WO2017162208A1 - 靶向pd-1的多肽及其应用 - Google Patents

靶向pd-1的多肽及其应用 Download PDF

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WO2017162208A1
WO2017162208A1 PCT/CN2017/078162 CN2017078162W WO2017162208A1 WO 2017162208 A1 WO2017162208 A1 WO 2017162208A1 CN 2017078162 W CN2017078162 W CN 2017078162W WO 2017162208 A1 WO2017162208 A1 WO 2017162208A1
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cancer
polypeptide
protein
lys
binding
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French (fr)
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李洪林
刘晓峰
朱丽丽
李巧
全丽娜
吴方舒
赵振江
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华东理工大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention belongs to the field of biotechnology.
  • the invention relates to polypeptides that target human PD-1 proteins and uses thereof.
  • T cell function There are numerous co-stimulatory signals and co-suppressive signals in the immune system that precisely regulate the intensity and quality of T cell responses. These inhibition signals are called immunological checkpoints. Tumor cells often overexpress these immune checkpoint proteins, thereby inhibiting T cell activation and evading immune system killing. Enhancing the activity of T cells through different strategies is of great significance for tumor immunotherapy. Blocking the immune checkpoint is one of the effective strategies to enhance the activity of T cells.
  • immunosuppressive molecules on the surface of T cells such as PD-1, CTLA-4, Tim-3, SLAM, etc. These immunosuppressive molecules can bind to their corresponding ligands to activate the immunosuppressive regulatory pathway, which leads to The failure of T cell function, of which PD-1/PD-L1 pathway is the current research hotspot.
  • PD-1 is an important inhibitory receptor on the surface of T cells and is named for its initial discovery in apoptotic T-cell lymphoma and its ability to promote programmed cell death.
  • the PD-1 protein is inducibly expressed in activated T cells, B cells, macrophages, dendritic cells, monocytes, and the like.
  • PD-L1 is more widely distributed than PD-L2.
  • PD-L1 can be expressed in B cells, DCs, macrophages, bone marrow-derived mast cells, T cells, fibroblasts, epithelial cells, etc., while PD-L2 is only It is expressed in cells such as DC, macrophages, and memory B cells. Therefore, blocking the PD-1/PD-L1 pathway is more effective than blocking the PD-1/PD-L2 pathway.
  • the mechanism by which PD-1/PD-L1 inhibits the immune pathway is as follows: tumor cells have a large number of gene mutations and abnormal expression of proteins, which can act as tumor antigens to activate T cells.
  • Tumor-specific cytotoxic T cells reach the tumor site, recognize tumor cells through T cell antigen receptor (TCR), release interferon gamma (IFN- ⁇ ) and T cell particles, and kill tumor cells.
  • TCR T cell antigen receptor
  • IFN- ⁇ interferon gamma
  • T cell antigen receptor TCR
  • IFN- ⁇ interferon gamma
  • T cell particles T cell particles
  • kill tumor cells in addition to its anti-tumor effect, IFN- ⁇ can also induce tumor cells to express PD-L1.
  • PD-L1 bind to PD-1 in activated T cells and inhibit the anti-tumor effect of T cells.
  • polypeptide molecules Compared with antibody drugs, polypeptide molecules have become the most attractive antibody replacement because of their high specificity and tissue affinity, low production cost, low immunogenicity and good tissue permeability. Things. Therefore, research and development of some low molecular weight organic small molecules or peptide drugs have become blocked An important research direction of the PD-1/PD-L1 pathway.
  • the invention provides a polypeptide of formula I:
  • [Xaa0] is a peptide consisting of Gly Asn, Thr Glu, or Phe Asn, or none;
  • [Xaa1] is Trp, Lys or Met
  • [Xaa2] is Asn Ser Gln, Arg His Gly Asn Ile, Ser Leu Glu Glu Leu, Ser Trp Lys Ser Glu, Gln Asn, Cys Pro Cys, Cys His Gly Pro Cys, Cys Ala Glu Pro Leu, Ser Trp Cys Pro Cys Peptide segment
  • [Xaa3] is a peptide consisting of Ala Gln Leu, Met Lys Leu Ala, Glu Lys Ala Lys, Leu Lys Glu Ala, or Glu Lys Cys Lys;
  • [Xaa4] is Asn Gln, Asp Leu, Lys, Asp Leu, or Arg;
  • polypeptide has PD-1 binding activity, and the polypeptide is 15-18 amino acids in length.
  • polypeptide is selected from the group consisting of:
  • the polypeptide has a binding KD value of 1-5 ⁇ M, preferably 1-4 ⁇ M, more preferably 1-3 ⁇ M, and most preferably a binding KD value to native PD-L1 and PD-1. .
  • polypeptide is selected from the group consisting of a polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs: 1-9.
  • polypeptide is a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 or 6.
  • the invention provides an isolated nucleic acid molecule encoding the polypeptide of the first aspect of the invention.
  • the invention provides a host cell comprising the isolated nucleic acid molecule of the second aspect of the invention.
  • the host cell includes, but is not limited to, bacteria, yeast, plant cells, Kun Worm cells or mammalian cells.
  • the bacteria include, but are not limited to, Escherichia coli, Bacillus subtilis; and the yeast includes, but is not limited to, Pichia pastoris, Saccharomyces cerevisiae.
  • the present invention provides a pharmaceutical composition comprising the polypeptide of the first aspect of the invention and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition further comprises: (c) a pharmaceutically acceptable other drug that blocks the PD-1/PD-L1 pathway.
  • the other drugs that block the PD-1/PD-L1 pathway include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, which targets PD-1 protein, and MPDL3280A, which targets PD-L1. , MDX1105, MEDI4736 and so on.
  • the pharmaceutical composition may further comprise other anti-tumor drugs including, but not limited to, Ipilimumab, ramucirumab, trametinib, ceritini, dabrafenib, and the like.
  • the dosage form of the pharmaceutical composition is suitable for the following modes of administration, including but not limited to: injection administration, infusion administration, intraperitoneal administration, intratumoral administration, intramuscular administration. medicine.
  • the present invention provides the use of the polypeptide of the first aspect of the invention for the preparation of inhibiting PD-1 binding to PD-L1 or inhibiting tumors, or treating infections caused by bacteria, viruses or fungi or treating inflammatory The drug of the disease.
  • the tumor includes, but is not limited to, melanoma, lung cancer (preferably non-small cell lung cancer), kidney cancer, ovarian cancer, prostate cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer , head and neck cancer, uterine cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophagus Cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, pediatric Solid tumor, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal pelvic cancer, central nervous system
  • lung cancer
  • the viruses include, but are not limited to, hepatitis viruses (types A, B, and C), herpes viruses, influenza viruses, adenoviruses, coronaviruses, measles viruses, dengue viruses, polioviruses, rabies viruses, and the like;
  • the bacteria include, but are not limited to, chlamydia, rickettsia, mycobacteria, staphylococcus, pneumococcus, cholera, tetanus, and the like;
  • the fungus includes, but is not limited to, Candida, Aspergillus, dermatitis, etc.; or
  • the inflammatory diseases include, but are not limited to, ankylosing spondylitis, autoimmune hemolytic anemia, arthritis, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis, pernicious anemia, polymyositis, and the like.
