WO2017166804A1 - 抗pd-1的单克隆抗体 - Google Patents

抗pd-1的单克隆抗体 Download PDF

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WO2017166804A1
WO2017166804A1 PCT/CN2016/103814 CN2016103814W WO2017166804A1 WO 2017166804 A1 WO2017166804 A1 WO 2017166804A1 CN 2016103814 W CN2016103814 W CN 2016103814W WO 2017166804 A1 WO2017166804 A1 WO 2017166804A1
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antibody
antigen
amino acid
seq
acid sequence
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French (fr)
Inventor
夏瑜
李百勇
王忠民
张发明
席甘
黄莺
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Wuhan Hanxiong Biotech Inc
Akeso Pharmaceuticals Inc
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Wuhan Hanxiong Biotech Inc
Akeso Biopharma Inc
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Priority to ES16896573T priority Critical patent/ES2906650T3/es
Priority to EP16896573.9A priority patent/EP3441086B1/en
Priority to DK16896573.9T priority patent/DK3441086T3/da
Priority to JP2019502126A priority patent/JP6857232B2/ja
Publication of WO2017166804A1 publication Critical patent/WO2017166804A1/zh
Priority to US16/146,925 priority patent/US11104734B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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 [IG], 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • 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
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
    • 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
    • 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

Definitions

  • the present invention relates to the field of biomedicine, and more particularly, to a monoclonal antibody against PD-1.
  • PD-1 Programmed death factor 1
  • CD279 also known as CD279; gene name PDCD1; accession number NP_005009
  • PD-1 is essential for regulating the balance between stimulatory and inhibitory signals of the immune system and maintaining peripheral tolerance The role of cell surface receptors. It is an inhibitory member of the immunoglobulin superfamily that shares homology with CD28.
  • the structure of PD-1 is a monomeric type I transmembrane protein that is transformed by an immunoglobulin variable region-like extracellular domain and an immunoreceptor tyrosine inhibition motif (ITIM) and an immunoreceptor tyrosine.
  • ITIM immunoreceptor tyrosine inhibition motif
  • ITMS immunoreceptor tyrosine
  • PD-1 expression is inducible on T cells, B cells, natural killer (NK) cells, and monocytes, for example, by T cell receptor (TCR) or B cell receptor (BCR) signaling. After lymphocytes.
  • TCR T cell receptor
  • BCR B cell receptor
  • PD-1 has two known ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), which are members of the cell surface expression of the B7 family.
  • phosphatases such as SHP-1 and SHP-2 to its intracellular tyrosine motif, which then detaches the effector molecule activated by TCR or BCR signaling. Phosphoric acid.
  • PD-1 can transduce inhibitory signals into T cells and B cells only when they are simultaneously engaged with TCR or BCR.
  • the present invention is directed to solving at least some of the above technical problems or at least providing a useful commercial choice.
  • one object of the present invention is to propose a monoclonal antibody against PD-1.
  • the invention provides a monoclonal antibody against PD-1 or an antigen binding fragment thereof.
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having at least one of the following amino acid sequences: (1) the amino acid sequence set forth in SEQ ID NO: 1, and (2) SEQ ID NO An amino acid sequence represented by 3, (3) an amino acid sequence represented by SEQ ID NO: 5, and (4) an amino acid sequence having one or more amino acid conservative mutations compared to (1) to (3).
  • the antibody or antigen-binding fragment thereof may further comprise: a heavy chain variable region having at least one of the following amino acid sequences: (5) an amino acid sequence represented by SEQ ID NO: 2, (6) SEQ ID NO: an amino acid sequence represented by 4, (7) an amino acid sequence represented by SEQ ID NO: 6, and (8) an amino acid sequence having one or more amino acid conservative mutations compared to (5) to (7).
  • the antibody or antigen-binding fragment thereof has the amino acid sequence set forth in SEQ ID NO: (EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQTPEKGLDWVATISGGGRDTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCARQKGEAWFAYWGQGTLVTVSS) a heavy chain variable region having SEQ ID NO: amino acid sequence (DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTLNIHPMEENDTAMYFCQQSKEVPWTFGGGTKLEIK) light chain variable region shown in Fig.
  • the antibody or antigen binding fragment thereof having the SEQ ID NO: 3 the amino acid sequence shown in (EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQAPGKGLDWVATISGGGRDTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCARQKGEAWFAYWGQGTLVTVSS) a heavy chain variable region having SEQ ID NO: amino acid sequence (DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTLNINPMEENDTAMYFCQQSKEVPWTFGGGTKLEIK) a heavy chain 4 Variable zone.
  • the antibody or antigen binding fragment thereof having the SEQ ID NO: amino acid sequence (EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQAPGKGLDWVATISGGGRDTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQKGEAWFAYWGQGTLVTVSS) of FIG. 5 having a heavy chain variable region SEQ ID NO: amino acid sequence (DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWYQQKPGQPPKLLIYAASNKATGVPARFSGSGSGTDFTLNINPMEANDTAVYFCQQSKEVPWTFGGGTKLEIK) of the light chain shown in FIG. 6 Variable zone.
  • the antibody of the antibody or antigen-binding fragment thereof is a monoclonal antibody.
  • the antibody or antigen-binding fragment thereof can bind PD-1 specifically and efficiently, and can be used for: promoting activation and proliferation of T cells, regulating expression and secretion of cytokines, or stimulating resistance. Tumor cells produce a stronger immune response.
  • an antibody or antigen-binding fragment thereof is capable of specifically recognizing PD-1, and is capable of promoting activation and proliferation of T cells, and regulating the expression and secretion of cytokines, and is used to stimulate antitumor.
  • the cells produce a stronger immune response.
  • the invention provides an isolated polynucleotide which, according to an embodiment of the invention, encodes an antibody or antigen-binding fragment thereof as described above.
  • the polynucleotide described above has a nucleotide sequence as shown in one of the following.
  • the polynucleotide has a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1 (the nucleotide sequence of H1L1) or its complement: SEQ ID NO:7 (GAAGTGCAGCTGGTGCAGAGCGGAGGGGGACTGGTGCAGCCCGGCGGGTCTCTGAAGCTGAGTTGCGCCGCTTCAGGATTCACTTTTAGCTCCTACGGCATGTCCTGGGTGCGACAGACCCCCGAGAAAGGGCTGGACTGGGTCGCTACCATCTCTGGAGGCGGGAGAGACACATACTATCCTGATAGTGTCAAGGGCCGGTTCACAATTAGCAGAGACAACTCCAAAAACAATCTGTATCTGCAGATGAATAGCCTGAGGGCAGAAGATACCGCCCTGTACTATTGTGCCCGCCAGAAAGGAGAGGCTTGGTTTGCATACTGGGGACAGGACACTGGTCACCGTCAGCAGC).
  • the polynucleotide encoding SEQ ID NO: amino acid sequence is shown in the nucleotide sequence or its complementary sequence: SEQ ID NO: 10 (GATATTGTGCTGACTCAGAGCCCTGCTTCCCTGGCCGTGTCTCCAGGACAGCGAGCTACCATCACATGCAGAGCATCTGAGAGTGTGGACAACTACGGAATTAGTTTCATGAATTGGTTTCAGCAGAAGCCCGGCCAGCCCCCTAAACTGCTGATCTATGCCGCTAGCAACAAGGGCACCGGGGTGCCTGCTCGATTCTCAGGAAGCGGCTCCGGGACAGACTTTACTCTGAACATTCACCCAATGGAGGAAAATGATACAGCAATGTACTTCTGCCAGCAGAGCAAGG AGGTGCCCTGGACCTTTGGCGGGGGAACAAAGCTGGAAATCAAA).
