunoblotting, Western blotting, immunofluorescence, immunoprecipitation, flow cytometry, and the like.
  • the kit may contain other components, packaging, instructions or other materials to aid in the detection of the PD-1 protein.
  • mAb monoclonal antibody
  • monoclonal antibodies differ from conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody being directed against a single determinant on the antigen.
  • monoclonal antibodies are also advantageous in that they are obtained by hybridoma or recombinant engineered cell culture without intermixing with other immunoglobulins.
  • the modifier 'monoclonal' indicates the identity of the antibody and is obtained from a homogeneous population of antibodies, which should not be construed as requiring any particular method to produce the antibody.
  • the term 'humanized monoclonal antibody' as used herein The amino acid sequence of the murine monoclonal antibody is divided into complementarity-determining regions (complementarity-determining regions, In addition to CDRs, all or most of the sequences (including the framework region sequences in the variable region) replace the amino acid sequence of the adult immunoglobulin to minimize the immunogenicity of the murine monoclonal antibody by genetic engineering. .
  • 'antibody' and 'immunoglobulin' as used herein are isomeric polysaccharide proteins of the same structural feature of about 150,000 daltons, which consist of two identical light chains (L) and two identical heavy chains ( H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (V H ). This is followed by a plurality of constant regions.
  • V H variable region
  • Each light chain has a variable region (V L ) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain and the variable region of the heavy chain relatively. Particular amino acid residues form an interface between the variable regions of the light and heavy chains.
  • variable regions of an antibody differ in sequence, which form the binding and specificity of various specific antibodies to their particular antigen.
  • variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in the light and heavy chain variable regions into three segments in the complementarity determining region (CDR) or hypervariable region.
  • CDR complementarity determining region
  • FR framework region
  • the variable regions of the antibody heavy and light chains each comprise four FR regions which are substantially in a beta-sheet configuration and are joined by three CDRs forming a linker loop, in some cases forming a partial beta sheet structure.
  • the CDRs in each chain are tightly bound together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH). Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).
  • Antibody constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cytotoxicity (antibody-dependent) Cellular cytotoxicity, ADCC) or complemnt-dependent cytotoxicity (CDC).
  • the antibody of the present invention can usually be produced by the following method.
  • a gene containing an antibody encoding the present invention is inserted into an expression vector containing a suitable expression control sequence.
  • expression control sequence' as used herein generally refers to a sequence involved in controlling the expression of a gene.
  • Expression control sequences include promoter and termination signals operably linked to a gene of interest.
  • Gene encoding the antibody of the present invention (DNA The sequences can be obtained by conventional means well known to those skilled in the art, such as artificial synthesis of the protein sequences disclosed in accordance with the present invention or by PCR. Thereafter, synthetic or PCR amplification can be carried out by various methods well known in the art.
  • the DNA fragment is inserted into a suitable expression vector.
  • the expression vector used in the present invention may be a commercially available expression vector known to those skilled in the art, such as pCDNA3.1 of Invitrogen Corporation. Expression vector.
  • Suitable host cells for receiving expression vector transformation typically include prokaryotic cells and eukaryotic cells.
  • prokaryotic host cells include Escherichia coli, Bacillus subtilis and the like.
  • eukaryotic host cells include yeast cells, insect cells, mammalian cells, and the like.
  • preferred host cells are mammalian cells, especially the Chinese hamster ovary ( CHO) cells.
  • the host cell transformed with the expression vector is cultured under suitable conditions (for example, adherent culture in a cell culture flask or a bioreactor in a serum-free medium), and the culture supernatant is harvested, and then the protein-A is included.
  • suitable conditions for example, adherent culture in a cell culture flask or a bioreactor in a serum-free medium
  • the culture supernatant is harvested, and then the protein-A is included.
  • the antibodies of the invention are obtained by affinity chromatography, ion exchange chromatography, filtration, etc., by conventional separation procedures or means well known to those skilled in the art.
  • the purified antibody of the present invention can be dissolved in a suitable solvent such as a physiological saline solution, and the solubility can be prepared from 0.01 to 100. Between mg/ml, the ideal final solubility can be prepared between 0.1 and 20 mg/ml.
  • the present invention selects a recombinant human PD-1 extracellular protein expressed by a mammal as an immune antigen, by repeatedly applying a small dose
  • the mouse is subcutaneously immunized to obtain a polyclonal antibody secreting anti-PD-1 protein; the mouse containing the high titer antibody is picked from the mouse, and the spleen cells are taken, and the mouse myeloma cells are fused in vitro, and then drug screening and sub- A plurality of hybridoma monoclonal cells stably stabilizing antibodies against human PD-1 protein were established by cloning and the like.
  • mouse hybridoma cell lines which is code-named AB7, was identified by ELISA, immunoblotting, immunohistochemistry, etc., and it was confirmed that the secreted monoclonal antibody not only specifically binds to human PD-1 protein, but also blocks Binding of the PD-1 protein to its ligand (PD-L1, PD-L2) was inhibited.
  • a gene fragment encoding the heavy chain variable region and the light chain variable region of the mouse antibody is obtained by genetic engineering or the like, and the human-mouse chimeric antibody gene of the antibody and the expression vector thereof are constructed based thereon.
  • the expression vector is transfected into Chinese hamster ovary (CHO) cells to obtain recombinant engineered cells secreting human-mouse chimeric antibody, and isolated and purified from recombinant engineering cell culture medium to obtain biologically active human-mouse chimeric Antibody protein.
  • the present invention selects a recombinant human PD-1 extracellular protein expressed by a mammal as an immune antigen, by repeatedly applying a small dose
  • the mouse is subcutaneously immunized to obtain a polyclonal antibody secreting anti-PD-1 protein; the mouse containing the high titer antibody is picked from the mouse, and the spleen cells are taken, and the mouse myeloma cells are fused in vitro, and then drug screening and sub- A plurality of hybridoma monoclonal cells stably stabilizing antibodies against human PD-1 protein were established by cloning and the like.
  • mouse hybridoma cell lines which is code-named AB7, was identified by ELISA, immunoblotting, immunohistochemistry, etc., and it was confirmed that the secreted monoclonal antibody not only specifically binds to human PD-1 protein, but also blocks Binding of the PD-1 protein to its ligand (PD-L1, PD-L2) was inhibited.
  • a gene fragment encoding the heavy chain variable region and the light chain variable region of the mouse antibody is obtained by genetic engineering or the like, and the human-mouse chimeric antibody gene of the antibody and the expression vector thereof are constructed based thereon.
  • the expression vector is transfected into Chinese hamster ovary (CHO) cells to obtain recombinant engineered cells secreting human-mouse chimeric antibody, and isolated and purified from recombinant engineering cell culture medium to obtain biologically active human-mouse chimeric Antibody protein.
  • FIG 1 is a schematic view of the results of an outer membrane protein on the binding of recombinant human PD-1 in 96-well plates in an ELISA assay mouse hybridoma (AB7) and culture supernatants in Example 1 the package of the present invention.
  • the unfused SP2/0 myeloma cell culture supernatant was a negative control sample.
  • FIG. 2 is a schematic diagram showing the results of binding of antibodies and PD-1 gene transfected and untransfected CHO cells in the culture supernatant of AB7 hybridoma cells by flow cytometry in Example 2.
  • the unfused SP2/0 myeloma cell culture supernatant was a negative control; wherein, FIG. 2A represents the CHO cells in which the PD-1 gene was not transfected in the SP2/0 negative control supernatant sample, and FIG. 2B represents the hybridization in AB7.
  • FIG. 2A represents the PD-1 gene transfected CHO cells in the SP2/0 negative control supernatant sample, and FIG. 2D represents the AB7 hybridoma cell culture.
  • the PD-1 gene transfected CHO cells in the supernatant samples The unfused SP2/0 myeloma cell culture supernatant was a negative control; wherein, FIG. 2A represents the CHO cells in which the PD-1 gene was not transfected in the SP2/
  • Fig. 3 is a view showing the results of analyzing the antibody protein obtained by affinity chromatography in AB7 hybridoma cell culture supernatant by SDS-PAGE analysis in Example 3 of the present invention.
  • Lane 1 is an unreduced AB7 antibody protein sample
  • Lane 2 is a DTT-reduced AB7 antibody protein sample
  • M is a protein molecular weight marker.
  • Figure 4 is a graph showing the results of the analysis of the binding of the mouse AB7 monoclonal antibody to the PHA-activated human Jurkat T lymphocytes by flow cytometry in Example 4. Among them, a non-related mouse antibody IgG sample was used as a negative control. 4A represents a non-related mouse antibody IgG negative control sample, and FIG. 4B represents a sample of an AB7 antibody.
  • Figure 5 is an in vitro competitive ELISA assay in Example 5 of the present invention to demonstrate that the AB7 antibody antagonizes the biotinylated human PDL1-Fc protein (Biotin-PDL1-Fc) and the human PD-coated on a 96-well plate.
  • the AB7&Biotin-PDL1-Fc group was a competition sample of AB7 antibody and Biotin-PDL1-Fc
  • the MouseIg&Biotin-PDL1 Fc group was a non-related mouse antibody and Biotin-PDL1-Fc competition sample as a negative control.
  • Figure 6 is an in vitro competitive ELISA assay in Example 5 of the present invention to demonstrate that the AB7 antibody antagonizes the biotinylated human PDL2-Fc protein (Biotin-PDL2-Fc) and the human PD-coated on a 96-well plate.
  • the AB7&Biotin-PDL2-Fc group was a competition sample of AB7 antibody and Biotin-PDL2-Fc
  • the MouseIg&Biotin-PDL2Fc group was a non-related mouse antibody and Biotin-PDL2-Fc competition sample as a negative control.
  • Figure 7 is a diagram showing the results of direct ELISA assay for the expression of CHO cell expression supernatant transfected with an expression vector containing the human-mouse chimeric antibody (cAB7) gene to maintain binding to human PD-1 protein. .
  • Mouse hybridoma cell line codenamed AB7 (derived from mouse Mus musculus) was published in 2013 Deposited on October 17th at the General Microbiology Center of China Microbial Culture Collection Management Committee (CMSCC No. 8351; preservation location: No. 1 Beichen West Road, Chaoyang District, Beijing, China) No. 3, Institute of Microbiology, Chinese Academy of Sciences).
  • Example 1 Establishment and screening of mouse hybridoma cell lines secreting anti-PD-1 antibody
  • Step 1 Acquisition of recombinant human PD-1 protein (immune antigen) and animal immunization
  • the human PD-1 protein used for immunization is a recombinant human PD-1 extracellular protein expressed by a mammal (product of Beijing Yiqiao Shenzhou Co., Ltd.). After the recombinant human PD-1 protein was mixed with Freund's complete adjuvant (product of Sigma, USA), Balb/c mice were injected subcutaneously (100). Ll/only, a total of 5-10 ⁇ g PD-1 protein). First immunization After 2-3 weeks, the mice were given a subcutaneous injection of a mixture of PD-1 protein and Freund's incomplete adjuvant (American Sigma) to strengthen the vaccine 2-3.
  • mice spleen cell suspension was prepared aseptically, and mouse SP2/0 myeloma cells (purchased from the Shanghai Institute of Bioscience, Chinese Academy of Sciences), in a ratio of 5:1 at 50% PEG- Fusion under the influence of 1000 (American Sigma product).
  • the fusion was carried out according to a conventional method (Kohler G. and Milstein C: Nature 1975; 256: 495-497), and the amount of PEG was 1 ml, which was slowly added over 1 minute.