  • the present invention provides a method of inhibiting the binding of PD-1 to PD-L1, comprising the steps of: inhibiting PD- using the polypeptide of the first aspect of the invention or the pharmaceutical composition of the fourth aspect of the invention 1 combined with PD-L1.
  • the method of inhibiting PD-1 binding to PD-L1 is non-therapeutic in vitro.
  • Figure 1 shows the results of PCR identification of the human PD-1 recombinant plasmid constructed in Example 3, wherein the marker represents the DNA molecular weight marker; the band appearing in the lane is the target band (357 bp).
  • Figure 2 shows the results of SDS-PAGE electrophoresis of purified human PD-1 protein of Example 4, wherein M represents the protein molecular weight marker; Lane 1 represents the human PD-1 protein, the target band size is 13 KDa, and the theoretical value Consistent.
  • Figure 3a shows a SPR map of the binding of the human PD-1 protein to the polypeptide of amino acid sequence SEQ ID NO: 1, and the KD value of the polypeptide and PD-1 protein was determined to be 1.38 ⁇ M.
  • Figure 3b shows a SPR map of the binding of the human PD-1 protein to the polypeptide of amino acid sequence SEQ ID NO: 2, and the KD value of the polypeptide and PD-1 protein was determined to be 3.13 ⁇ M.
  • Figure 3c shows a SPR map of the binding of the human PD-1 protein to the polypeptide of amino acid sequence SEQ ID NO: 3, and the KD value of the polypeptide and PD-1 protein was determined to be 3.14 ⁇ M.
  • Figure 3d shows a SPR map of the binding of the human PD-1 protein to the polypeptide of amino acid sequence SEQ ID NO: 4, and the KD value of the strip of the polypeptide and the PD-1 protein was determined to be 3.39 ⁇ M.
  • Figure 3e shows a SPR map of the binding of the human PD-1 protein to the polypeptide having the amino acid sequence of SEQ ID NO: 5, and the KD value of the polypeptide and PD-1 protein was determined to be 3.32 ⁇ M.
  • Figure 3f shows a SPR map of binding of human PD-1 protein to human PD-L1 protein, and the KD value of human PD-L1 protein and PD-1 protein was determined to be 1.15 ⁇ M.
  • Figure 4 shows a SPR map of the human PD-L1 protein competes with the polypeptide of amino acid sequence SEQ ID NO: 1 for binding to human PD-1 protein.
  • Figure 5a shows a SPR map of the binding of the human PD-1 protein to the polypeptide of amino acid sequence SEQ ID NO: 6, and the KD value of the polypeptide and PD-1 protein was determined to be 2.08 ⁇ M.
  • Figure 5b shows a SPR map of the binding of the human PD-1 protein to the polypeptide of amino acid sequence SEQ ID NO: 7, and the KD value of the strip of the polypeptide and the PD-1 protein was determined to be 3.96 ⁇ M.
  • Figure 5c shows a SPR map of the binding of the human PD-1 protein to the polypeptide of amino acid sequence SEQ ID NO: 8, and the KD value of the polypeptide and PD-1 protein was determined to be 3.11 ⁇ M.
  • Figure 5d shows the SPR map of the binding of the human PD-1 protein to the polypeptide of amino acid sequence SEQ ID NO:9, The KD value of the peptide and PD-1 protein obtained was 4.81 ⁇ M.
  • PD-1 protein refers to an important inhibitory receptor on the surface of T cells in humans. It has two ligands, PD-L1 and PD-L2. PD-L1 binds to PD-1 in activated T cells and acts to inhibit the anti-tumor effect of T cells. Therefore, blocking the PD-1/PD-L1 pathway has a positive effect on inhibiting or killing tumors.
  • the PD-1 protein of the present invention includes a PD-1 protein comprising a certain mutation as long as the mutated PD-1 protein performs the same function as wild-type PD-1 in humans.
  • the PD-1 protein of the present invention has the amino acid sequence set forth in SEQ ID NO: 14.
  • the PD-1 protein has a ligand (PD-L1) that specifically binds thereto.
  • PD-L1 of the present invention has the amino acid sequence shown in SEQ ID NO: 12.
  • the present invention provides polypeptides that are capable of competitively binding to human PD-1 proteins, which have structural similarities, i.e., have specific amino acid residues at specific positions.
  • the invention provides a polypeptide of Formula I:
  • [Xaa0] is a peptide consisting of Gly Asn, Thr Glu, or Phe Asn, or none;
  • [Xaa1] is Trp, Lys or Met;
  • [Xaa2] is Asn Ser Gln, Arg His Gly Asn Ile, Ser Leu Glu Peptides composed of Glu Leu, Ser Trp Lys Ser Glu, Gln Asn, Cys Pro Cys, Cys His Gly Pro Cys, Cys Ala Glu Pro Leu, Ser Trp Cys Pro Cys;
  • [Xaa3] is Ala Gln Leu, Met Lys Leu Ala Glu Lys Ala Lys, A peptide consisting of Leu Lys Glu Ala, or Glu Lys Cys Lys;
  • [Xaa4] is Asn Gln, Asp Leu, Lys, Asp Leu, or Arg; these polypeptides are 15-18 amino acids in length.
  • polypeptides have good binding affinity to the human PD-1 protein, wherein the polypeptide with the highest binding affinity is comparable to the natural ligand PD-L1 of the human PD-1 protein.
  • the polypeptide is a polypeptide having the amino acid sequence of any one of SEQ ID NOS: 1-9; or the polypeptide having the amino acid sequence of any one of SEQ ID NOS: 1-9 is subjected to 1 a derivative polypeptide formed by substitution, deletion or addition of -6, (preferably 1-4) amino acid residues, and having PD-1 binding activity.
  • the polypeptide of the invention has a binding KD value to PD-1 of 1-5 ⁇ M, preferably 1-4 ⁇ M, more preferably 1-3 ⁇ M, most preferably a binding KD value to native PD-L1 and PD-1. approximate.
  • polypeptide of the invention is selected from the group consisting of the amino acid sequence as set forth in any one of SEQ ID NOs: 1-9; more preferably the polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 or 6.
  • the invention provides an isolated nucleic acid molecule encoding a polypeptide of the invention, and a host cell comprising the isolated nucleic acid molecule.
  • a host cell for example, including, but not limited to, bacteria, yeast, plant cells, insect cells, or mammalian cells.
  • bacteria include, but are not limited to, Escherichia coli, Bacillus subtilis; and the yeast includes, but is not limited to, Pichia pastoris, Saccharomyces cerevisiae.
  • the present invention also provides a pharmaceutical composition comprising a polypeptide of the present invention and a pharmaceutically acceptable excipient.
  • the polypeptides of the invention may also be used in combination with other mechanisms of blocking the PD-1/PD-L1 pathway as well as other anti-tumor agents to enhance each other's effects.
  • other pharmaceutically acceptable drugs that block the PD-1/PD-L1 pathway may also be included in the above pharmaceutical compositions.