  • an antibody or antigen-binding fragment thereof which specifically recognizes PD-1 according to an embodiment of the present invention can be efficiently synthesized by employing the polynucleotide according to the present invention.
  • antibodies or antigen-binding fragments thereof that specifically recognize PD-1 the features and advantages described above are equally applicable to the polynucleotide, and are not described herein.
  • the invention provides an expression vector comprising the polynucleotide described above.
  • the above expression vector further comprises:
  • a control element operably linked to the polynucleotide for controlling expression of the polynucleotide in a host cell.
  • the host cell described above may be a mammalian cell.
  • the mammalian cell can be a human renal epithelial cell line cell.
  • the human renal epithelial cell line cell is a 293T cell.
  • control element comprises at least one of the following:
  • CMV promoter early CMV enhancer
  • SV polyA terminator Promoters, enhancers, and terminators
  • the promoter is a CMV promoter
  • the enhancer is an early CMV enhancer
  • the terminator is an SV polyA terminator
  • the invention provides a recombinant cell comprising the expression vector described above.
  • the invention provides a method of preparing an antibody or antigen-binding fragment thereof as described above. According to an embodiment of the invention, comprising: culturing the recombinant cells described above.
  • an antibody or antigen-binding fragment thereof which specifically recognizes PD-1 according to an embodiment of the present invention can be efficiently synthesized by culturing the above recombinant cells.
  • antibodies or antigen-binding fragments thereof that specifically recognize PD-1 the features and advantages described above are equally applicable to the method and will not be described herein.
  • the invention also provides the use of a polynucleotide, expression vector, or recombinant cell as described above for the preparation of an antibody or antigen-binding fragment thereof which specifically binds to PD-1.
  • a polynucleotide, expression vector, or recombinant cell as described above for the preparation of an antibody or antigen-binding fragment thereof which specifically binds to PD-1.
  • the inventors have found that an antibody or an antigen-binding fragment thereof capable of specifically binding to PD-1 can be efficiently produced by using the above polynucleotide, expression vector, or recombinant cell.
  • the prepared antibody or antigen-binding fragment thereof is used to effectively block the binding of PD-1 to its receptor, thereby blocking the PD-1 receptor, such as the SHP1/2-related signaling pathway, thereby effectively inhibiting tumor growth.
  • the invention provides a hybridoma deposited with the China Center for Type Culture Collection (CCTCC).
  • CTCC China Center for Type Culture Collection
  • an antibody or antigen-binding fragment thereof which specifically recognizes PD-1 according to an embodiment of the present invention can be efficiently synthesized.
  • antibodies or antigen-binding fragments thereof that specifically recognize PD-1 the features and advantages described above are equally applicable to the hybridoma, and are not described herein.
  • the invention provides the use of the hybridomas described above for the preparation of monoclonal antibodies.
  • an antibody or antigen-binding fragment thereof which specifically recognizes PD-1 according to an embodiment of the present invention can be efficiently synthesized.
  • antibodies or antigen-binding fragments thereof that specifically recognize PD-1 the features and advantages described above are equally applicable to this versatile application and will not be described herein.
  • the present invention provides the use of the aforementioned antibody or antigen-binding fragment thereof, the foregoing polynucleotide, expression vector, recombinant cell or the hybridoma described above for the preparation of a medicament,
  • the medicament is used for promoting the activation and proliferation of T cells and regulating the expression and secretion of cytokines, which are used to stimulate anti-tumor cells to produce a stronger immune response.
  • the invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises an antibody or antigen-binding fragment thereof as described above, a preceding polynucleotide, a preceding expression vector, a preceding recombinant cell or a hybridoma as described above.
  • the invention proposes a method for identifying a drug capable of interacting with PD-1.
  • the method comprises:
  • the antibody or antigen-binding fragment thereof described above is contacted with the antigen, and the antibody is determined a first binding amount of the antigen or antigen-binding fragment thereof to the antigen, wherein the antigen is PD-1 or a fragment thereof;
  • the second amount is greater than the first amount is an indication that the candidate drug is capable of binding to PD-1.
  • a candidate drug that binds to PD-1 can be screened.
  • the invention proposes a drug combination.
  • the pharmaceutical combination comprises:
  • the immunopotentiating drug different from (1) comprises at least one selected from the group consisting of anti-CTLA-4 antibody, anti-CD40 antibody, Budesonide (budesonide), salicylate, and any
  • the salicylate comprises at least one of sulfasalazine, olsalazine, balsalazide, and mesalamine.
  • Combined blockade of PD-1 and CTLA-4 will be used in conjunction with standard tumor therapy.
  • the combined blockade of PD-1 and CTLA-4 will effectively bind to the tissue of chemotherapy.
  • Tests have shown that by combining anti-PD-1 antibodies with anti-CTLA-4 antibodies, the same effect can be achieved by reducing the dose of chemotherapeutic drugs.
  • An example of published literature Anti-PD-1 antibodies and anti-CTLA-4 antibodies were used in combination with decarbazine (docetaxel, an anticancer drug) to document melanoma.
  • Another example uses an anti-PD-1 antibody and an anti-CTLA-4 antibody with IL-2 (interleukin-2) to treat melanoma.
  • IL-2 interleukin-2
  • the principle of the above combination is that cell death is the result of cytotoxic effects in many chemotherapeutic drugs, and the level of pathways in which tumor cells express antigens is increased.
  • Another combination therapy is a combination of anti-PD-1 antibody and anti-CTLA-4 antibody to increase the synergistic effect of radiation therapy, surgery, hormone therapy, and the like. Each method increases the source of the antigen in the body.
  • Angiogenesis inhibitors can also be used in combination with anti-PD-1 antibodies and anti-CTLA-4 antibody blockers to inhibit vascular proliferation and thereby inhibit tumor cell growth. This may also be by increasing the expression of tumor cell antigens in the body.
  • FIG. 2 is a graph showing the results of competition Elisa for inhibition of PD-1 and PdL1 by H1L1, H2L2, and H3L3, in accordance with one embodiment of the present invention
  • Figure 3 is a graph showing the results of competition Elisa for inhibition of PD-1 and PdL2 by H2L2, in accordance with one embodiment of the present invention
  • FIG. 4 is a diagram showing the detection results of the dynamic characteristic parameters of the H1L1 according to an embodiment of the present invention.
  • FIG. 5 is a diagram showing detection results of H2L2 dynamic characteristic parameters according to an embodiment of the present invention.
  • FIG. 6 is a diagram showing detection results of dynamic characteristic parameters of H3L3 according to an embodiment of the present invention.
  • Figure 7 is a graph showing the levels of IL-2 and IFNgamma secreted by T cells stimulated by antibodies H1L1, H2L2 and H3L3 by blocking PD-1 protein function activation, in accordance with one embodiment of the present invention
  • Figure 8 is a fluorescent diagram of binding of antibodies H1L1, H2L2 and H3L3 to BB007 cells, in accordance with one embodiment of the present invention.
  • Figure 9 is a graph showing fluorescence intensity analysis of antibodies H1L1, H2L2 and H3L3 binding to BB007 cells, in accordance with one embodiment of the present invention.