  • reaction was stopped in serum-free RPMI-1640 medium, centrifuged at 1000 rpm for 10 min, the supernatant was removed, and the cells under centrifugation were further subjected to 10% HAT (H is hypoxanthine, A-amino oxime). , T thymidine, Sigma Sigma product, RPMI 1640-10% FCS medium, adjust the cell concentration to 1 ⁇ 10 6 /ml, add 96-well flat-bottomed cell culture plate (200 ⁇ l per well), at 37 ° C, 5 Incubate for 2-3 weeks in a % CO2 incubator.
  • HAT hypoxanthine, A-amino oxime
  • Step 3 Enzyme-linked immunosorbent assay (ELISA) for screening antibody-positive mouse hybridoma cells
  • the plate was coated with recombinant human PD-1 protein (2 ⁇ g/ml, pH 9.6, 0.1 M NaHCO3 solution), coated at 37 ° C for 2 hours or 4 ° C overnight; 2% bovine serum albumin (BSA) blocked at 4 ° C overnight.
  • BSA bovine serum albumin
  • the positive cells obtained by the above screening were RPMI-1640-10%. Dilute the FCS medium to 1-10 cells per well, place it on a 96-well cell culture plate, and incubate at 37 ° C, 5% CO2 incubator 2-3 Week. To be cloned, the supernatant was taken for further detection by ELISA to identify the secretion of anti-PD-1 antibody. After detection and identification, a plurality of antibody-secreting positive cell lines were obtained. After subcloning, one of the strains AB7 hybridoma cell line stably secreted and expressed anti-PD-1 monoclonal antibody.
  • Figure 1 shows the ELISA
  • the results of the detection of the binding of the supernatant of AB7 hybridoma cells to recombinant human PD-1 protein showed that the supernatant of the hybridoma cells contained antibodies with high titer against human PD-1 protein. This antibody was identified as an IgG class.
  • the hybridoma cell line was further expanded and cultured for a long time and deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee on October 17, 2013 (the deposit number is CGMCC). No.8351. Deposit location: China, Beijing).
  • the AB7 antibody was used as a primary antibody, and FITC fluorescently labeled goat anti-mouse IgG was used as a secondary antibody, and the binding of the AB7 antibody to the human PD-1 gene-transfected CHO cells was analyzed by flow cytometry.
  • total mRNA was extracted from human peripheral blood lymphocytes (PBL), and then amplified in vitro by reverse transcription-polymerase chain reaction (RT-PCR) to obtain full-length cDNA encoding PD-1 (including extracellular and cDNA fragment of the intracellular region).
  • PBL peripheral blood lymphocytes
  • RT-PCR reverse transcription-polymerase chain reaction
  • Primers for RT-PCR were PD-1 forward primers: ATTAAGCTTGAGCAGTGGAGAAGGCGGCA, Seq ID No: 11 , PD-1 reverse primer: AATTGGATCCCTCCTGAGGAAATGGGCTGA, Seq ID No: 12 ).
  • the cDNA fragment was amplified by the pair of primers to contain the entire coding region of the PD-1 protein, and the length was 989 bp.
  • This DNA was subjected to restriction endonuclease, and then inserted into the expression plasmid pCDNA3.1 (Invitrogen, USA) to obtain a recombinant plasmid pCDNA3.1-PD-1 containing the PD-1 cDNA.
  • the expression plasmid was mixed with Fugen-6 liposome (product of Roche) and co-transfected into CHO cells (purchased from Invitrogen, USA). After transfection for 2 days, the cells were separated by centrifugation and then redissolved in mice. The solution of the source AB7 monoclonal supernatant (for the test sample) or the SP2/0 supernatant (for the negative control) was incubated at 4 ° C for 1 hour; after washing with PBS-0.1% Tween 20 solution, the FITC-labeled goat was added.
  • Murine IgG (product of Sigma) was incubated at 4 ° C for 1 hour; after washing with PBS-0.1% Tween 20 solution, the sample was loaded to Cytomics FC500 MCL) flow cytometry (Beckman Coulter, USA).
  • FIG. 2 is a schematic diagram of the results of the flow cytometry test. As shown in Figure 2, nearly 30% of the samples transfected with the human PD-1 gene The cells can specifically bind to the AB7 monoclonal antibody (Fig. 2D); In the SP2/0 negative control supernatant samples, neither transfected (Fig. 2C) nor untransfected CHO cells (Fig. 2A) showed significant binding; untransfected CHO cells did not bind significantly to AB7 antibody. (Fig. 2B), indicating that the flow cytometry results have higher specificity.
  • mouse anti-human PD-1 monoclonal antibody (AB7) protein was isolated and purified by affinity chromatography.
  • the purification steps are as follows:
  • the AB7 hybridoma cells were expanded, inoculated into 200-500 ml serum-free 1640 medium, cultured at 37 ° C for 5 days, and then the culture supernatant was collected and passed through 0.45. After filtration, the ⁇ m filter was applied to an affinity chromatography column containing Protein G-Sepharose Fast Flow (available from GE GE); the column was first rinsed with PBS solution to remove the impurity protein. The adsorbed AB7 antibody protein was eluted with a low pH (2.7-3.0) glycine (0.1 M) solution.
  • FIG. 1 is an unreduced intact AB7 antibody sample with a molecular weight of about 150 kD; Lane 2 is a DTT-reduced AB7 antibody sample, separated into two main bands, wherein the molecular weight is about 50 kD.
  • the upper band is the AB7 antibody heavy chain, and the lower band having a molecular weight of about 25 kD is the AB7 antibody light chain.
  • the AB7 monoclonal antibody was used as a primary antibody and FITC fluorescently labeled goat anti-mouse IgG as a secondary antibody for detecting and analyzing the PD-1 protein expressed on human T lymphocytes.
  • the human T lymphocytes are Jurkat cells (purchased from the Cell Collection Center of the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences). To this end, first Jurkat The cells were placed in RPMI-10% FCS containing T lymphocyte activating factor PHA (product of Sigma, USA, final concentration of PHA: 3 ⁇ g/mL). Culture in culture medium to activate and induce expression of PD-1 protein.
  • the cells were centrifuged and re-dissolved in the purified test sample AB7 antibody (final solubility: 5 ⁇ g/ml) or non-related mouse IgG. (Mouse IgG, negative control sample, final solubility: 5 ⁇ g / ml) in a solution, incubate at 4 ° C for 1 hour; PBS-0.1% Tween20 After washing with liquid, FITC-labeled goat anti-mouse IgG (Sigma, 1:200) was added and incubated at 4 ° C for 1 hour; then PBS-0.1%. After washing with Tween 20 solution, the samples were tested by Cytomics FC500 MCL flow cytometry (Beckman Coulter, USA). Flow cytometry analysis.
  • FIG 4 is a graph showing the results of the flow cytometry analysis.
  • AB7 monoclonal antibody sample can be associated with PHA-activated Jurkat The cells bind and have no obvious binding reaction with unactivated Jurkat cells.
  • non-related mouse IgG negative control samples Fig. 4A
  • activated or unactivated Jurkat There were no obvious binding reactions in the cells.
  • Bioassay of AB7 monoclonal antibody competitive ELISA assay to identify mouse AB7 monoclonal antibody blocking the binding of PD-L1 and PD-L2 to PD-1 protein in vitro
  • One of the methods for identifying the biological activity of the AB7 monoclonal antibody in vitro is to detect whether the antibody can block the binding of PD-1 to its PD-1 ligand (PD-L1 and PD-L2) by competitive ELISA.
  • the basic principle and process of the competitive ELISA method is: first, biotin-labeled human PD-1 ligand protein (such as PDL1-Fc or PDL2-Fc fusion protein) is mixed with monoclonal antibodies of different solubility, and then The mixture was transferred to a 96-well plate pre-coated with PD-1 protein, and after incubation and elution, enzyme-labeled Avidin (such as horseradish peroxidase-labeled Avidin) was added; After incubation and elution, the substrate was added and the OD value was determined.
  • biotin-labeled human PD-1 ligand protein such as PDL1-Fc or PDL2-Fc fusion protein
  • Figure 5 is a representative result of the in vitro competition of the AB7 antibody with the biotinylated PDL1-Fc protein (Biotin-PDL1Fc) for binding to the PD-1 protein.
  • Biotin-PDL1Fc biotinylated PDL1-Fc protein
  • Figure 6 is a representative result of the competition of the AB7 antibody with the biotin-labeled PDL2-Fc protein (Biotin-PDL2Fc) for binding to the PD-1 protein in vitro.
  • the color reaction of each well was performed in a sample in which the AB7 antibody was competed with the biotinylated PDL2-Fc protein (AB7 & Biotin-PDL2Fc).
  • the OD value is inversely related to the amount of AB7 antibody protein added: the higher the amount of AB7 antibody added, the lower the OD value.
  • Primers reverse transcription-polymerase chain reaction (RT-PCR) method (Wang Y And so on: Degenerated primer design to amplify the heavy chain variable region from Immunoglobulin cDNA.
  • RT-PCR reverse transcription-polymerase chain reaction
  • Step 1 using a kit (Pik days door Jiangsu Company) mRNA was extracted from mouse hybridoma AB7;
  • Step 2 A cDNA template was obtained on an eppendorf tube by reverse transcription PCR (RT-PCR).
  • the sequence for the AB7 antibody light chain variable region reverse transcription PCR primer (AB7-L) is: GTG CTG GAG GGG ACA GTC ACT (SEQ ID NO.: 13);
  • AB7 antibody heavy chain variable region reverse transcription PCR primer (AB7-H) sequence: CTG AGG CAC CTC CAG ATG TT TGTCGTTCACTGCCATCAAT ( SEQ ID NO.: 14 );
  • the RT-PCR reaction system is as follows:
  • the reaction was carried out at a temperature of 42 ° C for 1 hour, then the temperature was raised to 75 ° C, and the cDNA obtained after 15 minutes of inactivation was obtained. Store at -20 ° C and store for later use.
  • Step 3 PCR cloning and amplification of the light chain variable region and heavy chain variable region genes of AB7 antibody
  • a pair of primers for cloning and amplifying the light chain variable region gene of the AB7 antibody by PCR method are:
  • Reverse primer AAT TGG ATC CAG TTG GTG CAG CAT CAG C (SEQ ID NO.: 16)
  • Reverse primer AAT TGG ATC CTG GGG GTG TCG TTT TGG C (SEQ ID NO.: 18)
  • the DNA product obtained by PCR amplification was analyzed by electrophoresis in 1% agarose gel. After the end of the electrophoresis, the isolated DNA bands were excised and sequenced separately to obtain the nucleotide sequence of the antibody heavy chain variable region DNA.
  • the nucleotide sequence of the variable light region variable region DNA of the antibody is shown in SEQ ID NO.: 3
  • the amino acid sequence of the antibody light chain variable region predicted by the DNA nucleotide sequence is shown in SEQ ID NO.:1.
  • Complementarity-determining regions (CDRs) of the light chain antigen The amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO.: 5, SEQ ID NO.: 6 and SEQ ID NO.: 7, respectively.
  • the nucleotide sequence of the antibody heavy chain variable region DNA was determined as shown in SEQ ID. NO.: 4, the amino acid sequence of the antibody heavy chain variable region deduced from the nucleotide sequence of the DNA is shown in SEQ ID NO.: 2.
  • Complementary region of the heavy chain antigen The amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO.: 8, SEQ ID NO.: 9 and SEQ ID NO.: 10, respectively.
  • the AB7 antibody light chain variable region gene and heavy chain variable region gene obtained by clonal amplification in Example 6 were fused to human-kappa light chain constant region (C-domain) and human IgG1-heavy chain constant region gene fragment, respectively. , obtaining a human-mouse chimeric light chain gene ( cAB7L) and human-mouse chimeric heavy chain gene (cAB7H).