  • the other drugs that block the PD-1/PD-L1 pathway include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, which target PD-1 protein, and MPDL3280A, MDX1105, MEDI4736, etc., which target PD-L1.
  • the pharmaceutical composition may further comprise other anti-tumor drugs including, but not limited to, Ipilimumab, ramucirumab, trametinib, ceritini, dabrafenib, and the like.
  • administration forms of the pharmaceutical compositions of the present invention include, but are not limited to, injection administration, infusion administration, intraperitoneal administration, intratumoral administration, intramuscular administration, and the like.
  • the content of the active ingredient in the pharmaceutical composition can be determined autonomously by a person skilled in the medical field, such as the patient's gender, age, general health status and the like.
  • the tumor includes, but is not limited to, melanoma, lung cancer (preferably non-small cell lung cancer), kidney cancer, ovarian cancer, prostate cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer , head and neck cancer, uterine cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophagus Cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myelogenous leukemia,
  • the medicament that inhibits PD-1 binding to PD-L1 can also be used to treat bacterial, viral or fungal infections.
  • infections caused by the following viruses hepatitis viruses (types A, B and C), herpesviruses, influenza viruses, adenoviruses, coronaviruses, measles viruses, dengue viruses, polioviruses, rabies viruses, etc.; Infection caused by bacteria: Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Pneumococcal, Cholera, Tetanus, etc.; or pathogenic infections caused by the following fungi: Candida, Aspergillus, dermatitis, etc.
  • the medicament for inhibiting PD-1 binding to PD-L1 is also useful for treating inflammatory diseases including, but not limited to, ankylosing spondylitis, autoimmune hemolytic anemia, arthritis, myasthenia gravis , systemic lupus erythematosus, rheumatoid arthritis, pernicious anemia, polymyositis, etc.
  • inflammatory diseases including, but not limited to, ankylosing spondylitis, autoimmune hemolytic anemia, arthritis, myasthenia gravis , systemic lupus erythematosus, rheumatoid arthritis, pernicious anemia, polymyositis, etc.
  • the present invention further provides a method of inhibiting the binding of PD-1 to PD-L1, comprising inhibiting PD-1 and PD-L1 using the polypeptide or pharmaceutical composition of the present invention.
  • the method of inhibiting PD-1 binding to PD-L1 is non-therapeutic in vitro.
  • the invention provides a polypeptide capable of competitively binding to a human PD-1 protein
  • polypeptide of the present invention binds well to human PD-1 protein, wherein the binding affinity of the polypeptide with the highest binding affinity to human PD-1 protein is the same as that of the native PD-L1 protein;
  • the polypeptide of the present invention has high specificity, tissue affinity, difficulty in producing immunogenicity, and good tissue permeability;
  • the production of the polypeptide of the present invention is low in cost.
  • DMF Dimethylformamide
  • DCM dichloromethane
  • HBTU benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate Salt
  • TIS triisopropylsilane
  • EDT ethanedithiol
  • NMM N-methylmorpholine
  • methanol ethanol, 20 amino acids
  • peptide solid phase synthesis tube mass spectrometer MicrOTOF-Q11 (BrukerDaltonics).
  • the amino acid sequence shown in SEQ ID NO: 1-9 was used to take the corresponding amino acid and HBTU, and dissolved in a small amount of DMF, and put into a solid phase synthesis tube, 10 times of NMM was added, and the reaction was stirred. After 1-2 hours, a small amount of resin was taken from the solid phase synthesis tube in a test tube, washed twice with ethanol, and detected by the ninhydrin method. The liquid in the solid phase synthesis tube was removed, and washed twice with DMF, methanol, and DCM to obtain a peptide resin obtained by condensation of the first amino acid.
  • the gene of interest is the nucleic acid encoding amino acids 34-150 on the PD-1 protein.
  • the target fragment was cloned into the pET-28a vector (Novagen) using specific primers designed with two restriction sites of NcoI and NdeI.
  • the forward primer was: 5'-TTTTCCATGGGTCCCCCCACCTTCTCCCCAG-3' (SEQ ID NO: 10); the reverse primer was: 5'-GCCGCCGCATATGTTATTCTGCCCTTCTCTCTG-3' (SEQ ID NO: 11).
  • PCR amplification was carried out using the human PD-1 gene as a template.
  • the PCR system was: 2 ⁇ L PD-1 plasmid, 2 ⁇ L forward primer, 2 ⁇ L reverse primer, 5 ⁇ L 10 ⁇ buffer solution, 4 ⁇ L dNTP, 1 ⁇ L pfu polymerase, 34 ⁇ L ddH 2 O.
  • PCR amplification conditions pre-denaturation at 94 ° C for 4 min; denaturation at 94 ° C for 30 s, annealing at 60 ° C for 30 s, extension at 72 ° C for 48 s, 30 cycles; 72 ° C for 10 min.
  • nucleic acid electrophoresis is carried out, the target band is cut out, and the PCR product is recovered by a PCR product recovery kit.
  • the obtained PCR product and the plasmid pET-28a vector were digested with NcoI and NdeI restriction endonucleases, respectively, and ligated under the action of T4 ligase, and transformed into E. coli DH5 ⁇ competent cells. After overnight culture, the monoclonal was picked for PCR analysis (see Figure 1), and then the positive recombinants were sequenced to retain the strain and glycerol.
  • the human PD-1 constructed by the recombinant plasmid has the nucleotide sequence shown as SEQ ID NO: 13.
  • the human PD-1 protein used in the present invention is obtained after prokaryotic expression and purification.
  • the specific experimental procedure is as follows: the correctly sequenced recombinant plasmid was transformed into E. coli BL21 competent cells for induction expression. A single colony was picked and inoculated into TB medium containing kanamycin, and cultured overnight at 37 ° C with shaking. The next day, the small culture product was expanded into 1 L of TB medium, cultured to an OD 600 of 0.5-0.6, and the inducer IPTG was added to a final concentration of 0.5 mM, and induced at 37 ° C for 5-7 h.
  • the bacteria were collected at 4,000 rpm, and the bacteria were first lysed with a lysis buffer solution (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM DTT, 0.5 mM EDTA, 5% glycerol), then crushed by high pressure, and centrifuged at 12,000 rpm. precipitation. The mixture was washed twice with a washing buffer (20 mM Tris-HCl, pH 8.0, 2 M urea, 2.5% Triton X-100), and a precipitate was taken. The protein was then dissolved in a lysis buffer solution (20 mM Tris-HCl, pH 8.0, 8 M urea) and the supernatant was centrifuged.
  • a lysis buffer solution 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM DTT, 0.5 mM EDTA, 5% glycerol
  • renaturation buffer 50mM Tris-HCl, pH 8.0, 50mM L-Arg, 24mM NaCl, 1mM KCl, 1mM EDTA.
  • the dilution method was renatured at 4 ° C for 24 h.
  • the protein was concentrated with a 3 kDa concentrating tube to about 20 mL, and the protein was placed in a dialysis bag, dialyzed against dialysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM DTT) overnight, and concentrated.
  • the obtained protein supercation exchange column and molecular sieve were purified.