  • a fusion protein PD-1-mIgGFc having the following amino acid sequence was prepared according to a molecular biological method:
  • the PD-1-mIgGFc fusion protein prepared by the above method was used as an antigen, and BALB/C mice were emulsified and immunized with Freund's adjuvant. After the mouse produces an immune response, the spleen cells are fused with mouse myeloma cells to form hybridoma cells, and the resulting hybridoma cells are cultured using a 96-well plate.
  • PD-1-hFc is used as an antigen coating enzyme label, and contains A 1% BSA PBS buffer was used to block the plate, and a good plate was coated for indirect ELISA to screen for hybridoma cells secreting a new antibody that specifically binds to PD-1.
  • hybridoma cells obtained by indirect ELISA screening hybridoma cells capable of secreting monoclonal antibodies that compete with PD-L1 for binding to PD-1 were screened by competition ELISA.
  • Ligand 2 ⁇ g/ml of PDL1-hIgG1 Fc solution, 50 ⁇ l/well, and incubated at 37 ° C for 1 hour;
  • a PD-1 18A10 hybridoma cell line was selected, and the secreted monoclonal antibody was named 18A10.
  • subcloning is required to screen for a monoclonal stable cell line secreting a PD-1 binding to PD-L1 for specific binding to the PD-1 antibody.
  • the specific practices are as follows:
  • Live cells were counted for cells that need to be subcloned, and inoculated into 96-well cell culture plates by dilution with IMDM medium containing 15% fetal bovine serum according to the number of viable cells. The theoretically inoculated cell density was 1 cell/well. . After the cells were grown into monoclonal cell clusters, they were screened by ELISA. After multiple subcloning and screening, 18A10 stable monoclonal cell lines were obtained.
  • the 18A10 stable cell line was cultured with 10% low IgG fetal bovine serum, and after 7 to 10 days of culture, the cell culture supernatant was collected for antibody purification to obtain 18A10 antibody.
  • the mRNA was extracted from the 18A10 hybridoma cell line according to the instructions of the Tiangen Cultured Cell Bacterial Total RNA Extraction Kit (Cat. No. DP430).
  • M13 universal primers were used for PCR identification, and positive clones were selected for sequencing.
  • variable region amino acid sequence of the murine 18A10 antibody was aligned with a human variable region gene sequence.
  • Three humanized antibodies were designed by selectively mutating a part of the murine amino acid sequence to the humanized amino acid sequence.
  • the designed humanized antibodies were named H1L2, H2L2 and H3L3 according to the degree of humanization.
  • the heavy chain variable region sequence of the humanized antibody H1L1 is:
  • amino acid sequence of the light chain variable region of the humanized antibody H1L1 is:
  • the heavy chain variable region sequence of the humanized antibody H2L2 is:
  • amino acid sequence of the light chain variable region of the humanized antibody H2L2 is:
  • the heavy chain variable region sequence of the humanized antibody H3L3 is:
  • amino acid sequence of the light chain variable region of the humanized antibody H3L3 is:
  • the nucleic acid sequence encoding the humanized antibodies H1LI, H2L2, H3L3 is fully gene-generated and constructed into an expression vector.
  • the expression vector DNA was extracted and transfected into mammalian cell 293 cells. After transfection, the antibody is expressed in mammalian cells and secreted outside the cell.
  • the humanized antibody H1L2, H2L2, and H3L3 proteins can be obtained by purifying the expressed antibody by an antibody A affinity chromatography column.
  • the humanized design was carried out, and then these humanized antibodies were produced by recombinant production for a series of comparative experiments including, but not limited to, ELISA binding experiments and competition ELISA experiments.
  • the above three antibodies H1L1, H2L2 and H3L3 have strong affinity for PD-1.
  • Ligand Add PDL1-mIgG2aFc solution 0.3 ⁇ g/ml, 50 ⁇ l/well, and incubate at 37 ° C for 1 hour;
  • Ligand Add PDL2-his tag solution 1.0 ⁇ g/ml, 50 ⁇ l/well, and incubate at 37 ° C for 1 hour;
  • the biotin-labeled antigen PD-1 was immobilized on the surface of the SA sensor, and after equilibration in PBST, it was bound to the antibody H1L1, and H1L1 was diluted three times with PBST at concentrations of 200, 66.67, 22.22, 7.41, 2.47, 0.82, 0.27, 0 nM. Dissociated in PBST.
  • the detection methods of H2L2 and H3L3 are the same as those of H1L1.
  • the kinetic parameters of H1L1, H2L2 and H3L3 are shown in Table 4. The results are shown in Figure 4, Figure 5 and Figure 6.
  • the mixed lymphocyte reaction (MLR) assay was used to detect the secretion of IL-2 and IFNgamma by T lymphocytes by antibodies H1L1, H2L2 and H3L3.
  • MLR experiments were performed by mixing human-derived T cells (TC) and dendritic cells (DC), and using DC cell antibody-presenting ability to stimulate T cells to secrete IL-2 and IFNgamma.
  • TC human-derived T cells
  • DC dendritic cells
  • TC and DC were mixed in a 96-well plate, and TC 1 ⁇ 105 and DC 1 ⁇ 104 were added per well, and antibody concentrations of 1 nM, 10 nM, and 100 nM were set.
  • the IL-2 content of the supernatant was quantitatively detected by the IL-2 test kit, and the IFNgamma content was quantitatively detected by the IFNgamm test kit.
  • Fig. 7 The secretion levels of IL-2 and IFNgamma secreted by T cells by antibodies H1L1, H2L2 and H3L34 are shown in Fig. 7. As can be seen from Figure 7, antibodies H1L1, H2L2 and H3L3 were effective in stimulating T cells to secrete IL-2 and IFNgamma in a dose-dependent manner.
  • BB007 cells The binding activity of the antibodies H1L1, H2L2 and H3L3 to the surface antigen of the cells stably expressing PD-1 (designated BB007 cells) in Example N was determined by a flow method.
  • BB007 cells were obtained by routine trypsin digestion, washed once with PBS, and divided into several tubes, the number of cells per tube was 2*10 5 ; the concentration was 20 nM, 10 nM, 5 nM, 1 nM, 0.1 nM with PBS (1% BSA).
  • H1L1, H2L2 and H3L3 antibody dilutions 100 ⁇ l of each concentration; incubate with cells for 1 hour on ice; wash once with PBS; add 100 ⁇ l FITC-Goat-Anti-Human IgG (1:500) per tube to ice Incubate for 1 hour; fluorescence signal was detected on the flow cytometer using a FITC channel by adding 300 ⁇ l of PBS per tube.
  • the EC50 of the binding efficiency of H1L1, H2L2 and H3L3 was 3.38nM, 1.59nM, 1.68nM (Fig. 9).