  • the light chain chimeric gene and the heavy chain chimeric gene were subsequently cloned into pcDNA3.1, respectively.
  • the expression plasmid was transferred to E. coli for amplification, and a large number of expression plasmids containing the human-mouse chimeric antibody gene were isolated.
  • the expression plasmid containing the human-mouse chimeric antibody gene was further mixed with Fugen-6 liposome (Roche) and co-transfected into CHO cells. 2-3 days after cell transfection, the culture supernatant was taken and used with a 96-well plate coated with human PD-1 protein. HRP-encoded Goat-anti-human-IgG is a secondary antibody (purchased from Shanghai Xitang Biotechnology Co., Ltd.) for the detection of secondary antibodies.
  • the chimeric antibody (cAB7) in the supernatant was detected by ELISA to bind to human PD-1 protein. Table 1 and Figure 7 show the representative results of the ELISA. As shown in Table 1 and Figure 7, the human-murine chimeric antibody gene expression plasmid transfected CHO cell supernatant contains protein specific for human PD-1. Binding chimeric antibodies.
  • the supernatant of the above transfected cells can be loaded into the Protein after centrifugation and filtration through a 0.45 ⁇ m filter.
  • a affinity layer column (geneA-Sepharose Fast Flow) is used to separate and purify antibody proteins.
  • the present invention selects a recombinant human PD-1 extracellular protein expressed by a mammal as an immune antigen, by repeatedly applying a small dose
  • the mouse is subcutaneously immunized to obtain a polyclonal antibody secreting anti-PD-1 protein; the mouse containing the high titer antibody is picked from the mouse, and the spleen cells are taken, and the mouse myeloma cells are fused in vitro, and then drug screening and sub- A plurality of hybridoma monoclonal cells stably stabilizing antibodies against human PD-1 protein were established by cloning and the like.
  • mouse hybridoma cell lines which is code-named AB7, was identified by ELISA, immunoblotting, immunohistochemistry, etc., and it was confirmed that the secreted monoclonal antibody not only specifically binds to human PD-1 protein, but also blocks Binding of the PD-1 protein to its ligand (PD-L1, PD-L2) was inhibited.
  • a gene fragment encoding the heavy chain variable region and the light chain variable region of the mouse antibody is obtained by genetic engineering or the like, and the human-mouse chimeric antibody gene of the antibody and the expression vector thereof are constructed based thereon.
  • the expression vector is transfected into Chinese hamster ovary (CHO) cells to obtain recombinant engineered cells secreting human-mouse chimeric antibody, and isolated and purified from recombinant engineering cell culture medium to obtain biologically active human-mouse chimeric Antibody protein.

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Abstract

本发明公开了一种拮抗抑制程序性死亡受体（programmed death-1， PD-1）与其配体结合的小鼠单克隆抗体及其重链可变区与轻链可变区氨基酸序列。本发明还公开了编码该抗体重链可变区与轻链可变区的DNA分子核苷酸序列。本发明还公开了该抗体的人-鼠嵌合抗体及其衍生体的制备方法，以及其在PD-1蛋白检测上的应用。

Description

拮抗抑制程序性死亡受体 PD-1 与其配体结合的单克隆抗体及其编码序列与用途 技术领域
本发明属于生物技术-单克隆抗体领域。本发明涉及一种拮抗抑制程序性死亡受体PD-1（programmed death-1）与其配体结合的单克隆抗体及其编码序列与用途。
背景技术
长期以来，生物医学界一直认为肿瘤的发生发展与机体内的免疫功能状态息息相关。体内免疫功能在正常状态下发挥着监控变异的肿瘤细胞与抑制肿瘤发生发展的作用。但当体内免疫功能状态出现低下或功能受到抑制时，肿瘤细胞的变异与增长则会加快，严重时更危急生命。因此以提高免疫功能为目的的免疫疗法（Immunotherapy）已成为当今国际肿瘤临床治疗中的一大目标与手段（见综述：Topalian SL 等: Cancer immunotherapy comes of age. JCO 2011, 29:4828-4836; Pardoll D和 Drake C：Immunotherapy earns its spot in the ranks of cancer therapy，J Exp Med 2012，209：201-209）。免疫疗法的近期突破与其广泛应用则得益于免疫学基础研究进展及以杂交瘤/单克隆抗体等为代表的现代生物技术的发展。
现代免疫学研究表明由胸腺发育而来的T 淋巴细胞在识别与杀灭肿瘤细胞中起着非常关键的作用。 T 淋巴细胞在功能上大致可分为两大类：即以调控免疫功能为主的辅助T 细胞（T helper cells）和直接参与识别靶抗原与杀伤靶细胞的细胞毒性T 细胞（cytotoxic T cells， CTL）。体内这两类T细胞的活化与增生一般都需要两个信号通路的协同作用：其中第1信号通路是通过T细胞上的可特异识别抗原的受体蛋白(T-cell receptor，TcR)与表达在肿瘤等靶细胞或抗原提呈细胞（antigen-presenting cells, APC）上相应的抗原多肽-MHC (major histocompatibility complex)复合物的结合而传导，该信号通路为抗原特异性的；第2信号通路是通过T细胞上的协同刺激（co-stimulation）或协同抑制（co-inhibition）因子与表达在肿瘤等靶细胞或抗原提呈细胞上的配体结合而传导，该信号通路为抗原非特异性的。T 淋巴细胞只有在第1与第2信号通路都存在的状态下才发生活化与增生。
在第2信号通路中发挥协同刺激作用（即正向或上调免疫功能）的代表性因子是CD28与其配体B7-1、B7-2；而发挥协同抑制作用（即负向或下调免疫功能）的代表性因子则是CTLA-4与其配体B7-1、B7-2，及程序性死亡受体（programmed death-1）PD-1与其配体PD-L1和PD-L2；这些协同刺激/协同抑制因子在结构上都很相似，均属于免疫球蛋白超家族成员 (见综述：Chen LP：Co-inhibitory molecules of the B7-CD28 family in the control of T cell immunity, Nature Immunol 2004, 336-347)。
理论上，至少可以通过两大不同途径达到提高体内免疫功能的目的：
途径1是通过提高CD28等协同刺激因子的表达而直接达到上调免疫功能的效果;
途径2则是通过降低或阻断CTLA-4或 PD-1/PD-L1等协同抑制因子表达，从而去除或降低肿瘤靶细胞带来的免疫抑制，进而间接达到上调免疫功能的效果。
直接提高CD28等协同刺激因子的表达尽管可以达到上调免疫功能的效果，但由于2006年在英国进行的一涉及代号为TGN1412的抗CD28单克隆抗体药物的临床I期研究中，6位健康受试者在接受注射该抗体药物的当天即发生了极其严重的不良反应（Suntharalingam G等： Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412。NEJM 2006,355：1018-1028），因此目前科学界普遍认为激动型抗CD28抗体类药物在临床应用上具有很高的安全风险与不确定性，对其未来开发上市的前景也存在很多担忧。相反的，以降低或阻断CTLA-4或PD-1/PD-L1等协同抑制因子表达与活性的拮抗型单克隆抗体药物，近年来由于其在美国已开展的多个早期临床研究中都表现出明显的抗肿瘤疗效及可接受的安全性，现反而已成为国际肿瘤药物领域的研究与开发热点（见综述：Quezada SA and Peggs KS：Exploiting CTLA-4, PD-1 and PD-L1 to reactivate the host immune response against cancer。 British Journal of Cancer 2013， 108：1560-1565；Flemming A. Cancer: PD-1 makes waves in anticancer immunotherapy. Nat Rev Drug Discov. 2012;11:601）。
PD-1 基因最早是由日本科学家Tasuku Honjo及其同事在1992年发现与克隆的，其胞外区有1个IgV样区，与CTLA-4有23%的同源性（Ishida, Y., Agata, Y., Shibahara, K. and Honjo, T. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J. 1992，11:3887；综述见：Okazaki T和Honjo T： PD-1 and PD-1 ligands：from discovery to clinical application. International Immunology 2007， 19：813-824）。PD-1主要表达于活化的T淋巴细胞 、 B 淋巴细胞及单核细胞上（Yasutoshi Agata等Internationl Immunology1996，8：675）。 PD-1的受体或配体已知有两个：分别为PD-L1（Freeman GJ 等，JEM 2000,192：1027-1034），或又称B7-H1（Dong H 等， Nature Medicine 1999，5：1365-1369）和PD-L2 （ Latchman Y 等，Nat. Immunol.2001，2： 261-268 ),或又称B7-DC （Tseng SY等，JEM 2001，193：839-845）。 PD-L1及PD-L2主要表达在肿瘤等靶细胞或抗原提呈细胞上（Thompson RH 等， Cancer Res 2006；66：3881-3885）。PD-1参与负向调控体内免疫功能的这一重要事实最早是由Tasuku Honjo及其同事在研究PD-1基因敲除小鼠中观察到的。他们发现PD-1基因敲除的小鼠在C57BL/6基因背景下，发生狼疮样肾小球肾炎和关节炎（Nishimura H 等，Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor.Immunity 1999，11:141）；而在Balb/c基因背景下，则产生高滴度的抗心肌组织抗体并由此引发严重的自身免疫性心肌病（Nishimura, H.等， Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science 2001，291:319）。