  • the protein after the molecular sieve was subjected to 14% SDS-PAGE gel electrophoresis to identify the purity of the purified protein.
  • the obtained electropherogram is shown in Figure 2.
  • the protein size is about 13KDa, which is consistent with the theoretical value of human PD-1 protein, indicating that the human PD-1 protein was successfully obtained.
  • Example 5 SPR binding experiment of human PD-1 protein and polypeptide of the present invention
  • Binding of the polypeptide of the present invention to human PD-1 protein was determined by Surface Plasmon Resonances (SPR) assay using Biacore T200.
  • SPR Surface Plasmon Resonances
  • the specific experimental procedure was as follows: the purified PD-1 protein was diluted to 50 ⁇ g/ml with 10 mM sodium acetate solution, and the PD-1 protein was coupled to the CM5 chip using an amino coupling kit at a flow rate of 10 ⁇ l/min. For 420 s, the final coupling amount is approximately 5000 RU.
  • the binding experiments were carried out in a PBS solution (137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4) containing 0.05% of Surfactant P20.
  • the experimental temperature was set to 25 ° C
  • the flow rate was 30 ⁇ l / min
  • the binding time was 90 s
  • the dissociation time was 120 s.
  • After dissolving the polypeptide in the running solution it was diluted to different concentrations and bound to PD-1 with the running solution.
  • the present inventors simultaneously examined the binding affinity K D value of the human PD-1 protein to the human PD-L1 protein, which was 1.15 ⁇ M (as shown in Fig. 3f).
  • Example 6 SPR assay for competitive binding of human PD-L1 protein to the polypeptide of SEQ ID NO: 1 to human PD-1 protein
  • the polypeptide of SEQ ID NO: 1 had the highest binding affinity to human PD-1 protein.
  • the inventors have taken the polypeptide of SEQ ID NO: 1 as an example to investigate the ability of the peptide to block the binding of human PD-1 protein to human PD-L1 protein.
  • Human PD-L1 protein was purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.
  • the specific experimental procedure was as follows: First, 50 ⁇ g/ml human PD-L1 protein was coupled to the surface of CM5 chip by amino coupling; then different concentrations of the polypeptide represented by SEQ ID NO: 1 (125 nM, 62.5 nM, 31.25 nM, 15.60 nM, 0 nM) were incubated with 100 nM human PD-1 protein for 15 min on ice and flowed through running buffer solution (10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl and 0.05% surfactant P20, pH 7.4). On the surface of the chip, the flow rate was 30 ⁇ l/min, the binding time was 90 s, and the dissociation time was 120 s.
  • the present invention provides a polypeptide that targets human PD-1 protein, that is, a polypeptide capable of competitively binding to a human PD-1 protein, each polypeptide having a specific amino acid residue at a specific position, thereby enabling a similar structure.
  • These polypeptides all bind well to human PD-1 protein, wherein the polypeptide of SEQ ID NO: 1 has the highest binding affinity to human PD-1 protein, and its K D value is 1.38 ⁇ M with PD-L1 and PD- The combined K D value of 1 is comparable.
  • these peptides can act as inhibitors of PD-1/PD-L1 binding or modulators of PD-1/PD-L1 binding, as well as to study PD-1/PD-L1 immunological checkpoint blockers and designs.