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Abstract

提出了抗PD-1的单克隆抗体或其抗原结合片段，包括具有下列至少之一氨基酸序列的重链可变区：(1)SEQ ID NO：1所示的氨基酸序列，(2)SEQ ID NO：3所示的氨基酸序列，(3)SEQ ID NO：5所示的氨基酸序列，(4)与(1)～(3)相比具有一个以上氨基酸保守突变的氨基酸序列。

Description

抗PD-1的单克隆抗体 技术领域
本发明涉及生物医药领域，更具体的，本发明涉及一种抗PD-1的单克隆抗体。
背景技术
程序化死亡因子1(PD-1)，也称为CD279；基因名称PDCD1；登录号NP_005009为在调节免疫系统的刺激性与抑制性信号之间的平衡和维持外周耐受性中具有至关重要的作用的细胞表面受体。其为与CD28具有同源性的免疫球蛋白超家族的抑制性成员。PD-1的结构为单体I型跨膜蛋白，其由一个免疫球蛋白可变区样细胞外结构域以及含有免疫受体酪氨酸抑制基序(ITIM)和免疫受体酪氨酸转换基序(ITSM)的细胞质结构域组成。PD-1的表达在T细胞、B细胞、天然杀伤(NK)细胞和单核细胞上是可诱导的，例如在通过T细胞受体(TCR)或B细胞受体(BCR)信号转导活化淋巴细胞后。PD-1具有两个已知的配体，PD-L1(B7-H1,CD274)和PD-L2(B7-DC,CD273)，它们为B7家族的细胞表面表达的成员。当衔接配体时，PD-1将磷酸酶例如SHP-1和SHP-2招募至其细胞内酪氨酸基序，所述基序随后使被TCR或BCR信号转导激活的效应分子脱去磷酸。由此，仅当其同时与TCR或BCR衔接时，PD-1才可将抑制性信号转导入T细胞和B细胞。
然而，目前特异性识别PD-1的抗体仍有待改进。
发明内容
本发明旨在至少在一定程度上解决上述技术问题之一或至少提供一种有用的商业选择。为此，本发明的一个目的在于提出一种抗PD-1的单克隆抗体。
在本发明的第一方面，本发明提出了一种抗PD-1的单克隆抗体或其抗原结合片段。根据本发明的实施例，该抗体或其抗原结合片段包括：具有下列至少之一氨基酸序列的重链可变区：(1)SEQ ID NO：1所示的氨基酸序列，(2)SEQ ID NO：3所示的氨基酸序列，(3)SEQ ID NO：5所示的氨基酸序列，(4)与(1)～(3)相比具有一个以上氨基酸保守突变的氨基酸序列。
根据本发明的实施例，该抗体或其抗原结合片段可以进一步包括：具有下列至少之一氨基酸序列的重链可变区：(5)SEQ ID NO：2所示的氨基酸序列，(6)SEQ ID NO：4所示的氨基酸序列，(7)SEQ ID NO：6所示的氨基酸序列，(8)与(5)～(7)相比具有一个以上氨基酸保守突变的氨基酸序列。
根据本发明的实施例，该抗体或其抗原结合片段具有SEQ ID NO：1所示氨基酸序列 (EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQTPEKGLDWVATISGGGRDTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCARQKGEAWFAYWGQGTLVTVSS)的重链可变区和具有SEQ ID NO：2所示氨基酸序列(DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTLNIHPMEENDTAMYFCQQSKEVPWTFGGGTKLEIK)的轻链可变区。
根据本发明的实施例，该抗体或其抗原结合片段具有SEQ ID NO：3所示氨基酸序列(EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQAPGKGLDWVATISGGGRDTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCARQKGEAWFAYWGQGTLVTVSS)的重链可变区和具有SEQ ID NO：4所示氨基酸序列(DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTLNINPMEENDTAMYFCQQSKEVPWTFGGGTKLEIK)的轻链可变区。
根据本发明的实施例，该抗体或其抗原结合片段具有SEQ ID NO：5所示氨基酸序列(EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQAPGKGLDWVATISGGGRDTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQKGEAWFAYWGQGTLVTVSS)的重链可变区和具有SEQ ID NO：6所示氨基酸序列(DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWYQQKPGQPPKLLIYAASNKATGVPARFSGSGSGTDFTLNINPMEANDTAVYFCQQSKEVPWTFGGGTKLEIK)的轻链可变区。
根据本发明的实施例，该抗体或其抗原结合片段的抗体为单克隆抗体。
根据本发明的实施例，所述抗体或其抗原结合片段可以特异性的高效的结合PD-1，并且能够用于：促进T细胞的活化和增殖，调节细胞因子的表达和分泌，或者刺激抗肿瘤细胞产生更强的免疫应答。
发明人意外发现，根据本发明实施例的抗体或其抗原结合片段，能够特异性识别PD-1，并且能够促进T细胞的活化和增殖，并调节细胞因子的表达和分泌，使用来刺激抗肿瘤细胞产生更强的免疫应答。
在本发明的第二方面，本发明提出了一种分离的多核苷酸，根据本发明的实施例，该多核苷酸编码前面所述的抗体或其抗原结合片段。
根据本发明的实施例，前面所描述的多核苷酸，具有下列之一所示的核苷酸序列。
根据本发明的具体实施例，该多核苷酸具有编码SEQ ID NO：1所示的氨基酸序列(H1L1的核苷酸序列)的核苷酸序列或其互补序列：SEQ ID NO：7 (GAAGTGCAGCTGGTGCAGAGCGGAGGGGGACTGGTGCAGCCCGGCGGGTCTCTGAAGCTGAGTTGCGCCGCTTCAGGATTCACTTTTAGCTCCTACGGCATGTCCTGGGTGCGACAGACCCCCGAGAAAGGGCTGGACTGGGTCGCTACCATCTCTGGAGGCGGGAGAGACACATACTATCCTGATAGTGTCAAGGGCCGGTTCACAATTAGCAGAGACAACTCCAAAAACAATCTGTATCTGCAGATGAATAGCCTGAGGGCAGAAGATACCGCCCTGTACTATTGTGCCCGCCAGAAAGGAGAGGCTTGGTTTGCATACTGGGGACAGGGGACACTGGTCACCGTCAGCAGC)。
根据本发明的具体实施例，该多核苷酸具有编码SEQ ID NO：3所示的氨基酸序列(H2L2的核苷酸序列)的核苷酸序列或其互补序列：SEQ ID NO：8(GAGGTGCAGCTGGTGCAGTCTGGCGGCGGACTGGTGCAGCCCGGCGGGTCACTGAAGCTGAGCTGCGCCGCTTCCGGCTTCACCTTTAGCTCCTACGGAATGTCCTGGGTGCGACAGGCACCCGGGAAGGGGCTGGACTGGGTCGCTACTATCTCAGGAGGCGGGAGAGACACCTACTATCCTGATAGCGTCAAGGGCCGGTTCACAATTAGCCGGGACAACAGCAAGAACAATCTGTACCTGCAGATGAACAGCCTGAGGGCTGAGGATACTGCACTGTACTATTGTGCCCGCCAGAAGGGCGAAGCATGGTTTGCCTATTGGGGCCAGGGAACCCTGGTGACAGTCTCTAGT)。