正常组织细胞中PD-L1基因的表达而介导的免疫抑制或免疫耐受，是体内保护其正常组织免于受周围自身淋巴细胞攻击与杀伤的重要机制（Keir M.E. 等， Tissue expression of PD-L1 mediates peripheral T cell tolerance. J Exp Med 2006，203：883-895）。而肿瘤细胞则能够借通过调高PD-L1或PD-L2蛋白的表达及与其表达在淋巴细胞上的PD-1受体的相互作用而传导出抑制信号，使体内免疫出现抑制或耐受而逃避淋巴细胞的识别与攻击（Dong H等， Nature Medicine 2002，8：793-800； Azuma T 等， Blood 2008，111：3635-3643）；而如给予PD-1或PD-L1 抗体以阻断PD-1/PD-L1之间相互作用，则可打破或消除因肿瘤导致的免疫抑制或免疫耐受，恢复体内T淋巴细胞识别与攻击肿瘤靶细胞的能力，进而达到抗肿瘤生长与转移的良好效果（Iwai Y 等， PNAS 2002， 99：1229； Hirano F 等， Cancer Res 2005，65：1089-1096）。基于这些前期基础研究与临床前动物实验结果，近年来国际上已有多家制药企业如美国Bristol-Myers Squibb （BMS）/Medarex，Merck 公司及Roche/Genentech等公司正在加紧研制各种以阻断PD-1与其配体结合为目的的拮抗类抗体新药，并为此申请了相关专利。
例如，美国Medarex公司 及日本 Ono Pharmaceutical 公司在 专利号 为No 8008449 美国发明专利 （ 发明人 Korma AJ 等，发明名称：Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics）中公开了由转染CHO细胞及其表达的含全长或含胞膜外区人PD-1蛋白作为混合抗原，交叉免疫具人免疫球蛋白（Ig）转基因的工程小鼠（HuMAb Moues）中筛选获得的多个抗人PD-1抗体的杂交瘤及其分泌的抗体蛋白 、 编码抗体蛋白的核苷酸序列，及该抗体用于PD-1蛋白检测及治疗肿瘤等疾病的应用。
Merck Sharp & Dohme 公司在 专利号 为No 8168757 美国发明专利 （ 发明人 Finnefrock AC 等，发明名称PD-1 binding proteins） 中 ，也公开了其由含PD-1基因的DNA分子免疫小鼠后筛选获得的多个抗人PD-1抗体的杂交瘤及其抗体蛋白 、 其编码核苷酸序列，及该抗体用于通过增强机体内免疫功能而达到治疗肿瘤及感染性疾病的目的。
Genentech 公司在专 利号 为No 8217149的 美国发明专利 （ 发明人 Irving B 等，发明名称Anti-PD-L1 antibodies, compositions and articles of manufacture）中，公开了从噬菌体抗体展示基因库（phage-display）中筛选扩增获得的多个抗人PD-L1抗体的抗体蛋白片段 、 其编码核苷酸序列及其用途。
美国Weythe（惠氏）公司及Medimmune（免疫医疗有限公司）在其中国专利申请号为201010170022.4 、 发明名称为'抗PD-1抗体及其用途'的专利文件中披露了从噬菌粒scFv基因展示库筛选获得的抗人PD-1抗体的片段 、 其编码DNA序列及该类抗体作为药物成分，用于治疗包括自身免疫病、变态反应、癌症等免疫系统相关疾病的应用。
美国Medarex（米德列斯）公司则在其一国际 PCT/US2006/026046进入中国的申请号为200680028238.9 、 发明名称为'抗程序性死亡配体1（PD-L1）的人单克隆抗体'的专利文件中披露了其从免疫小鼠中筛选获得的高亲和力特异结合人PD-L1的单克隆抗体 、 其编码DNA序列及其用该类抗体用于治疗包括癌症和传染病在内的各种疾病的方法。
在PD-1/ PD-L1抗体药物研发领域，目前进展最为靠前的当属美国Bristol-Myers Squibb/Medarex公司的抗-PD1抗体Nivolumab（代号BMS-936558,曾用代号MDX-1106）及美国Merck 公司的抗-PD1抗体lambrolizumab（代号MK-3475），目前这两种抗体药物在美国都已进入临床II/III期研究阶段。进展其次的是Roche/Genentech公司的抗PD-L1抗体（代号MPDL3280A），该药物目前在美国已进入临床I/II期研究阶段。这些抗体药物主要用于治疗肺癌 、 黑色素瘤 、 肾癌等多种晚期肿瘤，其中早期临床试验（I期）结果表明抗PD-1或抗PD-L1抗体药物不但具有抑制肿瘤增长及延长患者生存期的客观疗效，而且其长期使用后安全性也在可接受范围（Brahmer JR 等， JCO 2010，28：3167-3175；Topalian S 等，NEJM 2012，366：2443-2454； Brahmer JR 等，NEJM 2012，366：2455-2465；Hamid O等，NEJM2013，369:134-144）。如Topalian等在2012年6月报道：在一项有296例晚期癌症患者参与的I期临床研究中，每2周给予患者静脉注射美国BMS公司的名为Nivolumab（代号为BMS-93655）的抗PD-1抗体后，结果有高达28%的黑色素瘤患者、27%的肾细胞癌患者及出乎意料之高的18%非小细胞肺癌（NSCLC）患者，其肿瘤出现抑制或缩小；此外，该抗体药物在临床疗效上还表现出长期可持续的特点：如在已随访1年以上的31位患者中，有20位（64.5%）临床上表现依然有效（Topalian S等,NEJM2012,366：2443-2454）。
类似的，Hamid 等于2013年7月报道：在另一项有135例晚期黑色素瘤患者参与的I期临床研究中，每2至3周给予患者静脉注射Merck 公司的名为Lambrolizumab（代号为MK-3475）的抗PD-1抗体后，结果有高达38%的患者肿瘤出现抑制或缩小；该抗体药物在临床疗效上也表现出长期可持续的特点：如在随访中位数时间为11个月的52位肿瘤患者中，有42位（81%）临床上依然有效，并还在继续接受该药物治疗（Hamid O 等，N EJM 2013，369:134-144）。
鉴于PD-1与其受体PD-L1或PD-L2之间的结合具有位点多样复杂、涉及的氨基酸数目多达几十个以上等特点，因此理论上推测应可以制备针对不同结合位点或氨基酸序列的各种新的抗PD-1单克隆抗体。这些结合不同位点的单克隆抗体应具有新的用途与价值：如该类抗体一方面可与目前上述的已处于临床研究阶段的抗PD-1或抗PD-L1抗体药物合并使用，达到增强免疫功能及更好的抗肿瘤疗效；另一方面，该类抗体也有望开发成为独立使用的新型免疫功能增强剂或抗肿瘤药物。
因此，研发更多及更新的抗PD-1单克隆抗体及分泌它的杂交瘤细胞系就显得很有意义与必要。
 技术问题
本发明要解决的技术问题之一提供一种拮抗抑制程序性死亡受体PD-1（programmed death-1）与其配体（PD-L1及PD-L2）结合的鼠源单克隆抗体或其衍生体。该鼠源单克隆抗体的衍生体包括人-鼠嵌合抗体 、 人源化抗体 、 抗体Fab片段 、 单链抗体等。
本发明要解决的技术问题之二是提供编码上述抗体的DNA分子或基因。
本发明要解决的技术问题之三是提供制备上述抗体的方法。
本发明要解决的技术问题之四是提供该抗体的用途，包括将上述抗体作为检测试剂，用于定量及定性分析生物样品中PD-1蛋白。
 技术解决方案
为了获得更多及更新的抗PD-1单克隆抗体，在本发明中选取哺乳动物表达的重组人PD-1胞膜外蛋白为免疫抗原，采用传统小鼠杂交瘤技术与重组基因工程方法获得了多个新的抗PD-1的单克隆抗体。其中一代号为AB7的杂交瘤，其分泌的单克隆抗体不但可与人PD-1蛋白特异结合，而且可以拮抗抑制PD-1与其受体（PD-L1及PD-L2）的结合。本发明一方面提供了该杂交瘤株及其分泌的抗体；本发明另一发面公开了该抗体的重链可变区与轻链可变区氨基酸序列及其编码DNA核苷酸序列;本发明还提供了该抗体的人-鼠嵌合抗体及其衍生体;本发明还提供了表达与制备该人-鼠嵌合抗体及其衍生体的表达载体 、工程细胞及其方法。
为解决上述技术问题，本发明一方面提供了一种拮抗抑制PD-1（programmed death-1）与其配体结合的鼠源单克隆抗体，该抗体的特征是其轻链可变区具有SEQ ID NO.:1所示的氨基酸序列，重链可变区具有SEQ ID NO.:2所示的氨基酸序列。 该鼠源单克隆抗体来源于代号为 AB7 的小鼠杂交瘤细胞株，该杂交瘤细胞株已于 2013 年 10 月 17 日 保藏在中国微生物菌种保藏管理委员会普通微生物中心（保藏编号为 CGMCC No. 8351. 保藏地点：中国，北京）。 此外，本发明还提供编码上述鼠源单克隆抗体的DNA分子, 其轻链可变区具有SEQ ID NO.：3所示的核苷酸序列，重链可变区具有SEQ ID NO.：4的核苷酸序列。
本发明另一方面提供了来源于上述鼠源单克隆抗体的衍生体如人-鼠嵌合抗体 、 人源化抗体 、 抗体Fab片段 、 单链抗体等。与鼠源单克隆抗体相比，人-鼠嵌合抗体或人源化抗体作为治疗药物具有在人体中半衰期长（可长达20天）及免疫原性低等优点，便于长期或反复多次体内使用。本发明中将上述 鼠源单克隆抗体 的重链可变区和轻链可变区基因分别与人抗体重链恒定区和轻链恒定区基因相连，获得人-鼠嵌合抗体cAB7。该人-鼠嵌合抗体 或其衍生体 的特征是其 轻链抗原互补决定区具有 SEQ ID NO ： 5 ， SEQ ID NO ： 6 及 SEQ ID NO ： 7 的氨基酸序列；其重链抗原互补决定区具有 SEQ ID NO ： 8 ， SEQ ID NO ： 9 及 SEQ ID NO ： 10 的氨基酸序列。 此外，本发明还提供编码上述 人 - 鼠嵌合抗体或其衍生体的 DNA 分子或基因，其轻链可变区具有 SEQ ID NO ： 3 所示的核苷酸序列，重链可变区具有 SEQ ID NO ： 4 的核苷酸序列。
本发明第三方面是提供了一种表达载体， 它含有编码上述 人-鼠嵌合抗体 或其衍生体的 DNA 分子序列以及与该序列操作性相连的表达调控序列。
本发明第四方面提 供了一种重组宿主细胞，它由上述表达载体转化而成。该重组宿主细胞或其子代细胞表达上述 人-鼠嵌合抗体 或其衍生体 。
本发明的第五方面提 供了一种制备所述的人 - 鼠嵌合抗体或其衍生体的方法，该方法包括如下步骤：
a) 提供一表达载体，该表达载体含有权利要求 4 所述的 DNA 分子序列以及与该序列操作性相连的表达调控序列；
b) 用步骤 a ）所述的表达载体转化宿主细胞；
c) 在适合所述人源化单克隆抗体表达的条件下培养步骤 b) 所得的宿主细胞：和
d) 采用亲合层析从宿主细胞培养液中分离纯化获得所述抗体。
本发明的第六方面提 供了一种用于定量或定性检测分析样品中的 PD-1 蛋白的 检测试剂或试剂盒，它 包括上述 AB7 鼠源单克隆抗体或 人 - 鼠嵌合抗体，优选地，它 以未标记的 AB7 抗体作为第一抗体，以化学标记（如荧光素 、 酶 、 生物素等）的第二抗体如羊抗小鼠IgG合并使用作为试剂盒，用于定量及定性检测分析各类生物样品中的PD-1蛋白。采用的定量及定性检测法是本领域众所周知的，包括如，ELISA、免疫印迹、蛋白印迹、免疫荧光、免疫沉淀、流式细胞仪等。所述的试剂盒可含有其它组分、包装、说明书或其它材料，以辅助检测所述PD-1蛋白。
本文所采用的术语'单克隆抗体（单抗）'指从一纯系细胞得到的免疫球蛋白，具有相同的结构和化学特性，对单一抗原决定簇有特异性。单克隆抗体与常规多克隆抗体制剂（通常是具有针对不同决定簇的不同抗体）不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们是通过杂交瘤或重组工程细胞培养获得，不会混杂有其它免疫球蛋白。修饰语'单克隆'表示了抗体的特性，是从均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本文所采用的术语'人源化单克隆抗体' 系将鼠源单克隆抗体的氨基酸序列除保留互补决定区（complementarity-determining regions， CDR）外，其它序列（包括可变区中的框架区序列）全部或大部分替换成人免疫球蛋白的氨基酸序列，以达到通过基因工程手段最大限度地降低鼠源性单克隆抗体的免疫原性。
本文所用的术语'抗体'和'免疫球蛋白'是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链（L）和两个相同的重链（H）组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区（VH）。其后是多个恒定区。每条轻链的一端有可变区（VL），另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
本文所用的术语'可变'表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中成为互补决定区（CDR）或超变区中的三个片段中。可变区中较保守的部分称为构架区（Framework regions， FR）。抗体重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位（参见Kabat等,NIH Publ.No.91-3242,卷1,647-669页（1991））。抗体恒定区不直接参与抗体与抗原的结合,但是它们表现出不同的效应功能,例如参与抗体的依赖于抗体的细胞毒性(antibody-dependent cellular cytotoxicity,ADCC)或补体介导毒性(complemnt-dependent cytotoxicity,CDC)。
本发明的抗体通常可以通过以下方法来制备。