  • the development of blocking the PD-1/PD-L1 pathway provides peptide-based lead drugs.

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Abstract

靶向人PD-1的多肽及其应用，所述靶向人PD-1多肽的制备方法以及包含这些多肽的药物组合物。所述多肽能够与人PD-1蛋白结合，从而能作为开发人PD-1受体抑制剂的先导肽，为抗肿瘤药物的发展提供基础。所述靶向人PD-1的多肽可以采用化学合成的方法制备得到，其具有纯度高、分子量小、安全可靠等优势。

Description

靶向PD-1的多肽及其应用 技术领域
本发明属于生物技术领域。具体地说，本发明涉及靶向人PD-1蛋白的多肽及其应用。
背景技术
免疫系统中存在着众多的共刺激信号和共抑制信号精确调控T细胞反应的强度和质量，这些抑制信号被称为免疫检查点。肿瘤细胞通常会过表达这些免疫检查点蛋白，从而抑制T细胞的激活，逃避免疫系统的杀伤。通过不同的策略增强T细胞的活性对肿瘤免疫治疗具有重要意义，其中对免疫检查点进行阻断是增强T细胞活性的有效策略之一。T细胞表面存在着一系列的免疫抑制性分子，如PD-1、CTLA-4、Tim-3、SLAM等，这些免疫抑制性分子可以与其相应的配体结合从而激活免疫抑制调节通路，进而导致T细胞功能的衰竭，其中，PD-1/PD-L1通路是目前的研究热点。
PD-1是T细胞表面一个重要的抑制性受体，因最初在凋亡的T细胞淋巴瘤中发现，并能促进程序性细胞死亡而得名。PD-1蛋白可诱导性地表达于活化的T细胞、B细胞、巨噬细胞、树突状细胞以及单核细胞等。其配体有两个，分别为PD-L1(CD274，B7-H1)和PD-L2(CD273，B7-DC)。PD-L1比PD-L2的分布更加广泛，PD-L1可以在B细胞、DC、巨噬细胞、骨髓源肥大细胞、T细胞、成纤维细胞、上皮细胞等细胞中表达，而PD-L2仅在DC、巨噬细胞、记忆性B细胞等细胞中表达，因此，阻断PD-1/PD-L1通路比阻断PD-1/PD-L2通路更为有效。PD-1/PD-L1抑制免疫通路的机理如下：肿瘤细胞存在着大量的基因突变以及蛋白的异常表达，可以作为肿瘤抗原活化T细胞。肿瘤特异性细胞毒T细胞到达肿瘤部位后，通过T细胞抗原受体(TCR)识别肿瘤细胞，释放干扰素γ(IFN-γ)以及T细胞颗粒，杀伤肿瘤细胞。但IFN-γ除具有抗肿瘤作用外，还能够诱导肿瘤细胞表达PD-L1。这些PD-L1与活化的T细胞中的PD-1结合后，抑制T细胞的抗肿瘤作用。
虽然目前已经有很多阻断PD-1/PD-L1通路的药物进入临床试验或者个别药物已经获得美国FDA的审批，例如：靶向PD-1蛋白的nivolumab、pembrolizumab和pidilizumab，以及靶向与PD-L1的MPDL3280A、MDX1105、MEDI4736等，但是这些药物大部分都是抗体类药物，具有生产成本高、组织渗透性差、易产生免疫原性等缺点。
与抗体类药物相比，多肽分子因具有较高的特异性和组织亲和性、生产成本低、不易产生免疫原性及较好的组织渗透性等优点，而成为最有吸引力的抗体替代物。因而，研究并开发出一些低分子量的有机小分子或者多肽类药物成为阻断 PD-1/PD-L1通路的重要研究方向。
综上所述，本领域急需低分子量的有机小分子或者多肽作为阻断PD-1/PD-L1通路的候选药物。
发明内容
本发明的目的是提供能够阻断PD-1/PD-L1通路的低分子量的有机小分子或者多肽类。
在第一方面，本发明提供一种式I表示的多肽：
[Xaa0]-[Xaa1]-Asp-Tyr-[Xaa2]-Arg-[Xaa3]-Tyr-[Xaa4](I)
其中，
[Xaa0]为Gly Asn、Thr Glu、或Phe Asn构成的肽段、或无；
[Xaa1]为Trp、Lys或Met；
[Xaa2]为Asn Ser Gln、Arg His Gly Asn Ile、Ser Leu Glu Glu Leu、Ser Trp Lys Ser Glu、Gln Asn、Cys Pro Cys、Cys His Gly Pro Cys、Cys Ala Glu Pro Leu、Ser Trp Cys Pro Cys构成的肽段；
[Xaa3]为Ala Gln Leu、Met Lys Leu Ala、Glu Lys Ala Lys、Leu Lys Glu Ala、或Glu Lys Cys Lys构成的肽段；
[Xaa4]为Asn Gln、Asp Leu、Lys、Asp Leu、或Arg；
并且所述多肽具有PD-1结合活性，所述多肽的长度为15-18个氨基酸。
在具体的实施方式中，所述多肽选自下组：
(a)氨基酸序列如SEQ ID NO:1-9中任一所示的多肽；
(b)将(a)所述的多肽经过1-6个(较佳地1-4个)氨基酸残基的取代、缺失或添加而形成的，且具有PD-1结合活性的由(a)衍生的多肽。
在优选的实施方式中，所述多肽与PD-1的结合KD值为1-5μM，优选1-4μM、更优选1-3μM，最优选与天然PD-L1与PD-1的结合KD值近似。
在具体的实施方式中，所述多肽选自氨基酸序列如SEQ ID NO:1-9中任一所示的多肽。
在具体的实施方式中，所述多肽是氨基酸序列如SEQ ID NO:1或6所示的多肽。
在第二方面，本发明提供一种分离的核酸分子，所述核酸分子编码本发明第一方面所述的多肽。
在第三方面，本发明提供一种宿主细胞，所述宿主细胞包含本发明第二方面所述的分离的核酸分子。
在优选的实施方式中，所述宿主细胞包括但不限于：细菌、酵母、植物细胞、昆 虫细胞或哺乳动物细胞。
在优选的实施方式中，所述细菌包括但不限于：大肠杆菌、枯草杆菌；所述酵母包括但不限于：毕赤酵母、酿酒酵母。
在第四方面，本发明提供一种药物组合物，所述药物组合物包含本发明第一方面所述的多肽以及药学上可接受的赋形剂。
在优选的实施方式中，所述的药物组合物还包含：(c)药学上可接受的其它阻断PD-1/PD-L1通路的药物。
在另一优选的实施方式中，所述其它阻断PD-1/PD-L1通路的药物包括但不限于：靶向PD-1蛋白的nivolumab、pembrolizumab、pidilizumab，以及靶向PD-L1的MPDL3280A、MDX1105、MEDI4736等。
在优选的实施方式中，所述的药物组合物还可包含其它抗肿瘤药物，所述抗肿瘤药物包括但不限于：Ipilimumab、ramucirumab、trametinib、ceritini、dabrafenib等。
在另一优选的实施方式中，所述药物组合物的剂型适用于以下给药方式，包括但不限于：注射给药、输注给药、腹膜内给药、瘤内给药、肌肉内给药。
在第五方面，本发明提供本发明第一方面所述的多肽的用途，用于制备抑制PD-1与PD-L1结合或抑制肿瘤、或治疗细菌、病毒或真菌引起的感染或治疗炎性疾病的药物。
在具体的实施方式中，所述肿瘤包括但不限于：黑色素瘤、肺癌(优选非小细胞肺癌)、肾癌、卵巢癌、前列腺癌、乳腺癌、结肠癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、直肠癌、肛门癌、胃癌、睾丸癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、何杰金氏病、非何杰金淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、急性髓性白血病、慢性髓性白血病、急性淋巴细胞白血病、慢性淋巴细胞白血病、小儿实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管生成、脊轴瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤等；