根据本发明的具体实施例，该多核苷酸具有编码SEQ ID NO：5所示的氨基酸序列(H3L3的核苷酸序列)的核苷酸序列或其互补序列：SEQ ID NO：9(GAGGTGCAGCTGGTGCAGAGTGGAGGCGGGCTGGTGCAGCCCGGCGGGTCACTGAAGCTGAGCTGCGCCGCTTCCGGCTTCACCTTTAGCTCCTACGGAATGTCCTGGGTGCGACAGGCACCCGGGAAGGGGCTGGACTGGGTCGCTACTATCTCAGGAGGCGGGAGAGACACCTACTATCCTGATAGCGTGAAGGGCCGGTTCACAATTAGCCGGGACAACAGCAAGAACACTCTGTACCTGCAGATGAACTCTCTGAGGGCTGAGGATACAGCAGTCTACTATTGTGCCCGCCAGAAGGGCGAAGCATGGTTTGCCTATTGGGGCCAGGGAACCCTGGTGACAGTCTCTAGT)。
根据本发明的具体实施例，该多核苷酸具有编码SEQ ID NO：2所示的氨基酸序列(H1L1的核苷酸序列)的核苷酸序列或其互补序列：SEQ ID NO：10(GATATTGTGCTGACTCAGAGCCCTGCTTCCCTGGCCGTGTCTCCAGGACAGCGAGCTACCATCACATGCAGAGCATCTGAGAGTGTGGACAACTACGGAATTAGTTTCATGAATTGGTTTCAGCAGAAGCCCGGCCAGCCCCCTAAACTGCTGATCTATGCCGCTAGCAACAAGGGCACCGGGGTGCCTGCTCGATTCTCAGGAAGCGGCTCCGGGACAGACTTTACTCTGAACATTCACCCAATGGAGGAAAATGATACAGCAATGTACTTCTGCCAGCAGAGCAAGG AGGTGCCCTGGACCTTTGGCGGGGGAACAAAGCTGGAAATCAAA)。
根据本发明的具体实施例，该多核苷酸具有编码SEQ ID NO：4所示的氨基酸序列(H2L2的核苷酸序列)的核苷酸序列或其互补序列：SEQ ID NO：11(GATATTGTGCTGACTCAGAGCCCTGCTTCCCTGGCCGTGTCTCCAGGACAGCGAGCTACCATCACATGCAGAGCATCTGAGAGTGTGGACAACTACGGAATTAGTTTCATGAATTGGTTTCAGCAGAAGCCCGGCCAGCCCCCTAAACTGCTGATCTATGCCGCTAGCAACAAGGGCACCGGGGTGCCTGCTCGATTCTCAGGAAGCGGCTCCGGGACAGACTTTACTCTGAACATTAACCCAATGGAGGAAAATGATACAGCAATGTACTTCTGCCAGCAGAGCAAGGAGGTGCCCTGGACCTTTGGCGGGGGAACAAAGCTGGAAATCAAA)。
根据本发明的具体实施例，该多核苷酸具有编码SEQ ID NO：6所示的氨基酸序列(H3L3的核苷酸序列)的核苷酸序列或其互补序列：SEQ ID NO：12(GACATCGTCCTGACTCAGAGCCCTGCTTCCCTGGCCGTGAGCCCAGGCCAGCGAGCAACCATCACATGCAGAGCCTCAGAGAGCGTGGACAACTACGGCATTAGCTTCATGAATTGGTATCAGCAGAAGCCCGGGCAGCCTCCCAAGCTGCTGATCTACGCCGCTTCCAACAAGGCCACTGGGGTGCCTGCTCGATTCTCCGGCTCTGGGAGTGGAACAGACTTTACTCTGAACATTAATCCAATGGAAGCTAATGATACAGCAGTGTATTTCTGCCAGCAGAGCAAGGAGGTCCCATGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG)。
发明人发现，通过采用根据本发明的多核苷酸，能够有效地合成根据本发明实施例的特异性识别PD-1的抗体或其抗原结合片段。关于特异性识别PD-1的抗体或其抗原结合片段，前面所描述的特征和优点同样适用该多核苷酸，在此不再赘述。
在本发明的第三方面，本发明提出了一种表达载体，该表达载体包含前面所述的多核苷酸。
根据本发明的具体实施例，上述表达载体进一步包括：
控制元件，所述控制元件与所述多核苷酸可操纵地连接，用于控制所述多核苷酸在宿主细胞中的表达。
根据本发明的具体示例，上述宿主细胞可以为哺乳动物细胞。所述哺乳动物细胞可以为人肾上皮细胞系细胞。
根据本发明的具体示例，上述人肾上皮细胞系细胞为293T细胞。
根据本发明的具体实施例，所述控制元件包括下列至少之一：
CMV启动子，早期CMV增强子，SV polyA终止子。启动子、增强子和终止子，
任选地，所述启动子为CMV启动子，所述增强子为早期CMV增强子，所述终止子为SV polyA终止子。
在本发明的第四方面，本发明提出了一种重组细胞，该重组细胞包含前面所述的表达载体。
在本发明的第五方面，本发明提出了一种制备前面所述的抗体或其抗原结合片段的方法。根据本发明的实施例，包括：培养前面所述的重组细胞。
发明人发现，通过采用根据本发明的方法，能够通过培养上述重组细胞有效地合成根据本发明实施例的特异性识别PD-1的抗体或其抗原结合片段。关于特异性识别PD-1的抗体或其抗原结合片段，前面所描述的特征和优点同样适用该方法，在此不再赘述。
在本发明的第六方面，本发明还提出了前面所述的多核苷酸、表达载体、或重组细胞在制备抗体或其抗原结合片段的用途，所述抗体与PD-1特异性结合。由此，发明人发现，利用上述的多核苷酸、表达载体、或重组细胞可以有效地制备得到能够与PD-1特异性结合的抗体或其抗原结合片段。进而利用制备得到的抗体或其抗原结合片段有效地阻断PD-1与其受体的结合，进而阻断PD-1受体例如SHP1/2相关信号通路，从而可以有效地抑制肿瘤的增长。
在本发明的第七方面，本发明提出了一种杂交瘤，其保藏于中国典型培养物保藏中心(CCTCC)。
发明人发现，通过采用根据本发明的杂交瘤，能够有效地合成根据本发明实施例的特异性识别PD-1的抗体或其抗原结合片段。关于特异性识别PD-1的抗体或其抗原结合片段，前面所描述的特征和优点同样适用该杂交瘤，在此不再赘述。
在本发明的第八方面，本发明提出了前面所述的杂交瘤在制备单克隆抗体中的用途。
明人发现，通过采用根据本发明的杂交瘤，能够有效地合成根据本发明实施例的特异性识别PD-1的抗体或其抗原结合片段。关于特异性识别PD-1的抗体或其抗原结合片段，前面所描述的特征和优点同样适用该多用途，在此不再赘述。
在本发明的第就九方面，本发明提出了前面所述的抗体或其抗原结合片段、前面的多核苷酸、表达载体、重组细胞或前面所述的杂交瘤在制备药物中的用途，所述药物用于所述药物用于促进T细胞的活化和增殖，并调节细胞因子的表达和分泌，使用来刺激抗肿瘤细胞产生更强的免疫应答。
在本发明的第十方面，本发明提出了一种药物组合物。根据本发明的实施例，该药物组合物包含前面所述的抗体或其抗原结合片段、前面的多核苷酸、前面的表达载体、前面的重组细胞或前面所述的杂交瘤。
在本发明的第十一方面，本发明提出了一种用于鉴定能够与PD-1的药物的方法。根据本发明的实施例，该方法包括：