首先，将含有编码本发明的抗体的基因插入到含有合适的表达调控序列的表达载体中。
本文所用的术语'表达调控序列'通常指参与控制基因表达的序列。表达调控序列包括与目标基因操作性相连的启动子和终止信号。编码本发明抗体的基因（ DNA ）序列可用本领域技术人员熟知的常规手段，如根据本发明公开的蛋白质序列人工合成或用 PCR 法扩增得到。其后可用本领域熟知的各种方法将合成或 PCR 扩增得到的 DNA 片段插入到合适的表达载体中。本发明中所用的表达载体可以是本领域技术人员已知的市售表达载体，如 Invitrogen 公司的 pCDNA3.1 表达载体。
用于接纳表达载体转化的合适宿主细胞一般包括原核细胞和真核细胞。常用的原核宿主细胞的例子包括大肠杆菌、枯草杆菌等。常用的真核宿主细胞包括酵母细胞、昆虫细胞、哺乳动物细胞等。在本发明中，较佳的宿主细胞是哺乳动物细胞，尤其是中华仓鼠卵巢（ CHO ）细胞。
表达载体转化的宿主细胞在合适的条件下（如以无血清培养基在细胞培养瓶或生物反应器中贴壁或悬浮培养）培养后，收获培养上清液，然后用包括 protein-A 亲和层析、离子交换层析、过滤等本领域技术人员熟知的常规分离步骤或手段纯化得到本发明的抗体。
纯化得到的本发明抗体可以溶于适当的溶剂如生理盐水液体中，溶度可以制备成 0.01 至 100 mg/ml 之间，理想的最终溶度可以制备成 0.1 至 20 mg/ml 之间。
为获得一种拮抗抑制PD-1（programmed death-1）与其配体结合的鼠源单克隆抗体及分泌它的杂交瘤细胞系，本发明选取哺乳动物表达的重组人PD-1胞膜外蛋白为免疫抗原，通过反复多次小剂量的小鼠皮下免疫，获得分泌抗PD-1蛋白的多克隆抗体；再从中挑取含高效价抗体的小鼠，取其脾脏细胞，通过体外与小鼠骨髓瘤细胞融合、再经药物筛选及亚克隆等步骤而建立了多株稳定分泌抗人PD-1蛋白的抗体的杂交瘤单克隆细胞。 其中一代号为AB7的小鼠杂交瘤细胞株，经ELISA、免疫印迹、免疫组化等多种方法鉴定，证实其所分泌的单克隆抗体不但能够特异结合人PD-1蛋白，而且可阻断抑制PD-1蛋白与其配体（PD-L1，PD-L2）的结合。
本发明通过基因工程等手段获得了编码该鼠源抗体重链可变区及轻链可变区的基因片断，并在此基础上构建了该抗体的人-鼠嵌合抗体基因及其表达载体（pCDNA3.1-cAB7）。 该表达载体经转染进入中华仓鼠卵巢（CHO）细胞，获得分泌表达人-鼠嵌合抗体的重组工程细胞，并从重组工程细胞培养液中分离纯化获得到具有生物活性的人-鼠嵌合抗体蛋白。
 有益效果
为获得一种拮抗抑制PD-1（programmed death-1）与其配体结合的鼠源单克隆抗体及分泌它的杂交瘤细胞系，本发明选取哺乳动物表达的重组人PD-1胞膜外蛋白为免疫抗原，通过反复多次小剂量的小鼠皮下免疫，获得分泌抗PD-1蛋白的多克隆抗体；再从中挑取含高效价抗体的小鼠，取其脾脏细胞，通过体外与小鼠骨髓瘤细胞融合、再经药物筛选及亚克隆等步骤而建立了多株稳定分泌抗人PD-1蛋白的抗体的杂交瘤单克隆细胞。 其中一代号为AB7的小鼠杂交瘤细胞株，经ELISA、免疫印迹、免疫组化等多种方法鉴定，证实其所分泌的单克隆抗体不但能够特异结合人PD-1蛋白，而且可阻断抑制PD-1蛋白与其配体（PD-L1，PD-L2）的结合。
本发明通过基因工程等手段获得了编码该鼠源抗体重链可变区及轻链可变区的基因片断，并在此基础上构建了该抗体的人-鼠嵌合抗体基因及其表达载体（pCDNA3.1-cAB7）。 该表达载体经转染进入中华仓鼠卵巢（CHO）细胞，获得分泌表达人-鼠嵌合抗体的重组工程细胞，并从重组工程细胞培养液中分离纯化获得到具有生物活性的人-鼠嵌合抗体蛋白。
 附图说明
图1 为本发明实施例1中以ELISA法检测小鼠杂交瘤细胞（AB7）培养上清液与包被在96-孔板上的重组人PD-1胞膜外蛋白结合的结果示意图。其中未融合的SP2/0骨髓瘤细胞培养上清液为阴性对照样品。
图2 为实施例2 中以流式细胞仪检测AB7杂交瘤细胞培养上清液中抗体与PD-1基因转染及未转染的CHO细胞的结合结果示意图。其中未融合的SP2/0骨髓瘤细胞培养上清液为阴性对照；其中，图2A代表在SP2/0阴性对照上清样品中PD-1基因未转染的CHO细胞，图2B代表在AB7杂交瘤细胞培养上清样品中PD-1基因未转染的CHO细胞，图2A代表在SP2/0阴性对照上清样品中PD-1基因转染的CHO细胞，图2D代表在AB7杂交瘤细胞培养上清样品中PD-1基因转染的CHO细胞。
图3 为本发明实施例3中以SDS-PAGE 分析鉴定从AB7杂交瘤细胞培养上清液中亲合层析纯化获得的抗体蛋白的结果示意图。其中泳道1为未还原的AB7抗体蛋白样品，泳道2为DTT还原后的AB7抗体蛋白样品；M为蛋白质分子量标志。
图4为实施例4中流式细胞仪检测分析鼠源AB7单克隆抗体与PHA激活的人Jurkat T 淋巴细胞上PD-1蛋白结合的结果示意图。其中以非相关小鼠抗体IgG样品为阴性对照。其中，图4A代表非相关小鼠抗体IgG 阴性对照样品，图4B代表AB7抗体的样品。
图5 为本发明实施例5中以体外竞争ELISA 法检测证明AB7抗体可拮抗阻断生物素标记的人PDL1-Fc蛋白（Biotin-PDL1-Fc）与包被在96-孔板上人PD-1蛋白的结合的结果示意图。其中的AB7&Biotin-PDL1-Fc 组为AB7抗体与Biotin-PDL1-Fc竞争样品，MouseIg&Biotin-PDL1Fc组为非相关的小鼠抗体与Biotin-PDL1-Fc竞争样品，作为阴性对照。
图6 为本发明实施例5中以体外竞争ELISA 法检测证明AB7抗体可拮抗阻断生物素标记的人PDL2-Fc蛋白（Biotin-PDL2-Fc）与包被在96-孔板上人PD-1蛋白的结合的结果示意图。其中的AB7&Biotin-PDL2-Fc 组为AB7抗体与Biotin-PDL2-Fc竞争样品，MouseIg&Biotin-PDL2Fc组为非相关的小鼠抗体与Biotin-PDL2-Fc竞争样品，作为阴性对照。
图7 为本发明实施例7中以直接ELISA法检测证明由含人-鼠嵌合抗体（cAB7）基因的表达载体转染的CHO细胞表达上清保持与人PD-1蛋白结合的结果活性示意图。
代号为 AB7 的小鼠杂交瘤细胞株 ( 来源于小鼠 Mus musculus ) 已于 2013 年 10 月 17 日保藏在中国微生物菌种保藏管理委员会普通微生物中心（保藏编号为 CGMCC No. 8351 ；保藏地点：中国北京市朝阳区北辰西路 1 号院 3 号，中国科学院微生物研究所）。
本发明的最佳实施方式
本发明的实施方式
下面将结合实施实例来进一步描述本发明，这些实施例只是为了起说明作用，而不是用来限制本发明。
实施例1: 分泌抗 PD-1 抗体 的小鼠杂交瘤细胞系的建立与筛选鉴定
步骤1.重组人PD-1蛋白（免疫抗原）的获得与动物免疫
在本发明实施例中，用于免疫的人PD-1蛋白为由哺乳动物表达的重组人PD-1胞膜外蛋白（北京义翘神州公司产品）。该重组人PD-1蛋白与弗氏完全佐剂（美国Sigma公司产品）混合后，于皮下多点注射Balb/c小鼠（100 μl/只，共5-10μg PD-1蛋白）。首次免疫 2-3周后，小鼠再给予皮下多点注射含PD-1蛋白与弗氏不完全佐剂（美国Sigma公司产品）的混合物，加强免疫2-3 次后，取少量小鼠血清，用包被人PD-1蛋白的96-板以ELISA法检测小鼠血清中抗PD-1抗体的效价，取效价高者小鼠的脾细胞用于下一步的细胞融合。
步骤2、细胞融合
在末次免疫后3天，无菌制备小鼠脾细胞悬液，与小鼠SP2/0骨髓瘤细胞 (购自中国科学院上海生命科学院细胞保藏中心），以5：1的比例在50% PEG-1000（美国Sigma 公司产品）作用下融合。融合按常规法（Kohler G. and Milstein C: Nature 1975;256:495-497）,PEG用量1ml ，1分钟内缓慢加完。反应90秒后，以无血清的RPMI-1640培养基终止反应，1000rpm离心10 min,去除上清液，再将离心沉淀下的细胞以含10% HAT(H为次黄嘌呤、A氨基碟呤、T胸腺嘧啶核苷，为美国Sigma公司产品) 的RPMI 1640-10% FCS培养基将细胞浓度调节至1Х106/ml，加入96孔平底细胞培养板（每孔200μl），于37℃，5% CO2培养箱中培养2-3 周。
步骤3、酶联免疫吸附试验（ELISA）筛选抗体分泌阳性的小鼠杂交瘤细胞
以重组人PD-1蛋白（ 2 μg/ml， pH 9.6， 0.1 M NaHCO3 液）包被酶标板，37℃包被2小时或4℃过夜；2% 牛血清白蛋白（BSA）4℃封闭过夜。 经PBS-0.1% Tween20 液洗涤后加入待检杂交瘤细胞培养上清（以未融合的SP2/0骨髓瘤细培养上清为阴性对照）37℃孵育2小时； 经PBS-0.1% Tween20 液洗涤后，加入辣根过氧化物酶HRP-标记的羊抗小鼠IgG（美国Sigma公司产品），37℃孵育1小时；再经PBS-0.1% Tween20 液充分洗涤后，加入邻苯二胺（OPD）-0.1% H2O2底物液显色10-15min,以0.1MHCl终止反应。 在MK3-Multiskan酶标仪 (美国Thermo Scientific公司产品)中读取492nm 处OD值。测得的OD 492值比阴性对照高5-10倍的杂交瘤细胞再克隆化，并进行扩增冻存。
步骤4、阳性杂交瘤细胞的亚克隆-有限稀释法
将上述初筛得到的阳性细胞以RPMI-1640-10% FCS培养基稀释至每孔1-10个细胞，铺于96-孔细胞培养板，于37℃，5% CO2培养箱中培养2-3 周。待克隆长成，取上清液以ELISA再次检测鉴定抗PD-1抗体的分泌。经检测鉴定，获得多个抗体分泌阳性细胞株。经亚克隆鉴定，其中一株代号为AB7杂交瘤细胞株可稳定分泌表达抗PD-1单克隆抗体。图1为以ELISA 法检测测AB7杂交瘤细胞上清液与重组人PD-1蛋白结合的结果示意图，结果证明该杂交瘤细胞上清液含高效价抗人PD-1蛋白的抗体。该抗体经鉴定为IgG类。该杂交瘤细胞株再经大量扩增，长期传代培养并于2013年10月17日保藏在中国微生物菌种保藏管理委员会普通微生物中心（保藏编号为CGMCC No.8351. 保藏地点：中国，北京）。
实施例2 流式细胞仪检测分析鼠源AB7单克隆抗体与人PD-1基因转染的CHO细胞的结合
在本实施例中，以AB7抗体为一抗，以FITC荧光标记的羊抗小鼠IgG为二抗，采用流式细胞仪检测分析AB7抗体与人PD-1基因转染的CHO细胞结合。为此，先从人外周血淋巴细胞（PBL）中提取出总mRNA，再以Reverse transcription-polymerase chain reaction (RT-PCR)方法体外扩增获得含编码PD-1全长蛋白（包括胞外及胞内区）的 cDNA 片段。用于RT-PCR的引物为PD-1正向引物：ATTAAGCTTGAGCAGTGGAGAAGGCGGCA，Seq ID No:11，PD-1反向引物：AATTGGATCCCTCCTGAGGAAATGGGCTGA，Seq ID No:12）。 以该对引物扩增获得cDNA 片段包涵PD-1 蛋白全部编码区，长度为989bp。该DNA经限制性内切酶处理后，再插入至表达质粒pCDNA3.1（美国Invitrogen公司）中，从而获得含PD-1 cDNA的重组质粒pCDNA3.1-PD-1。随后将该表达质粒与Fugen-6脂质体（Roche公司产品）混合后共转染入CHO细胞(购自美国Invitrogen 公司），转染2天后，细胞经离心分离后，重溶于分别含鼠源AB7单克隆上清（为测试样品）或SP2/0上清（为阴性对照）的溶液中，4℃孵育1小时； 经PBS-0.1% Tween20 液洗涤后，加入FITC-标记的羊抗小鼠IgG（Sigma 公司产品），4℃孵育1小时； 再经PBS-0.1% Tween20 液洗涤后，将样品上样至Cytomics FC500 MCL）流式细胞仪检测（美国Beckman Coulter公司）。
图2为该流式细胞仪检测结果示意图。如图2所示：在转染人PD-1基因的样品中，有近30% 的细胞可与AB7单克隆抗体特异结合（图2D）； 而在SP2/0阴性对照上清样品中，无论是转染的（图2C）还是未转染的CHO细胞（图2A）均无明显结合；未转染的CHO细胞与AB7抗体也无明显结合（图2B），表明该流式细胞仪检测结果具有较高的特异性。
实施例3. 鼠源 抗人PD-1单克隆抗体（AB7）的体外制备及纯化
本实施例中，采用亲合层析法分离纯化获得鼠抗人PD-1单克隆抗体（AB7）蛋白。
其纯化步骤如下：
将AB7杂交瘤细胞扩增后，接种于200-500ml无血清的1640培养基中，37℃培养5天，随后收集培养上清，经过0.45 μm滤膜过滤后上样至含Protein G-Sepharose Fast Flow（购自通用电气GE公司）亲合层析柱；层析柱先以PBS液漂洗去除杂蛋白后, 再以低pH(2.7-3.0) 甘氨酸(0.1M) 液洗脱被吸附的AB7抗体蛋白。 洗脱液以1 mol/L Tris(pH 8.5-9.0)调节pH至7.0,再对10 倍体积的PBS液透析12～16后（期间换液2-3次）,透析后的样品再经0.45 μm滤膜过滤后即获得纯化的AB7抗体。
将纯化的AB7抗体按常规方法在DTT还原及未还原条件下进行经聚丙烯酰胺凝胶电泳分析（SDS-PAGE）（分离胶为10%，浓缩胶5%）。图3为该电泳分析结果图谱：其中泳道1为未还原的完整AB7抗体样品，分子量在150kD左右；泳道2为DTT还原后的AB7抗体样品，分离为2条主带,其中处于分子量在50kD左右的上面条带为AB7抗体重链,处于分子量在25kD左右的下面条带为AB7抗体轻链。
实施例4鼠源AB7单克隆抗体用于检测分析表达在人T 淋巴细胞上的PD-1蛋白