所述病毒包括但不限于：肝炎病毒(甲型、乙型和丙型)、孢疹病毒、流感病毒、腺病毒、冠状病毒、麻疹病毒、登革热病毒、脊髓灰质炎病毒、狂犬病病毒等；
所述细菌包括但不限于：衣原体、立克次氏体、分枝杆菌、葡萄球菌、肺炎球菌、霍乱、破伤风等；
所述真菌包括但不限于：假丝酵母、曲霉、皮炎芽酵母等；或者
所述炎性疾病包括但不限于：强直性脊柱炎、自身免疫性溶血性贫血、关节炎、重症肌无力、系统性红斑狼疮、类风湿性关节炎、恶性贫血、多肌炎等。
在第六方面，本发明提供一种抑制PD-1与PD-L1结合的方法，包括步骤：利用本发明第一方面所述的多肽或本发明第四方面所述的药物组合物抑制PD-1与PD-L1结合。
在优选的实施方式中，所述抑制PD-1与PD-L1结合的方法是体外非治疗性的。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示实施例3构建的人PD-1重组质粒的PCR鉴定结果，其中marker代表DNA分子量marker；泳道中出现的条带为目的条带(357bp)。
图2显示实施例4的经纯化人PD-1蛋白的SDS-PAGE电泳结果，其中M代表蛋白分子量marker；泳道1代表人的PD-1蛋白，该目的条带大小为13KDa，与理论值大小一致。
图3a显示人PD-1蛋白与氨基酸序列为SEQ ID NO:1的多肽结合的SPR图，测得的该条多肽与PD-1蛋白的KD值为1.38μM。
图3b显示人PD-1蛋白与氨基酸序列为SEQ ID NO:2的多肽结合的SPR图，测得的该条多肽与PD-1蛋白的KD值为3.13μM。
图3c显示人PD-1蛋白与氨基酸序列为SEQ ID NO:3的多肽结合的SPR图，测得的该条多肽与PD-1蛋白的KD值为3.14μM。
图3d显示人PD-1蛋白与氨基酸序列为SEQ ID NO:4的多肽结合的SPR图，测得的该条多肽与PD-1蛋白的KD值为3.39μM。
图3e显示人PD-1蛋白与氨基酸序列为SEQ ID NO:5的多肽结合的SPR图，测得的该条多肽与PD-1蛋白的KD值为3.32μM。
图3f显示人的PD-1蛋白与人PD-L1蛋白结合的SPR图，测得人PD-L1蛋白与PD-1蛋白的KD值为1.15μM。
图4显示人PD-L1蛋白与氨基酸序列为SEQ ID NO:1的多肽竞争结合人PD-1蛋白的SPR图。
图5a显示人PD-1蛋白与氨基酸序列为SEQ ID NO:6的多肽结合的SPR图，测得的该条多肽与PD-1蛋白的KD值为2.08μM。
图5b显示人PD-1蛋白与氨基酸序列为SEQ ID NO:7的多肽结合的SPR图，测得的该条多肽与PD-1蛋白的KD值为3.96μM。
图5c显示人PD-1蛋白与氨基酸序列为SEQ ID NO:8的多肽结合的SPR图，测得的该条多肽与PD-1蛋白的KD值为3.11μM。
图5d显示人PD-1蛋白与氨基酸序列为SEQ ID NO:9的多肽结合的SPR图，测 得的该条多肽与PD-1蛋白的KD值为4.81μM。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现了一组靶向人PD-1蛋白的多肽，该组多肽能够竞争性地与人PD-1蛋白结合，从而成为研究PD-1/PD-L1免疫检查点阻断剂的多肽类先导药物，进而为抗肿瘤药物的发展提供基础。在此基础上完成了本发明。
除非另有定义，本文中使用的所有技术和科学术语具有与所公开的发明所属领域的技术人员的普遍理解相同的含义。为便于理解本发明，对本发明涉及的相关术语作如下定义，但本发明的范围并不限于这些具体的定义。
PD-1蛋白
本文所用的术语“PD-1蛋白”是指人体内T细胞表面的一个重要的抑制性受体。其有两个配体，分别为PD-L1和PD-L2。PD-L1与活化的T细胞中的PD-1结合后，起到抑制T细胞的抗肿瘤作用。因此，阻断PD-1/PD-L1通路对于抑制或杀伤肿瘤有着积极作用。
本领域技术人员熟知，人体内的蛋白有可能产生一定突变，而仍保留其活性。因此，本发明所述的PD-1蛋白包括包含一定突变的PD-1蛋白，只要该突变的PD-1蛋白在人体内行使与野生型PD-1相同的功能。例如，在具体的实施方式中，本发明所述的PD-1蛋白具有SEQ ID NO:14所示氨基酸序列
(PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAE)，其相应的编码核苷酸序列如SEQ ID NO:13所示。
PD-1蛋白具有与其特异性结合的配体(PD-L1)。例如，在具体的实施方式中，本发明所述的PD-L1具有SEQ ID NO:12所示氨基酸序列。
本发明的多肽
本发明提供能够竞争性结合人PD-1蛋白的多肽，这些多肽具有结构上的相似性，即，在特定的位置具有特定的氨基酸残基。
在具体的实施方式中，本发明提供式I表示的多肽：
[Xaa0]-[Xaa1]-Asp-Tyr-[Xaa2]-Arg-[Xaa3]-Tyr-[Xaa4](I)
其中，[Xaa0]为Gly Asn、Thr Glu、或Phe Asn构成的肽段、或无；[Xaa1]为Trp、Lys或Met；[Xaa2]为Asn Ser Gln、Arg His Gly Asn Ile、Ser Leu Glu Glu Leu、Ser Trp Lys Ser Glu、Gln Asn、Cys Pro Cys、Cys His Gly Pro Cys、Cys Ala Glu Pro Leu、Ser Trp Cys Pro Cys构成的肽段；[Xaa3]为Ala Gln Leu、Met Lys Leu Ala、Glu Lys Ala Lys、 Leu Lys Glu Ala、或Glu Lys Cys Lys构成的肽段；[Xaa4]为Asn Gln、Asp Leu、Lys、Asp Leu、或Arg；这些多肽的长度为15-18个氨基酸。
这些多肽与人PD-1蛋白具有良好的结合亲和力，其中结合亲和力最高的多肽与人PD-1蛋白的天然配体PD-L1相当。在优选的实施方式中，所述多肽为氨基酸序列如SEQ ID NO:1-9中任一所示的多肽；或者将氨基酸序列如SEQ ID NO:1-9中任一所示的多肽经过1-6个，(较佳地1-4个)氨基酸残基的取代、缺失或添加而形成的，且具有PD-1结合活性的衍生多肽。
在具体的实施方式中，本发明的多肽与PD-1的结合KD值为1-5μM，优选1-4μM、更优选1-3μM，最优选与天然PD-L1与PD-1的结合KD值近似。
在优选的实施方式中，本发明的多肽选自氨基酸序列如SEQ ID NO:1-9中任一所示的多肽；更优选氨基酸序列如SEQ ID NO:1或6所示的多肽。
在本发明的多肽的基础上，本发明提供一种分离的核酸分子以及宿主细胞，所述核酸分子编码本发明的多肽，所述宿主细胞包含所述的分离的核酸分子。
本领域技术人员知晓如何选取宿主细胞并在其中进行蛋白质的表达，例如，所述宿主细胞包括但不限于：细菌、酵母、植物细胞、昆虫细胞或哺乳动物细胞。在具体的实施方式中，所述细菌包括但不限于：大肠杆菌、枯草杆菌；所述酵母包括但不限于：毕赤酵母、酿酒酵母。
由于本发明的多肽与人PD-1蛋白具有良好的结合亲和力，这些多肽能作为PD-1/PD-L1结合的抑制剂或调节PD-1/PD-L1结合程度的调节剂。因此，本发明还提供了一种药物组合物，所述药物组合物包含本发明的多肽以及药学上可接受的赋形剂。
本发明的多肽显然还可以与其它机理的阻断PD-1/PD-L1通路的药物以及其它抗肿瘤药物联用以增强彼此的作用。因此，上述药物组合物中还可包含药学上可接受的其它阻断PD-1/PD-L1通路的药物。例如，所述其它阻断PD-1/PD-L1通路的药物包括但不限于：靶向PD-1蛋白的nivolumab、pembrolizumab、pidilizumab，以及靶向PD-L1的MPDL3280A、MDX1105、MEDI4736等。在优选的实施方式中，所述的药物组合物还可包含其它抗肿瘤药物，所述抗肿瘤药物包括但不限于：Ipilimumab、ramucirumab、trametinib、ceritini、dabrafenib等。