在候选药物存在时，使前面所述的抗体或其抗原结合片段与抗原接触，并且确定所述抗 体或其抗原结合片段与所述抗原的第一结合量，其中，所述抗原为PD-1或其片段；以及
在不存在所述候选药物时，使前面所述的抗体或其抗原结合片段与抗原接触，并且确定所述抗体或其抗原结合片段与所述抗原的第二结合量，其中，所述抗原为PD-1或其片段，
其中，所述第二量大于所述第一量是所述候选药物能够与PD-1结合的指示。
由此，通过采用该方法，能够筛选与PD-1结合的候选药物。
在本发明的第十二方面，本发明提出了一种药物联合。根据本发明的实施例，该药物联合包括：
(1)前面所述的抗体或其抗原结合片段、前面的多核苷酸、前面的表达载体、前面的重组细胞或前面所述的杂交瘤；以及
2)与(1)不同的免疫增强药物。
根据本发明的实施例，所述与(1)不同的免疫增强药物包括选自下列的至少之一：抗CTLA-4抗体，抗CD40抗体，Budesonide(布地奈德)，水杨酸盐，任选地所述水杨酸盐类包括sulfasalazine、olsalazine、balsalazide以及mesalamine的至少之一。
PD-1和CTLA-4联合阻断将会与标准肿瘤治疗联合使用。例如PD-1和CTLA-4联合阻断会与化疗的组织有效的结合。试验表明，通过与抗PD-1抗体和抗CTLA-4抗体联合使用，降低化疗药物的剂量即可达到相同的疗效。文献发布的实例：用抗PD-1抗体和抗CTLA-4抗体与decarbazine(多西紫杉醇，一种抗癌药)联合使用来资料黑色素瘤。另一个实例用抗PD-1抗体和抗CTLA-4抗体与IL-2(白细胞介素-2)来治疗黑色素瘤。以上联合使用的原理为：细胞死亡是很多化疗药物中细胞毒素作用的结果，会使肿瘤细胞表达抗原的途径的水平增加。另外一个联合治疗是用抗PD-1抗体和抗CTLA-4抗体联合阻断来增加放射治疗、手术治疗、激素治疗等的协同作用。每一种方法都是增加了抗原在机体中的来源。血管增生抑制剂也可和抗PD-1抗体和抗CTLA-4抗体阻断联合使用，抑制血管增生进而抑制肿瘤细胞的增长。这可能也是通过增加机体内肿瘤细胞抗原的表达。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1为根据本发明一个实施例，H1L1,H2L2,和H3L3对PD-1结合Elisa的结果图；
图2为根据本发明一个实施例，H1L1,H2L2,和H3L3对PD-1和PdL1抑制的竞争Elisa的结果图；
图3为根据本发明一个实施例，H2L2对PD-1和PdL2抑制的竞争Elisa的结果图；
图4为根据本发明一个实施例，H1L1动力学特征参数检测结果图；
图5为根据本发明一个实施例，H2L2动力学特征参数检测结果图；
图6为根据本发明一个实施例，H3L3动力学特征参数检测结果图；
图7为根据本发明一个实施例，抗体H1L1、H2L2和H3L3通过阻断PD-1蛋白功能活化刺激T细胞分泌IL-2和IFNgamma水平图；
图8为根据本发明一个实施例，抗体H1L1、H2L2和H3L3与BB007细胞结合荧光图；
图9为根据本发明一个实施例，抗体H1L1、H2L2和H3L3结合BB007细胞后荧光强度分析图。
具体实施方式
下面详细描述本发明的实施例，需要说明的是下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。另外，如果没有明确说明，在下面的实施例中所采用的所有试剂均为市场上可以购得的，或者可以按照文本或已知的方法合成的，对于没有列出的反应条件，也均为本领域技术人员容易获得的。
实施例1PD-1 4G10杂交瘤细胞株的建立
根据分子生物学方法制备具有下列氨基酸序列的融合蛋白PD-1-mIgGFc：
Figure PCTCN2016103814-appb-000001
1.小鼠免疫及细胞融合
用上述的方法制备的PD-1-mIgGFc融合蛋白作为抗原，用弗氏佐剂进行乳化免疫BALB/C小鼠。小鼠产生免疫应答后取其脾细胞与小鼠骨髓瘤细胞进行细胞融合成杂交瘤细胞，形成的杂交瘤细胞使用96孔板进行培养。
2.间接ELISA
针对各杂交瘤细胞株细胞分泌的抗体，用PD-1-hFc作为抗原包被酶标板，用含 1％BSA的PBS缓冲液封闭酶标板，包被好的酶标板用于进行间接ELISA法筛选分泌与PD-1特异性结合的新的抗体的杂交瘤细胞。
具体步骤如下：
1)包被抗原：PD-1-hFc筛选原1μg/ml，50μl/孔，4℃包被过夜；
2)1％BSA(PBS稀释)37℃封闭2小时，1×PBST(Tween-20,1％)洗涤3次，轻轻拍干；
3)一抗：1μg/ml，1:3梯度稀释7个梯度浓度，空白对照组为PBS，37℃孵育1小时；
4)二抗：PBST洗涤3次，轻轻拍干，每孔50μl加入1:5000稀释的HRP酶标羊抗鼠IgG(H+L)二抗，37℃孵育1小时；
5)显色：PBST洗涤3次，轻轻拍干；每孔50μl加入TMB显色剂，室温反应5～10min；
6)显色终止：50μl/孔加入2M H2SO4溶液终止显色反应；
7)读数：在酶标仪上，用吸光度450nm检测各孔的吸光值。
3.竞争ELISA
对间接ELISA筛选得到的杂交瘤细胞，通过竞争ELISA法筛选出能够分泌与PD-L1竞争结合PD-1的单克隆抗体的杂交瘤细胞。
具体步骤如下：
1)包被抗原：在96孔酶标板上包被PD-1-mIgGFc抗原0.5μg/ml，50μl/孔，4℃包被过夜；
2)PBST洗板3次，轻轻拍干，加入1％BSA(PBS稀释)37℃封闭2小时，1×PBST(Tween-20,1％)洗涤3次；
3)一抗：3μg/ml，1:3梯度稀释7个梯度浓度，空白对照组为PBS，50μl/孔加入到包被好的酶标板上，室温孵育10min；
4)配体：加入PDL1-hIgG1Fc溶液2μg/ml，50μl/孔，37℃孵育1小时；
5)二抗：PBST洗涤3次，轻轻拍干；每孔50μl加入1:5000稀释的HRP酶标羊抗人IgG(H+L)二抗，37℃孵育1小时；
5)显色：PBST洗涤3次，轻轻拍干；每孔50μl加入TMB显色剂，室温反应5－10min；
6)终止：50μl/孔加入2M H2SO4终止显色反应；
7)读数：在酶标仪上，用吸光度450nm检测各孔的吸光值。
根据结果选出PD-1 18A10杂交瘤细胞株，其分泌的单克隆抗体命名为18A10。
4.亚克隆获得稳定细胞株
对于筛选得到的细胞株18A10，需要进行亚克隆，从而筛选得到分泌与PD-L1竞争特异性结合PD-1抗体的单克隆稳定细胞株。具体做法如下：
对需要进亚克隆的细胞用进行活细胞计数，根据活细胞数量用含15％胎牛血清的IMDM培养基进行稀释接种到96孔细胞培养板中培养，理论接种的细胞密度为1细胞/孔。待细胞长成单克隆细胞团后用ELISA方法进行筛选，经多次亚克隆及筛选后，得到18A10稳定单克隆细胞株。
5.18A10抗体生产
得到18A10稳定细胞株后，用含10％的低IgG胎牛血清对18A10稳定细胞株进行培养，在培养7～10天后收集细胞培养上清进行抗体纯化得到18A10抗体。
实施例2KF007杂交瘤细胞株cDNA序列的获得
1.按照Tiangen培养细胞细菌总RNA提取试剂盒(货号DP430)说明书从18A10杂交瘤细胞株中提取mRNA。
2.按照Invitrogen
Figure PCTCN2016103814-appb-000002
III First-Strand Synthesis System for RT-PCR试剂盒说明书合成第一条链，并做PCR扩增。
3.按照pEASY-T1Cloning Kit(Transgen CT101)试剂盒说明书进行TA克隆。
4.使用M13通用引物进行PCR鉴定，选取阳性克隆测序。