在本实施例中，AB7单克隆抗体作为一抗，FITC荧光标记的羊抗小鼠IgG作为二抗，用于检测分析表达在人T淋巴细胞上的PD-1蛋白。其中的人T淋巴细胞为Jurkat细胞(购自于中国科学院上海生命科学院细胞保藏中心）。为此，先将Jurkat 细胞置于含T淋巴细胞活化因子PHA（美国Sigma公司产品, PHA终浓度：3μg/mL）的RPMI-10%FCS 培养液中培养以激活与诱导PD-1蛋白的表达。激活与诱导 24-48小时后，细胞经离心分离后分别重溶于含纯化的测试样品AB7抗体（终溶度：5μg/ml）或非相关小鼠IgG (MouseIgG,阴性对照样品，终溶度：5μg /ml）的溶液中，4℃孵育1小时； 经PBS-0.1% Tween20 液洗涤后，加入FITC-标记的羊抗小鼠IgG （Sigma 公司产品,1:200），4℃孵育1小时；再经PBS-0.1% Tween20液洗涤后，将样品至Cytomics FC500 MCL流式细胞仪检测（美国Beckman Coulter公司）。流式细胞仪分析。
图4为该流式细胞仪检测分析的结果图谱。如图4B所示：AB7单克隆抗体样品可与PHA活化的Jurkat 细胞结合，与未活化的Jurkat 细胞无明显结合反应。在非相关小鼠IgG 阴性对照样品中（图4A），无论是活化的还是未活化的Jurkat 细胞，均无明显结合反应。
实施例5 AB7单克隆抗体生物活性测定：竞争性ELISA法检测鉴定鼠源AB7单克隆抗体体外阻断其受体PD-L1及PD-L2与PD-1蛋白的结合
体外鉴定AB7单克隆抗体的生物活性的方法之一是采用竞争性ELISA法检测该抗体是否可以阻断PD-1与其PD-1配体（PD-L1及PD-L2）的结合。该竞争性ELISA法的基本原理与过程是：先将生物素标记的人PD-1配体蛋白（如PDL1-Fc或PDL2-Fc融合蛋白）与不同溶度的单克隆抗体混合，之后再将混合物转入预先包被有PD-1蛋白的96-孔板，经孵育及洗脱后，加入酶标记的Avidin（如辣根过氧化物酶标记的Avidin）； 再经孵育及洗脱后，加入底物显示并测定OD值。
该竞争性ELISA法检测的具体步骤如下：
1 ）用重组人PD-1胞膜外蛋白（北京义翘神州公司产品）包被96-孔板（包被溶度：2 μg/ml,50 μl/孔)，4℃过夜；
2 ）经PBS液漂洗及2% BSA (稀释在 PBS-0.1% tween20液中) 室温封闭后,分别加入含固定溶度的生物素标记的PDL1-Fc蛋白或PDL2-Fc蛋白（PDL1-Fc蛋白及PDL2-Fc蛋白均为北京义翘神州公司产品）与不同溶度的AB7抗体，或非相关的小鼠抗体（mouse IgG）， 37℃孵育2 h；
3 ）经PBS-T洗脱后，加入辣根过氧化物酶标记的Avidin（1：5000），37℃孵育1 h；
4 ）经PBS-T洗脱后，加入显色液(邻苯二胺)-3% 双氧水，室温10min至显色；
5 ）加入HCL终止反应，以酶联免疫仪测定492 nm波长处各孔的吸光值。
图5为AB7抗体体外与生物素标记的PDL1-Fc蛋白（Biotin-PDL1Fc）竞争结合PD-1蛋白的代表性结果。如图5所示:在加入AB7抗体与生物素标记的PDL1-Fc蛋白（AB7&Biotin-PDL1Fc）竞争的样品中，各孔显色反应 OD值与加入的AB7抗体蛋白量成反比关系：即加入的AB7抗体的量越高，其OD值越低。而在加入非相关小鼠抗体与生物素标记的PDL1-Fc蛋白（MouseIgG&Biotin-PDL1Fc）竞争的样品中，各孔显色反应 OD值不受加入的抗体蛋白量影响。 此结果清楚表明AB7抗体可竞争阻断PD-L1（PDL1-Fc）与PD-1的结合。
图6则为AB7抗体体外与生物素标记的PDL2-Fc蛋白（Biotin-PDL2Fc）竞争结合PD-1蛋白的代表性结果。 如图6所示:在加入AB7抗体与生物素标记的PDL2-Fc蛋白（AB7&Biotin-PDL2Fc）竞争的样品中，各孔显色反应 OD值与加入的AB7抗体蛋白量成反比关系：即加入的AB7抗体的量越高，其OD值越低。而在加入非相关小鼠抗体与生物素标记的PDL2-Fc蛋白（MouseIgG&Biotin-PDL2Fc）竞争的样品中，各孔显色反应 OD值不受加入的抗体蛋白量影响。 此结果表明AB7抗体也可竞争阻断PD-L2（PDL2-Fc）与PD-1的结合。
实施例6.编码鼠源AB7抗体可变区基因的克隆
由于AB7抗体的重链及轻链均存在N-端封闭现象，常规Edman氨基酸降解法N-端测序未获结果。在此，先从小鼠AB7杂交瘤细胞中提取出总RNA中，再以该RNA为模板，采用兼并引物（degenerate primers），以Reverse transcription-polymerase chain reaction (RT-PCR)法(Wang Y 等：Degenerated primer design to amplify the heavy chain variable region from immunoglobulin cDNA。 BMC Bioinformatics. 2006; 7 Suppl （4): S9)分别克隆扩增获得AB7抗体重链可变区及轻链可变区的cDNA基因片段。
其中克隆步骤如下：
步骤1、 采用试剂盒（江苏海门碧云天公司产品）从小鼠AB7杂交瘤细胞中提取出mRNA；
步骤2、 采用逆转录PCR(RT-PCR)方法在 eppendorf 管 获得cDNA模板。
其中用于AB7抗体轻链可变区逆转录PCR引物（AB7-L）序列为：GTG CTG GAG GGG ACA GTC ACT（ SEQ ID NO.:13 ）；
用于AB7抗体重链可变区逆转录PCR引物（AB7-H）序列为：CTG AGG CAC CTC CAG ATG TT TGTCGTTCACTGCCATCAAT（ SEQ ID NO.:14 ） ；
RT-PCR 反应体系如下：
引物 2 µ l
RNA 模板 30 µ l
72℃ 孵育10分钟，然后冰上放置2分钟 。
随后加入：
5 ×RT-PCR反应 缓冲液 10 µ l
dNTPs 5 µ l
PrimeScript 逆转录酶 1.5 µ l
蒸馏水 1.5 µ l
总体积 50µl
于42℃温度下反应1小时，随后温度升至75℃，15分钟灭活后将获得的cDNA 置于-20℃，保存备用。
步骤3、AB7抗体轻链可变区及重链可变区基因的PCR克隆扩增
用于PCR法克隆扩增该AB7抗体轻链可变区基因的一对引物为：
正向引物：CCG AGA TCT GAC GTG CAG ATT TTC AGC T (SEQ ID NO.:15)
反向引物：AAT TGG ATC CAG TTG GTG CAG CAT CAG C (SEQ ID NO.:16)
而用于兼并引物（degenerate primers）PCR法克隆扩增AB7抗体重链可变区基因的一对引物为：
正向引物：GCG AAG CTT CAR CTG CAR CAR YCT G (SEQ ID NO.:17)
其中 Y=C或T， R= A or G 。
反向引物：AAT TGG ATC CTG GGG GTG TCG TTT TGG C (SEQ ID NO.:18)
PCR扩增得到的DNA产物在1%agarose胶中电泳分析。电泳结束后，将分离的DNA条带切下并分别进行测序获得抗体重链可变区DNA的核苷酸序列。测得的该抗体轻链可变区DNA的核苷酸序列见SEQ ID NO.:3，由该DNA核苷酸序列推测得到的抗体轻链可变区氨基酸序列见SEQ ID NO.:1。该轻链抗原互补决定区(complementarity-determining regions,CDR)的 CDR1、CDR2及CDR3的氨基酸序列分别见SEQ ID NO.:5、SEQ ID NO.:6 和SEQ ID NO.:7。
测得的该抗体重链可变区DNA的核苷酸序列见SEQ ID NO.:4，由该DNA的核苷酸序列推测得到的抗体重链可变区氨基酸序列见SEQ ID NO.:2。该重链抗原互补决定区的 CDR1、CDR2及CDR3的氨基酸序列分别见SEQ ID NO.:8、SEQ ID NO.:9 和SEQ ID NO.:10。
实施例7. 人-鼠嵌合抗体（cAB7）的制备
将实施例6中克隆扩增获得的AB7抗体轻链可变区基因和重链可变区基因分别与人-kappa轻链恒定区（C-domain）和人IgG1-重链恒定区基因片段融合，获得人-鼠嵌合轻链基因（ cAB7L ）及 人-鼠嵌合重链基因（ cAB7H ）。 其后将轻链嵌合基因与重链嵌合基因分别克隆至pcDNA3.1 表达质粒，转入大肠杆菌扩增，分离获得大量含人-鼠嵌合抗体基因的表达质粒。
含人-鼠嵌合抗体基因的表达质粒再与Fugen-6脂质体（Roche）混合后共转染入CHO细胞。细胞转染后2-3天，取培养上清液，用包被人PD-1蛋白的96-孔板，用 HRP 酶标记的 Goat-anti-human-IgG 为二抗（购自上海西塘生物公司 ) ， 为检测二抗， 以ELISA法检测上清中的嵌合抗体（cAB7）与人PD-1蛋白结合。下表1及图7为该ELISA代表性检测结果，如表1及图7所示：人-鼠嵌合抗体基因表达质粒转染的CHO细胞上清中含有可与人PD-1的蛋白特异结合的嵌合抗体。
表1以ELISA法检测瞬时转染细胞培养上清中抗体与人PD-1蛋白的结合活性
上清稀释倍数 1 2 4 8 16 32 64 128
OD492
显色值		 1.119 0.877 0.657 0.459 0.286 0.189 0.129 0.106
上述转染细胞上清经离心及0.45 μm滤膜过滤后，可上样至Protein A亲合层柱(通用电气GE公司proteinA-Sepharose Fast Flow)，用于分离纯化抗体蛋白。
 工业实用性
为获得一种拮抗抑制PD-1（programmed death-1）与其配体结合的鼠源单克隆抗体及分泌它的杂交瘤细胞系，本发明选取哺乳动物表达的重组人PD-1胞膜外蛋白为免疫抗原，通过反复多次小剂量的小鼠皮下免疫，获得分泌抗PD-1蛋白的多克隆抗体；再从中挑取含高效价抗体的小鼠，取其脾脏细胞，通过体外与小鼠骨髓瘤细胞融合、再经药物筛选及亚克隆等步骤而建立了多株稳定分泌抗人PD-1蛋白的抗体的杂交瘤单克隆细胞。 其中一代号为AB7的小鼠杂交瘤细胞株，经ELISA、免疫印迹、免疫组化等多种方法鉴定，证实其所分泌的单克隆抗体不但能够特异结合人PD-1蛋白，而且可阻断抑制PD-1蛋白与其配体（PD-L1，PD-L2）的结合。
本发明通过基因工程等手段获得了编码该鼠源抗体重链可变区及轻链可变区的基因片断，并在此基础上构建了该抗体的人-鼠嵌合抗体基因及其表达载体（pCDNA3.1-cAB7）。 该表达载体经转染进入中华仓鼠卵巢（CHO）细胞，获得分泌表达人-鼠嵌合抗体的重组工程细胞，并从重组工程细胞培养液中分离纯化获得到具有生物活性的人-鼠嵌合抗体蛋白。
 序列表自由内容
序列表
<110> 苏州思坦维生物技术有限责任公司
<120> 拮抗抑制程序性死亡受体PD-1与其配体结合的单克隆抗体及其编码序列与用途
<130> DAPCT-1841
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<170> PatentIn version 3.4
<210> 1
<211> 108
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<400> 1
QIVLTQSATI MSASLGERVT MTCTASSSVS SSYLHWYQQK PGSSPKLWIY STSNLASGVP 60
ARFSGSGSGT SYSLTISSME AEDAATYYCH QYHRSPLTFG AGTKLEIK 108
<210> 2
<211> 122
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<400> 2
QIQLVQSGPE LKKPGETVKI SCKASGYTFT DYSMHWVKQA PGKGLKWMGW IKVETGGPTY 60
AEDFKGRFAF SLETSARTAY LQINNLKNED TATYFCARDY YGNYYYAMDY WGQGTSVTVS 120
SA 122
<210> 3
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cagattgttc tcacccagtc tgcaacaatc atgtctgcat ctctagggga acgggtcacc 60
atgacctgca ctgccagctc aagtgtaagt tccagttact tgcactggta ccagcagaag 120
ccaggatcct cccccaaact ctggatttat agcacatcca acctggcttc tggagtccca 180
gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagtatggag 240
gctgaagatg ctgccactta ttactgccac cagtatcatc gttccccgct cacgttcggt 300
gctgggacca agctggaaat aaaa 324
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ccaggaaagg gtttaaagtg gatgggctgg ataaaagttg agaccggtgg gccaacatat 180
gcagaagact tcaagggacg gtttgccttc tctttggaaa cctctgccag aactgcctat 240
ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc tagagactac 300
tatggtaact attactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 360
tcagcc 366
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TASSSVSSSY LH 12
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Claims (1)

1 、一种拮抗抑制程序性死亡受体 PD-1 与其配体结合的 鼠源 单克隆抗体，其特征在于，所述抗体的轻链可变区具有 SEQ ID NO ： 1 所示的氨基酸序列，所述抗体的重链可变区具有 SEQ ID NO ： 2 的氨基酸序列。
2 、一种编码权利要求 1 所述抗体的 DNA 分子或基因，其特征在于，其轻链可变区具有 SEQ ID NO ： 3 所示的核苷酸序列，重链可变区具有 SEQ ID NO ： 4 的核苷酸序列。