本领域技术人员知熟知，本发明的药物组合物的具体剂型取决于要采用的给药方式。例如，本发明的药物组合物可采用的给药方式包括但不限于：注射给药、输注给药、腹膜内给药、瘤内给药、肌肉内给药，等等。而药物组合物中活性成分的含量可由医学领域的技术人员根据，例如患者的性别、年龄、总体健康状况等实际情况自主决定。
基于本发明的多肽的活性，本领域技术人员不难想到可将其制备成抑制PD-1与PD-L1结合的药物或抑制肿瘤的药物。在具体的实施方式中，所述肿瘤包括但不限于：黑色素瘤、肺癌(优选非小细胞肺癌)、肾癌、卵巢癌、前列腺癌、乳腺癌、结肠癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、直肠癌、肛门癌、胃癌、睾丸癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、何杰金氏病、非何杰金淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、急性髓性白血病、慢性髓性白血病、急性淋巴细胞白血病、慢性淋巴细胞白血病、小儿实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管生成、脊轴瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤。
在其它实施方式中，所述抑制PD-1与PD-L1结合的药物还可用于治疗细菌、病毒或真菌感染。例如，以下病毒引起的感染：肝炎病毒(甲型、乙型和丙型)、孢疹病毒、流感病毒、腺病毒、冠状病毒、麻疹病毒、登革热病毒、脊髓灰质炎病毒、狂犬病病毒等；以下细菌引起的感染：衣原体、立克次氏体、分枝杆菌、葡萄球菌、肺炎球菌、霍乱、破伤风等；或以下真菌引起的致病感染：假丝酵母、曲霉、皮炎芽酵母等。
在其它实施方式中，所述抑制PD-1与PD-L1结合的药物还可用于治疗炎性疾病，包括但不限于：强直性脊柱炎、自身免疫性溶血性贫血、关节炎、重症肌无力、系统性红斑狼疮、类风湿性关节炎、恶性贫血、多肌炎等。
在本发明的多肽或药物组合物的基础上，本发明进一步提供了一种抑制PD-1与PD-L1结合的方法，包括利用本发明的多肽或药物组合物抑制PD-1与PD-L1结合的步骤。在具体的实施方式中，所述抑制PD-1与PD-L1结合的方法是体外非治疗性的。
本发明的优点：
1.本发明提供了能够竞争性结合人PD-1蛋白的多肽；
2.本发明的多肽能与人PD-1蛋白良好结合，其中，结合亲和力最高的多肽与人PD-1蛋白的结合情况与天然PD-L1蛋白无异；
3.本发明的多肽具有较高的特异性、组织亲和性、不易产生免疫原性以及较好的组织渗透性；和
4.本发明的多肽的生产成本低。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(Cold Spring Harbor Laboratory Press，2001)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例
实施例1.本发明多肽的合成
(1)实验仪器与材料
二甲基甲酰胺(DMF)、哌啶、树脂、二氯甲烷(DCM)、茚三酮反应试剂、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)、三异丙基硅烷(TIS)、乙二硫醇(EDT)、无水乙醚、三氟乙酸(TFA)、N-甲基吗啉(NMM)、甲醇、乙醇、20种氨基酸、多肽固相合成管、质谱仪器MicrOTOF-Q11(BrukerDaltonics)。
(2)实验步骤
称量树脂并投入到多肽固相合成管中，加入适量的DMF溶胀半小时以上。抽掉DMF，用脱保护液(哌啶:DMF＝1:4)进行Fmoc去保护反应，置于摇床上10分钟。抽掉脱保护液，用DMF、DCM洗涤三次，从固相合成管中取出少量树脂于试管中，用乙醇洗涤2次，茚三酮法检测并记录颜色，准备投料，进行氨基酸缩合反应。分别按照SEQ ID NO:1-9所示的氨基酸序列取相应氨基酸、HBTU，并用少量的DMF溶解，投入到固相合成管中，加入10倍的NMM，搅拌反应。1-2小时后，从固相合成管中取少量树脂于试管中，用乙醇洗涤2次，茚三酮法检测。抽掉固相合成管中的液体，用DMF、甲醇、DCM各洗涤2次，得到第一个氨基酸缩合后的肽树脂。对所得肽树脂重复进行以上“Fmoc去保护、氨基酸缩合”反应步骤，至最后一个氨基酸反应完毕，得到氨基酸序列如SEQ ID NO:1-9所示的肽。反应完毕后，DMF、甲醇、DCM各洗涤2次，继续抽干10-15分钟。固相合成管中取出合成完的部分肽树脂，于室温下在裂解液(TFA 94.5％；水2.5％；EDT 2.5％；TIS 1％)中裂解两小时。将树脂过滤后，于旋蒸仪中蒸干，用无水乙醚洗涤6次，然后常温挥干。粗肽使用分析级HPLC纯化，使用HPLC检测纯度>90％。所得的纯肽使用质谱鉴定。最后将纯化后的溶液冻干，即可得到纯品。表1列出了本发明多肽的分子量的理论值和质谱测定的实际值。
实施例3.人的PD-1重组质粒的构建
目的基因为PD-1蛋白上34-150位氨基酸的编码核酸。利用NcoI和NdeI两个酶切位点设计的特异性引物将目的片段克隆到pET-28a载体(Novagen公司)上。正向引物为：5’-TTTTCCATGGGTCCCCCCACCTTCTCCCCAG-3’(SEQ ID NO:10)；反向引物为：5’-GCCGCCGCATATGTTATTCTGCCCTTCTCTCTG-3’(SEQ ID NO:11)。以人的PD-1基因为模板，进行PCR扩增。PCR体系为：2μL PD-1质粒，2μL正向引物，2μL反向引物，5μL 10×缓冲溶液，4μL dNTP，1μL pfu聚合酶，34μL ddH2O。PCR扩增条件：94℃预变性4min；94℃变性30s，60℃退火30s，72℃延伸48s，30个循环；72℃ 10min。PCR扩增完毕后，进行核酸电泳，切下目的条带，用PCR产物回收试剂盒回收PCR产物。将所得的PCR 产物和质粒pET-28a载体分别用NcoI和NdeI两个限制性核酸内切酶进行双酶切，然后在T4连接酶的作用下连接，转化到大肠杆菌DH5α感受态细胞中。过夜培养后，挑取单克隆进行菌液PCR鉴定(结果见图1)，然后取阳性重组子测序，保留菌株和甘油菌。通过重组质粒构建的人的PD-1具有如SEQ ID NO:13所示的核苷酸序列。
实施例4.人的PD-1蛋白的表达与纯化
本发明所用到的人的PD-1蛋白是经原核生物表达、纯化后获得的。具体实验步骤如下：将测序正确的重组质粒转化到E.coli BL21感受态细胞中进行诱导表达。挑取单个菌落接种于含有卡那霉素的TB培养基中，于37℃振荡培养过夜。次日将小培产物扩培到1L的TB培养基中，培养至OD600为0.5-0.6，加入诱导剂IPTG至终浓度为0.5mM，37℃诱导5-7h。在4,000rpm下收菌，先用裂解缓冲溶液(50mM Tris-HCl，pH 8.0，50mM NaCl,，1mM DTT，0.5mM EDTA，5％甘油)裂解细菌，然后再高压破碎，在12,000rpm下离心取沉淀。用洗杂缓冲液(20mM Tris-HCl，pH 8.0，2M尿素，2.5％Triton X-100)洗杂两次，取沉淀。然后再用溶解缓冲溶液(20mM Tris-HCl，pH 8.0，8M尿素)溶解蛋白，离心取上清。取3kDa的蛋白超滤浓缩管浓缩蛋白至5mL左右，将其加入到1L的复性缓冲液(50mM Tris-HCl，pH 8.0，50mM L-Arg，24mM NaCl，1mM KCl，1mM EDTA)中，用稀释法在4℃下复性24h。用3kDa的浓缩管浓缩蛋白，至20mL左右，将蛋白装入透析袋中，在透析缓冲液(50mM Tris-HCl，pH 8.0，150mM NaCl，1mM DTT)中透析过夜，浓缩。对获得的蛋白过阳离子交换柱和分子筛进行纯化。将过完分子筛后的蛋白进行14％SDS-PAGE凝胶电泳，鉴定纯化后的蛋白纯度。所得电泳图如图2所示，蛋白大小约为13KDa，与人PD-1蛋白的理论值吻合，说明成功获得了人PD-1蛋白。