5.比对测序结果，选取正确序列。
实施例3抗体18A10人源化设计
为构建人源化抗体，将鼠源18A10抗体的可变区氨基酸序列与人的可变区基因序列进行比对。通过选择性突变部分鼠源性氨基酸序列至人源化氨基酸序列，分别设计出三种人源化抗体，设计的人源化抗体根据人源化程度的差异分别命名为H1L2、H2L2和H3L3。
人源化抗体H1L1的重链可变区序列为：
Figure PCTCN2016103814-appb-000003
人源化抗体H1L1的轻链可变区氨基酸序列为：
Figure PCTCN2016103814-appb-000004
人源化抗体H2L2的重链可变区序列为：
Figure PCTCN2016103814-appb-000005
人源化抗体H2L2的轻链可变区氨基酸序列为：
Figure PCTCN2016103814-appb-000006
人源化抗体H3L3的重链可变区序列为：
Figure PCTCN2016103814-appb-000007
人源化抗体H3L3的轻链可变区氨基酸序列为：
Figure PCTCN2016103814-appb-000008
实施例4人源化抗体H1L1、H2L2和H3L3蛋白表达
全基因合成编码人源化抗体H1LI、H2L2、H3L3的核酸序列，并构建到表达载体。提取表达载体DNA，转染哺乳动物细胞293细胞。细胞转染后，抗体在哺乳动物细胞内表达，并分泌到细胞外。通过抗体A亲和层析柱，纯化表达的抗体，即可获得人源化抗体H1L2、H2L2、H3L3蛋白。
实施例5 18A10重组人源化抗体ELISA实验
获得18A10DNA序列后，对其进行人源化设计，然后通过重组生产出这些人源化抗体进行一系列对比实验，当中包括但不限于ELISA结合实验和竞争ELISA实验。
1.18A10H1L1、18A10H2L2、18A10H3L3ELISA结合实验
具体步骤如下：
1)包被抗原：PD-1-mFc抗原0.5μg/ml，50μl/孔，4℃包被过夜；
2)1％BSA(PBS稀释)37℃封闭2小时，1×PBST(Tween-20,1％)洗涤3次，轻轻拍干；
3)一抗：1μg/ml，1:3梯度稀释7个梯度浓度，空白对照组为PBS，37℃孵育1小时；
4)二抗：PBST洗涤3次，轻轻拍干，每孔50μl加入1:5000稀释的HRP酶标羊抗人IgG(H+L)二抗，37℃孵育1小时；
5)显色：PBST洗涤3次，轻轻拍干；每孔50μl加入TMB显色剂，室温反应5～10min；
6)显色终止：50μl/孔加入2M H2SO4溶液终止显色反应；
7)读数：在酶标仪上，用吸光度450nm检测各孔的吸光值。
结果见图1，可计算出H1L1、H2L2和H3L3对PD-1的EC50值分别为0.156nM、0.111nM和0.144nM。
由此图1可知，上述三个抗体H1L1、H2L2和H3L3对PD-1具有较强的亲和力。
表1
Figure PCTCN2016103814-appb-000009
2.18A10H1L1、18A10H2L2、18A10H3L3与PDL1竞争ELISA实验
具体步骤如下：
1)包被抗原：在96孔酶标板上包被PD-1-mIgGFc抗原0.5μg/ml，50μl/孔，4℃包被过夜；
2)PBST洗板3次，轻轻拍干，加入1％BSA(PBS稀释)37℃封闭2小时，1×PBST(Tween-20,1％)洗涤3次；
3)一抗：3μg/ml，1:3梯度稀释7个梯度浓度，空白对照组为PBS，50μl/孔加入到包被好的酶标板上，室温孵育10min；
4)配体：加入PDL1-mIgG2aFc溶液0.3μg/ml，50μl/孔，37℃孵育1小时；
5)二抗：PBST洗涤3次，轻轻拍干；每孔50μl加入1:5000稀释的HRP酶标羊抗鼠IgG(H+L)二抗，37℃孵育1小时；
5)显色：PBST洗涤3次，轻轻拍干；每孔50μl加入TMB显色剂，室温反应5－10min；
6)终止：50μl/孔加入2M H2SO4终止显色反应；
7)读数：在酶标仪上，用吸光度450nm检测各孔的吸光值。
结果见图2，并且H1L1、H2L2和H3L3抑制Pd-1与PdL1的EC50值分别为0.992nM、0.838nM和1.194nM。
由此图2可知，上述三个抗体H1L1、H2L2和H3L3能够有效地抑制Pd-1与PdL1的结合。
表2
Figure PCTCN2016103814-appb-000010
3.18A10H2L2与PDL2竞争ELISA实验
具体步骤如下：
1)包被抗原：在96孔酶标板上包被PD-1-hIgGFc抗原1.0μg/ml，100μl/孔，4℃包被过夜；
2)PBST洗板3次，轻轻拍干，加入1％BSA(PBS稀释)37℃封闭2小时，1×PBST(Tween-20,1％)洗涤4次；
3)一抗：20μg/ml，1:3梯度稀释7个梯度浓度，空白对照组为PBS，50μl/孔加入到包被好的酶标板上，室温孵育10min；
4)配体：加入PDL2-his tag溶液1.0μg/ml，50μl/孔，37℃孵育1小时；
5)二抗：PBST洗涤5次，轻轻拍干；每孔50μl加入1:750稀释的HRP酶标抗his tag小鼠单克隆抗体二抗，37℃孵育1小时；
5)显色：PBST洗涤6次，轻轻拍干；每孔100μl加入TMB显色剂，室温反应30min；
6)终止：50μl/孔加入2M H2SO4终止显色反应；
7)读数：在酶标仪上，用吸光度450nm检测各孔的吸光值。
结果见图3，由此图3可知，上述三个抗体H2L2能够有效地抑制PD-1与PdL2结合。
表3
Figure PCTCN2016103814-appb-000011
实施例6
使用Fortebio分子相互作用仪测定KF007H1L1,H2L2,H3L3动力学参数：
生物素标记的抗原PD-1固定于SA传感器表面，在PBST中平衡后，与抗体H1L1结合，H1L1用PBST三倍稀释，浓度为200、66.67、22.22、7.41、2.47、0.82、0.27、0nM，于PBST中解离。H2L2和H3L3的检测方法与H1L1相同。H1L1,H2L2,H3L3动力学参数见表4，结果图见图4，图5，图6。
表4
Figure PCTCN2016103814-appb-000012
实施例7抗体H1L1、H2L2和H3L3刺激T细胞分泌IL-2和IFNgamma分析
利用混合淋巴细胞反应(MLR)实验检测抗体H1L1、H2L2和H3L3刺激T淋巴细胞分泌IL-2和IFNgamma分泌能力。MLR实验采用不同人来源的T细胞(TC)和树突状细胞(DC)进行混合，利用DC细胞抗体提呈能力刺激T细胞分泌IL-2和IFNgamma。首先 用细胞因子GM-CSF和IL-4诱导血液中单核细胞分化成树突状细胞，然后用TNFa刺激是未成熟的DC细胞成熟。成熟后的DC与同种异源的TC细胞进行混合5天后，检测细胞上清中的IL-2和IFNgamma的分泌水平。在96孔板中混合TC和DC，按每孔加入TC 1×105和DC 1×104，设置抗体浓度1nM、10nM和100nM。混合反应5天后，用IL-2检测试剂盒定量检测上清IL-2含量，用IFNgamm检测试剂盒定量检测IFNgamma含量。
抗体H1L1、H2L2和H3L34刺激T细胞分泌IL-2和IFNgamma分泌水平如图7所示。由图7可见，抗体H1L1、H2L2和H3L3能有效地刺激T细胞分泌IL-2和IFNgamma，并且呈剂量依赖关系。
实施例8抗体H1L1、H2L2和H3L3结合的EC50分析