3 、一种拮抗抑制程序性死亡受体 PD-1 与其配体结合的人 - 鼠嵌合抗体或其衍生体，其特征在于， 将权利要求1所述的重链可变区和来源于人的抗体重链恒定区相连，和将权利要求1所述的抗体轻链可变区基因与来源于人的抗体轻链恒定区相连， 所述人 - 鼠嵌合抗体或其衍生体的轻链抗原互补决定区具有 SEQ ID NO ： 5 ， SEQ ID NO ： 6 及 SEQ ID NO ： 7 的氨基酸序列；所述人 - 鼠嵌合抗体或其衍生体的重链抗原互补决定区具有 SEQ ID NO ： 8 ， SEQ ID NO ： 9 及 SEQ ID NO ： 10 的氨基酸序列。
4 、一种编码权利要求 3 所述人 - 鼠嵌合抗体或其衍生体的 DNA 分子或基因，其特征在于，其轻链可变区具有 SEQ ID NO ： 3 所示的核苷酸序列，重链可变区具有 SEQ ID NO ： 4 的核苷酸序列。
5 、一种表达载体，其特征在于，它含有权利要求 4 所述的 DNA 分子序列以及与该序列操作性相连的表达调控序列。
6 、一种重组宿主细胞，其特征在于，它由权利要求 5 所述的表达载体转化而成。
7 、根据权利要求 6 所述的重组宿主细胞，其特征在于，所述重组宿主细胞或其子代细胞表达权利要求 3 所述的人 - 鼠嵌合抗体或其衍生体。
8 、一种制备权利要求 3 所述的人 - 鼠嵌合抗体或其衍生体的方法，其特征在于，该方法包括如下步骤：
a) 提供一表达载体，该表达载体含有权利要求 4 所述的 DNA 分子序列以及与该序列操作性相连的表达调控序列；
b) 用步骤 a ）所述的表达载体转化宿主细胞；
c) 在适合所述人源化单克隆抗体表达的条件下培养步骤 b) 所得的宿主细胞：和
d) 采用亲合层析从宿主细胞培养液中分离纯化获得所述抗体。
9 、一种用于定量或定性检测分析样品中的 PD-1 蛋白的试剂或试剂盒，其特征在于，其包括如权利要求 1 或权利要求 3 所述的抗体。
10 、如权利要求 9 所述的试剂或试剂盒，其特征在于，其以如权利要求 1 或权利要求 3 所述的抗体为第一抗体，与化学标记的第二抗体合并使用。
PCT/CN2014/083780 2013-10-25 2014-08-06 拮抗抑制程序性死亡受体pd-1与其配体结合的单克隆抗体及其编码序列与用途 WO2015058573A1 (zh)

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Publication number Priority date Publication date Assignee Title
US9394365B1 (en) 2014-03-12 2016-07-19 Yeda Research And Development Co., Ltd Reducing systemic regulatory T cell levels or activity for treatment of alzheimer's disease
US9512227B2 (en) 2014-03-12 2016-12-06 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of Alzheimer's disease
WO2017205721A1 (en) 2016-05-27 2017-11-30 Agenus Inc. Anti-tim-3 antibodies and methods of use thereof
WO2017220989A1 (en) 2016-06-20 2017-12-28 Kymab Limited Anti-pd-l1 and il-2 cytokines
CN107686520A (zh) * 2016-08-04 2018-02-13 信达生物制药(苏州)有限公司 抗pd‑l1纳米抗体及其应用
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
WO2018071500A1 (en) 2016-10-11 2018-04-19 Agenus Inc. Anti-lag-3 antibodies and methods of use thereof
WO2018106864A1 (en) 2016-12-07 2018-06-14 Agenus Inc. Antibodies and methods of use thereof
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WO2018132739A2 (en) 2017-01-13 2018-07-19 Agenus Inc. T cell receptors that bind to ny-eso-1 and methods of use thereof
WO2018191502A2 (en) 2017-04-13 2018-10-18 Agenus Inc. Anti-cd137 antibodies and methods of use thereof
WO2018204363A1 (en) 2017-05-01 2018-11-08 Agenus Inc. Anti-tigit antibodies and methods of use thereof
US10144779B2 (en) 2015-05-29 2018-12-04 Agenus Inc. Anti-CTLA-4 antibodies and methods of use thereof
WO2018226580A3 (en) * 2017-06-05 2019-01-31 Janssen Biotech, Inc. ANTIBODIES SPECIFICALLY BINDING TO PD-1 AND THEIR METHODS OF USE
WO2019046856A1 (en) 2017-09-04 2019-03-07 Agenus Inc. T-CELL RECEPTORS THAT BIND TO SPECIFIC PHOSPHOPEPTIDES OF MIXED LINEAR LEUKEMIA (MLL) AND METHODS OF USE THEREOF
US10344090B2 (en) 2013-12-12 2019-07-09 Shanghai Hangrui Pharmaceutical Co., Ltd. PD-1 antibody, antigen-binding fragment thereof, and medical application thereof
US10428145B2 (en) 2015-09-29 2019-10-01 Celgene Corporation PD-1 binding proteins and methods of use thereof
WO2019210055A2 (en) 2018-04-26 2019-10-31 Agenus Inc. Heat shock protein-binding peptide compositions and methods of use thereof
US10513558B2 (en) 2015-07-13 2019-12-24 Cytomx Therapeutics, Inc. Anti-PD1 antibodies, activatable anti-PD1 antibodies, and methods of use thereof
US10519237B2 (en) 2014-03-12 2019-12-31 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
US10618963B2 (en) 2014-03-12 2020-04-14 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
US10751414B2 (en) 2016-09-19 2020-08-25 Celgene Corporation Methods of treating psoriasis using PD-1 binding antibodies
US10766958B2 (en) 2016-09-19 2020-09-08 Celgene Corporation Methods of treating vitiligo using PD-1 binding antibodies
US10894830B2 (en) 2015-11-03 2021-01-19 Janssen Biotech, Inc. Antibodies specifically binding PD-1, TIM-3 or PD-1 and TIM-3 and their uses
WO2021042019A1 (en) 2019-08-30 2021-03-04 Agenus Inc. Anti-cd96 antibodies and methods of use thereof
US10995141B2 (en) 2019-04-19 2021-05-04 ImmunoBrain Checkpoint, Inc. Modified anti-PD-L1 antibody and methods and uses for treating a neurodegenerative disease
WO2022254227A1 (en) 2021-06-04 2022-12-08 Kymab Limited Treatment of pd-l1 negative or low expressing cancer with anti-icos antibodies
US11564986B2 (en) 2015-07-16 2023-01-31 Onkosxcel Therapeutics, Llc Approach for treatment of cancer via immunomodulation by using talabostat
WO2024160721A1 (en) 2023-01-30 2024-08-08 Kymab Limited Antibodies

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3313883B1 (en) * 2015-06-23 2023-12-06 Memorial Sloan Kettering Cancer Center Novel pd-1 immune modulating agents
CN106397592A (zh) * 2015-07-31 2017-02-15 苏州康宁杰瑞生物科技有限公司 针对程序性死亡配体(pd-l1)的单域抗体及其衍生蛋白
CN107922503B (zh) 2016-03-04 2018-08-28 四川科伦博泰生物医药股份有限公司 一种pdl-1抗体、其药物组合物及其用途
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CN106939049B (zh) 2017-04-20 2019-10-01 苏州思坦维生物技术股份有限公司 拮抗抑制人pd-1抗原与其配体结合的单克隆抗体及其制备方法与应用
CN107446048B (zh) * 2017-09-13 2021-03-30 北京韩美药品有限公司 一种能够特异性地结合pd-1的抗体及其功能片段
CN108507992A (zh) * 2018-04-09 2018-09-07 苏州大学附属第医院 循环肿瘤细胞表面标志分子pd-l1的检测方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101213297A (zh) * 2005-05-09 2008-07-02 小野药品工业株式会社 程序性死亡-1(pd-1)的人单克隆抗体及单独使用或与其它免疫治疗剂联合使用抗pd-1抗体来治疗癌症的方法
CN102131828A (zh) * 2007-06-18 2011-07-20 奥根农股份公司 针对人程序性死亡受体pd-1的抗体
CN102892786A (zh) * 2010-03-11 2013-01-23 Ucb医药有限公司 Pd-1抗体

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE514713T1 (de) * 2002-12-23 2011-07-15 Wyeth Llc Antikörper gegen pd-1 und ihre verwendung
CN108997498A (zh) * 2008-12-09 2018-12-14 霍夫曼-拉罗奇有限公司 抗-pd-l1抗体及它们用于增强t细胞功能的用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101213297A (zh) * 2005-05-09 2008-07-02 小野药品工业株式会社 程序性死亡-1(pd-1)的人单克隆抗体及单独使用或与其它免疫治疗剂联合使用抗pd-1抗体来治疗癌症的方法
CN102131828A (zh) * 2007-06-18 2011-07-20 奥根农股份公司 针对人程序性死亡受体pd-1的抗体
CN102892786A (zh) * 2010-03-11 2013-01-23 Ucb医药有限公司 Pd-1抗体

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Publication number Priority date Publication date Assignee Title
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