实施例5.人PD-1蛋白与本发明多肽的SPR结合实验
本发明的多肽与人PD-1蛋白的结合通过Biacore T200进行表面等离子体共振(Surface Plasmon Resonances,SPR)实验测定。具体实验步骤如下：用10mM醋酸钠溶液将纯化好的PD-1蛋白稀释到50μg/ml，使用氨基偶联试剂盒将PD-1蛋白偶联到CM5芯片上，流速为10μl/min，结合时间为420s，最后的偶联量约为5000RU。结合实验是在含有0.05％的表面活性剂P20的PBS溶液(137mM NaCl，2.7mM KCl，8mM Na2HPO4，2mM KH2PO4，pH 7.4)中进行的。实验温度设定为25℃，流速为30μl/min，结合时间90s，解离时间120s。将多肽用运行溶液溶解后，成倍稀释成不同浓度，随着运行溶液结合到PD-1上。得到的结合数据用BIAevaluation2.0软件分析，利用稳态拟合得到亲和力KD值。本发明的多肽与人PD-1蛋白的SPR结合实验的实验结果如图3a-3e以及图5a-5d所示。表 1列出了本发明的多肽的序列及SPR实验测得的KD值。
本发明人同时检测了人PD-1蛋白与人PD-L1蛋白的结合亲和力KD值，其为1.15μM(如图3f所示)。
表1.本发明的多肽序列、分子量及它们与人PD-1蛋白结合的KD值
Figure PCTCN2017078162-appb-000001
实施例6.人PD-L1蛋白与SEQ ID NO:1所示多肽竞争性结合人PD-1蛋白的SPR实验
根据实施例5中的SPR实验结果，SEQ ID NO:1所示多肽与人PD-1蛋白的结合亲和力最高。因而，本发明人以SEQ ID NO:1所示多肽为例，探究了该肽阻断人PD-1蛋白与人PD-L1蛋白结合的能力。人PD-L1蛋白购自北京义翘神州生物技术有限公司。
具体实验步骤如下：首先将50μg/ml人PD-L1蛋白通过氨基偶联的方法偶联到CM5芯片表面；然后将不同浓度的SEQ ID NO:1所示多肽(125nM，62.5nM，31.25nM，15.60nM，0nM)分别与100nM的人PD-1蛋白在冰上孵育15min，随运行缓冲溶液(10mM磷酸盐缓冲液，2.7mM KCl，137mM NaCl和0.05％表面活性剂P20，pH 7.4)流经芯片表面，流速为30μl/min，结合时间为90s，解离时间为120s；最后用BIAevaluation2.0软件分析实验结果。实验结果如图4所示，随着多肽浓度的升高，溶液中未结合的PD-1蛋白浓度逐渐下降，从而使得PD-1与PD-L1的结合减弱，响应值随之下降。SPR实验结果如图4所示，该结果表明SEQ ID NO:1所示多肽能够阻断人PD-1与PD-L1的结合。
讨论：本发明提供了靶向人PD-1蛋白，即，能够竞争性结合人PD-1蛋白的多肽，各多肽在特定的位置具有特定的氨基酸残基，从而能够具备相似的结构。这些多肽均能与人PD-1蛋白发生良好的结合，其中，SEQ ID NO:1所示多肽与人PD-1蛋白的结合亲和力最高，其KD值为1.38μM与PD-L1和PD-1的结合KD值相当。因此，这些多肽能作为PD-1/PD-L1结合的抑制剂或调节PD-1/PD-L1结 合程度的调节剂，以及为研究PD-1/PD-L1免疫检查点阻断剂和设计、开发阻断PD-1/PD-L1通路提供了多肽类先导药物。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种式I表示的多肽：
    [Xaa0]-[Xaa1]-Asp-Tyr-[Xaa2]-Arg-[Xaa3]-Tyr-[Xaa4] (I)
    其中，
    [Xaa0]为Gly Asn、Thr Glu、或Phe Asn构成的肽段、或无；
    [Xaa1]为Trp、Lys或Met；
    [Xaa2]为Asn Ser Gln、Arg His Gly Asn Ile、Ser Leu Glu Glu Leu、Ser Trp Lys Ser Glu、Gln Asn、Cys Pro Cys、Cys His Gly Pro Cys、Cys Ala Glu Pro Leu、Ser Trp Cys Pro Cys构成的肽段；
    [Xaa3]为Ala Gln Leu、Met Lys Leu Ala、Glu Lys Ala Lys、Leu Lys Glu Ala、或Glu Lys Cys Lys构成的肽段；
    [Xaa4]为Asn Gln、Asp Leu、Lys、Asp Leu、或Arg；
    并且所述多肽具有PD-1结合活性，所述多肽的长度为15-18个氨基酸。
  2. 如权利要求1所述的多肽，其特征在于，所述多肽选自下组：
    (a)氨基酸序列如SEQ ID NO:1-9中任一所示的多肽；
    (b)将(a)所述的多肽经过1-6个(较佳地1-4个)氨基酸残基的取代、缺失或添加而形成的，且具有PD-1结合活性的由(a)衍生的多肽。
  3. 如权利要求1所述的多肽，其特征在于，所述多肽选自氨基酸序列如SEQ ID NO:1-9中任一所示的多肽。
  4. 如权利要求3所述的多肽，其特征在于，所述多肽是氨基酸序列如SEQ ID NO:1或6所示的多肽。
  5. 一种分离的核酸分子，所述核酸分子编码权利要求1-4中任一项所述的多肽。
  6. 一种宿主细胞，所述宿主细胞包含权利要求5所述的分离的核酸分子。
  7. 一种药物组合物，其特征在于，所述药物组合物包含权利要求1-4中任一项所述的多肽以及药学上可接受的赋形剂。
  8. 权利要求1-4中任一项所述的多肽的用途，用于制备抑制PD-1与PD-L1结合或抑制肿瘤、或治疗细菌、病毒或真菌引起的感染或治疗炎性疾病的药物。
  9. 如权利要求8所述的用途，其特征在于，所述肿瘤包括但不限于：黑色素瘤、肺癌(优选非小细胞肺癌)、肾癌、卵巢癌、前列腺癌、乳腺癌、结肠癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、直肠癌、肛门癌、胃癌、睾丸癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、何杰金氏病、非何杰金淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、急性髓性白血病、慢性髓性白血病、急性淋巴细胞白血病、慢性淋巴细胞白血病、小儿实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、 肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管生成、脊轴瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤；
    所述病毒包括但不限于：肝炎病毒(甲型、乙型和丙型)、孢疹病毒、流感病毒、腺病毒、冠状病毒、麻疹病毒、登革热病毒、脊髓灰质炎病毒、狂犬病病毒；
    所述细菌包括但不限于：衣原体、立克次氏体、分枝杆菌、葡萄球菌、肺炎球菌、霍乱、破伤风；
    所述真菌包括但不限于：假丝酵母、曲霉、皮炎芽酵母；或者
    所述炎性疾病包括但不限于：强直性脊柱炎、自身免疫性溶血性贫血、关节炎、重症肌无力、系统性红斑狼疮、类风湿性关节炎、恶性贫血、多肌炎。
  10. 一种抑制PD-1与PD-L1结合的方法，包括步骤：利用权利要求1-4中任一项所述的多肽或权利要求6所述的药物组合物抑制PD-1与PD-L1结合。
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