利用流式方法测定抗体H1L1、H2L2和H3L3与实施例N中稳定表达PD-1的细胞(命名为BB007细胞)表面抗原的结合活性。采用常规胰酶消化方法获取BB007细胞，PBS洗一次，分成若干管，每管细胞数为2*105；用PBS(1％BSA)配制浓度分别为20nM，10nM，5nM，1nM，0.1nM，0nM的H1L1、H2L2和H3L3抗体稀释液，每个浓度总体积为100μl；冰上与细胞孵育1小时；PBS洗一次；每管加入100μl FITC-Goat-Anti-Human IgG(1：500)冰上孵育1小时；每管加入300μl PBS在流式细胞仪上用FITC通道检测荧光信号。
H1L1、H2L2和H3L3抗体与BB007细胞的结合如图7所示。由图8可见，H1L1、H2L2和H3L3抗体能有效地结合BB007细胞表面的靶标PD-1蛋白，并且其结合效率呈剂量依赖关系。
表5 H1L1、H2L2和H3L3结合BB007细胞后荧光强度分析
Figure PCTCN2016103814-appb-000013
通过对结合的H1L1、H2L2和H3L3抗体进行荧光定量分析，曲线模拟H1L1、H2L2和H3L3的结合效率EC50分别为3.38nM，1.59nM，1.68nM(图9)
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必针对 的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (23)

  1. 一种抗PD-1的单克隆抗体或其抗原结合片段，其特征在于，包括：
    具有下列至少之一氨基酸序列的重链可变区：
    (1)SEQ ID NO：1所示的氨基酸序列，
    (2)SEQ ID NO：3所示的氨基酸序列，
    (3)SEQ ID NO：5所示的氨基酸序列，
    (4)与(1)～(3)相比具有一个以上氨基酸保守突变的氨基酸序列。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，进一步包括：
    具有下列至少之一氨基酸序列的轻链可变区：
    (5)SEQ ID NO：2所示的氨基酸序列，
    (6)SEQ ID NO：4所示的氨基酸序列，
    (7)SEQ ID NO：6所示的氨基酸序列，
    (8)与(5)～(7)相比具有一个以上氨基酸保守突变的氨基酸序列。
  3. 根据权利要求1或2所述的抗体或其抗原结合片段，其特征在于，包括下列之一：
    具有SEQ ID NO：1所示氨基酸序列的重链可变区和具有SEQ ID NO：2所示氨基酸序列的轻链可变区；
    具有SEQ ID NO：3所示氨基酸序列的重链可变区和具有SEQ ID NO：4所示氨基酸序列的轻链可变区；以及
    具有SEQ ID NO：5所示氨基酸序列的重链可变区和具有SEQ ID NO：6所示氨基酸序列的轻链可变区。
  4. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体为单克隆抗体。
  5. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，可以特异性的高效的结合PD-1，并且能够用于：
    促进T细胞的活化和增殖，调节细胞因子的表达和分泌，或者刺激抗肿瘤细胞产生更强的免疫应答。
  6. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码权利要求1～5任一项所述的抗体或其抗原结合片段。
  7. 根据权利要求6所述的多核苷酸，其特征在于，所述多核苷酸具有下列之一所示的核苷酸序列：
    SEQ ID NO：7所示的核苷酸序列或其互补序列；
    SEQ ID NO：8所示的核苷酸序列或其互补序列；
    SEQ ID NO：9所示的核苷酸序列或其互补序列；
    SEQ ID NO：10所示的核苷酸序列或其互补序列；
    SEQ ID NO：11所示的核苷酸序列或其互补序列；以及
    SEQ ID NO：12所示的核苷酸序列或其互补序列。
  8. 一种表达载体，其特征在于，包含权利要求6或7所述的多核苷酸。
  9. 根据权利要求8所述的表达载体，其特征在于，进一步包括：
    控制元件，所述控制元件与所述多核苷酸可操纵地连接，用于控制所述多核苷酸在宿主细胞中的表达。
  10. 根据权利要求9所述的表达载体，其特征在于，所述宿主细胞为哺乳动物细胞。
  11. 根据权利要求10所述的表达载体，其特征在于，所述哺乳动物细胞为人肾上皮细胞系细胞。
  12. 根据权利要求11所述的表达载体，其特征在于，所述人肾上皮细胞系细胞为293T细胞。
  13. 根据权利要求9所述的表达载体，其特征在于，所述控制元件包括下列至少之一：
    启动子、增强子和终止子，
    任选地，所述启动子为CMV启动子，所述增强子为早期CMV增强子，所述终止子为SV polyA终止子。
  14. 一种重组细胞，其特征在于，包含权利要求8～13任一项所述的表达载体。
  15. 一种制备权利要求1～5任一项所述的抗体或其抗原结合片段的方法，其特征在于，包括培养权利要求14所述的重组细胞。
  16. 权利要求6或7所述的多核苷酸、权利要求8～13任一项所述的表达载体、或权利要求14所述的重组细胞在制备抗体或其抗原结合片段的用途，所述抗体与PD-1特异性结合。
  17. 一种杂交瘤，其以保藏号CCTCC No:C201667，保藏于中国典型培养物保藏中心，保藏时间为2016年4月1日。
  18. 权利要求17所述的杂交瘤在制备单克隆抗体中的用途。
  19. 权利要求1～5任一项所述的抗体或其抗原结合片段、权利要求6或7所述的多核苷酸、权利要求8～13任一项所述的表达载体、权利要求14所述的重组细胞或权利要求17所述的杂交瘤在制备药物中的用途，所述药物用于促进T细胞的活化和增殖，并调节细胞因子的表达和分泌，使用来刺激抗肿瘤细胞产生更强的免疫应答。
  20. 一种药物组合物，其特征在于，包含权利要求1～5任一项所述的抗体或其抗原结 合片段、权利要求6或7所述的多核苷酸、权利要求8～13任一项所述的表达载体、权利要求14所述的重组细胞或权利要求17所述的杂交瘤。
  21. 一种用于鉴定能够与PD-1结合的药物的方法，其特征在于，包括：
    在候选药物存在时，使权利要求1～5任一项所述的抗体或其抗原结合片段与抗原接触，并且确定所述抗体或其抗原结合片段与所述抗原的第一结合量，其中，所述抗原为PD-1或其片段；以及
    在不存在所述候选药物时，使权利要求1～5任一项所述的抗体或其抗原结合片段与抗原接触，并且确定所述抗体或其抗原结合片段与所述抗原的第二结合量，其中，所述抗原为PD-1或其片段，
    其中，所述第二量大于所述第一量是所述候选药物能够与PD-1结合的指示。
  22. 一种药物联合，其特征在于，包括：
    (1)权利要求1～5任一项所述的抗体或其抗原结合片段、权利要求6或7所述的多核苷酸、权利要求8～13任一项所述的表达载体、权利要求14所述的重组细胞或权利要求17所述的杂交瘤；以及
    (2)与(1)不同的免疫增强药物。
  23. 根据权利要求16所述的药物联合，其特征在于，所述与(1)不同的免疫增强药物包括选自下列的至少之一：
    抗CTLA-4抗体，抗CD40抗体，Budesonide，水杨酸盐，任选地所述水杨酸盐类包括sulfasalazine、olsalazine、balsalazide以及mesalamine的至少之一。
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