US11365255B2 (en) 2013-12-12 2022-06-21 Suzhou Suncadia Biopharmaceuticals Co., Ltd. PD-1 antibody, antigen-binding fragment thereof, and medical application thereof
US10344090B2 (en) 2013-12-12 2019-07-09 Shanghai Hangrui Pharmaceutical Co., Ltd. PD-1 antibody, antigen-binding fragment thereof, and medical application thereof
US10618963B2 (en) 2014-03-12 2020-04-14 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
US11492405B2 (en) 2014-03-12 2022-11-08 Yeda Research And Development Co. Ltd Reducing systemic regulatory t cell levels or activity for treatment of disease and injury of the CNS
US11884727B2 (en) 2014-03-12 2024-01-30 Yeda Research And Development Co. Ltd. Reducing systemic regulatory T cell levels or activity for treatment of amyotrophic lateral sclerosis
US11643464B2 (en) 2014-03-12 2023-05-09 Yeda Research and Develpment & Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of a retinal degeneration disorder
US9512227B2 (en) 2014-03-12 2016-12-06 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of Alzheimer's disease
US9856318B2 (en) 2014-03-12 2018-01-02 Yeda Research And Development Co., Ltd. Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
US10981989B2 (en) 2014-03-12 2021-04-20 Yeda Research And Development Co. Ltd. Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
US9534052B2 (en) 2014-03-12 2017-01-03 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of Alzheimer's disease
US10961309B2 (en) 2014-03-12 2021-03-30 Yeda Research And Development Co. Ltd Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
US9982047B2 (en) 2014-03-12 2018-05-29 Yeda Research And Development Co. Ltd. Reducing systemic regulatory T cell levels or activity for treatment of Alzheimer's disease
US9982050B2 (en) 2014-03-12 2018-05-29 Yeda Research And Development Co. Ltd. Reducing systemic regulatory T cell levels or activity for treatment of alzheimer's disease
US9982049B2 (en) 2014-03-12 2018-05-29 Yeda Research And Development Co. Ltd. Reducing systemic regulatory T cell levels or activity for treatment of alzheimer's disease
US9982048B2 (en) 2014-03-12 2018-05-29 Yeda Research And Development Co. Ltd. Reducing systemic regulatory T cell levels or activity for treatment of Alzheimer's disease
US9982051B2 (en) 2014-03-12 2018-05-29 Yeda Research And Development Co. Ltd. Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS
US11884728B2 (en) 2014-03-12 2024-01-30 Yeda Research And Development Co. Ltd. Reducing systemic regulatory T cell levels or activity for treatment of amyotrophic lateral sclerosis
US9394365B1 (en) 2014-03-12 2016-07-19 Yeda Research And Development Co., Ltd Reducing systemic regulatory T cell levels or activity for treatment of alzheimer's disease
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CN107686520B (zh) * 2016-08-04 2023-01-03 信达生物制药(苏州)有限公司 抗pd-l1纳米抗体及其应用
CN107686520A (zh) * 2016-08-04 2018-02-13 信达生物制药(苏州)有限公司 抗pd‑l1纳米抗体及其应用
US10751414B2 (en) 2016-09-19 2020-08-25 Celgene Corporation Methods of treating psoriasis using PD-1 binding antibodies
US10766958B2 (en) 2016-09-19 2020-09-08 Celgene Corporation Methods of treating vitiligo using PD-1 binding antibodies
WO2018071500A1 (en) 2016-10-11 2018-04-19 Agenus Inc. Anti-lag-3 antibodies and methods of use thereof
US10882908B2 (en) 2016-10-11 2021-01-05 Agenus Inc. Anti-LAG-3 antibodies and methods of use thereof
US10844119B2 (en) 2016-10-11 2020-11-24 Agenus Inc. Anti-LAG-3 antibodies and methods of use thereof
US11993651B2 (en) 2016-10-11 2024-05-28 Agenus Inc. Anti-lag-3 antibodies and methods of use thereof
WO2018106862A1 (en) 2016-12-07 2018-06-14 Agenus Inc. Anti-ctla-4 antibodies and methods of use thereof
WO2018106864A1 (en) 2016-12-07 2018-06-14 Agenus Inc. Antibodies and methods of use thereof
EP4289484A2 (en) 2016-12-07 2023-12-13 Agenus Inc. Anti-ctla-4 antibodies and methods of use thereof
US11013802B2 (en) 2016-12-07 2021-05-25 Agenus Inc. Anti-CTLA-4 antibodies and methods of use thereof
US10912831B1 (en) 2016-12-07 2021-02-09 Agenus Inc. Anti-CTLA-4 antibodies and methods of use thereof
US11638755B2 (en) 2016-12-07 2023-05-02 Agenus Inc. Anti-CTLA-4 antibodies and methods of use thereof
WO2018132739A2 (en) 2017-01-13 2018-07-19 Agenus Inc. T cell receptors that bind to ny-eso-1 and methods of use thereof
WO2018191502A2 (en) 2017-04-13 2018-10-18 Agenus Inc. Anti-cd137 antibodies and methods of use thereof
EP4275698A2 (en) 2017-05-01 2023-11-15 Agenus Inc. Anti-tigit antibodies and methods of use thereof
WO2018204363A1 (en) 2017-05-01 2018-11-08 Agenus Inc. Anti-tigit antibodies and methods of use thereof
JP7235733B2 (ja) 2017-06-05 2023-03-08 ヤンセン バイオテツク，インコーポレーテツド Ｐｄ－１に特異的に結合する抗体、及び使用方法
TWI806873B (zh) * 2017-06-05 2023-07-01 美商健生生物科技公司 特異性結合pd-1之抗體及使用方法
US11746161B2 (en) 2017-06-05 2023-09-05 Janssen Biotech, Inc. Antibodies that specifically bind PD-1 and methods of use
US10995149B2 (en) 2017-06-05 2021-05-04 Janssen Biotech, Inc. Antibodies that specifically bind PD-1 and methods of use
JP2020522278A (ja) * 2017-06-05 2020-07-30 ヤンセン バイオテツク，インコーポレーテツド Ｐｄ−１に特異的に結合する抗体、及び使用方法
WO2018226580A3 (en) * 2017-06-05 2019-01-31 Janssen Biotech, Inc. ANTIBODIES SPECIFICALLY BINDING TO PD-1 AND THEIR METHODS OF USE
WO2019046856A1 (en) 2017-09-04 2019-03-07 Agenus Inc. T-CELL RECEPTORS THAT BIND TO SPECIFIC PHOSPHOPEPTIDES OF MIXED LINEAR LEUKEMIA (MLL) AND METHODS OF USE THEREOF
WO2019210055A2 (en) 2018-04-26 2019-10-31 Agenus Inc. Heat shock protein-binding peptide compositions and methods of use thereof
US11732046B2 (en) 2019-04-19 2023-08-22 ImmunoBrain Checkpoint, Inc. Modified anti-PD-L1 antibody and methods and uses for treating a neurodegenerative disease
US10995141B2 (en) 2019-04-19 2021-05-04 ImmunoBrain Checkpoint, Inc. Modified anti-PD-L1 antibody and methods and uses for treating a neurodegenerative disease
WO2021042019A1 (en) 2019-08-30 2021-03-04 Agenus Inc. Anti-cd96 antibodies and methods of use thereof
WO2022254227A1 (en) 2021-06-04 2022-12-08 Kymab Limited Treatment of pd-l1 negative or low expressing cancer with anti-icos antibodies
WO2024160721A1 (en) 2023-01-30 2024-08-08 Kymab Limited Antibodies

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