WO2021143695A1 - 一种抗肿瘤融合蛋白及其制法和应用 - Google Patents

一种抗肿瘤融合蛋白及其制法和应用 Download PDF

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WO2021143695A1
WO2021143695A1 PCT/CN2021/071335 CN2021071335W WO2021143695A1 WO 2021143695 A1 WO2021143695 A1 WO 2021143695A1 CN 2021071335 W CN2021071335 W CN 2021071335W WO 2021143695 A1 WO2021143695 A1 WO 2021143695A1
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fusion protein
tat
polypeptide
tumor
shp2
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PCT/CN2021/071335
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English (en)
French (fr)
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张英起
范富林
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广东泰禾医药科技有限公司
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Priority to JP2022543131A priority Critical patent/JP2023510893A/ja
Priority to US17/758,676 priority patent/US20230226157A1/en
Priority to EP21741405.1A priority patent/EP4101862A4/en
Publication of WO2021143695A1 publication Critical patent/WO2021143695A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03048—Protein-tyrosine-phosphatase (3.1.3.48)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/72—Fusion polypeptide containing domain for protein-protein interaction containing SH2 domain
    • C—CHEMISTRY; METALLURGY
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the present invention relates to the field of biology and medicine, and more specifically to an anti-tumor fusion protein and its preparation method and application.
  • Immune checkpoint blockers have become an important target for tumor immunotherapy.
  • Immune checkpoint blockade therapy targeting PD-1/PD-L1 uses the body's own immune system to resist and fight malignant tumors, which is substantially improved
  • multiple studies have reported that only 20%-30% of a small number of patients respond to the blocker, and some patients develop resistance after receiving drug treatment.
  • the development of new immune checkpoint blocking drugs and the combination strategy for different immune checkpoint drugs have become a new way to improve the response rate of patients.
  • the purpose of the present invention is to provide a more effective anti-tumor fusion protein.
  • a fusion protein in the first aspect of the present invention, has a structure shown in formula I or II from N-terminus to C-terminus:
  • Z1 is a CPP component
  • L is no or connecting element
  • Z2 is the SH2 domain of SHP2 and/or SHP1 or an active fragment thereof;
  • the Z1 and Z2 are connected in a head-to-head, head-to-tail, tail-to-head, or tail-to-tail manner.
  • the "head” refers to the N-terminus of the polypeptide or fragments thereof, especially the N-terminus of the wild-type polypeptide or fragments thereof.
  • the "tail” refers to the C-terminus of the polypeptide or fragments thereof, especially the C-terminus of the wild-type polypeptide or fragments thereof.
  • the Z1 and Z2 are D-type or L-type amino acids.
  • the Z1 and Z2 are L-type or D-type amino acids.
  • the Z1 and Z2 are L-type or D-type amino acids.
  • the CPP element is derived from a human or non-human mammal.
  • the CPP element includes wild type and mutant type.
  • the CPP element includes a full-length, mature form of CPP, or an active fragment thereof, preferably 5-30 amino acids in length.
  • the CPP element is a TAT penetrating peptide.
  • the CPP element is selected from the group consisting of polyarginine (R5-9), MPG, CADY, pVEC, Penetratin, Kaposi fibroblast growth factor peptide, VP22, or a combination thereof.
  • sequence of the CPP element is shown in any one of SEQ ID NO.: 1, 15-21.
  • L is composed of glycine and serine.
  • L has the following structure: -(Gly-Ser)n-; where n is 1-5, preferably 1-3.
  • L has the following structure: -(Gly-Ser-Ser-Ser-Ser)n-; where n is 1-5, preferably 1-3.
  • L contains 1-6 prolines, preferably 1-3 prolines.
  • the Z2 is derived from human or non-human mammals.
  • the Z2 includes wild type and mutant type.
  • the Z2 includes a full-length, mature form of SHP2 or SH2 domain of SHP1, or an active fragment thereof.
  • Z2 is selected from the following group: N-terminal SH2 domain, C-terminal SH2 domain, or a combination thereof.
  • Z2 also includes the ITIM motif binding region of the C-terminal SH2 domain.
  • Z2 is the N-terminal SH2 domain of SHP2, which has amino acids 1-99 or 9-51 in SEQ ID NO.: 2, and preferably contains the 9th amino acid in SEQ ID No.: 2. , 28, 49 and 51 amino acids, and the length is 43-99 amino acids.
  • Z2 is the C-terminal SH2 domain of SHP2, which has amino acids 27-39 or 29-37 in SEQ ID NO.: 3, and preferably contains amino acids 29 and 29 in SEQ ID NO.: 3. 31, 36 and 37 amino acids, and the length is 9-108 or 13-108 amino acids.
  • Z2 is the N-terminal SH2 domain of SHP1, which has amino acids 9-51 in SEQ ID NO.: 12, and preferably contains the 9th, 28th, 49th, and 49th amino acids in SEQ ID NO.: 12. 51 amino acids, and the length is 43-99 amino acids.
  • Z2 is the C-terminal SH2 domain of SHP1, which has amino acids 29-37 in SEQ ID NO.: 13, and preferably contains 29, 31, 36, and 29 in SEQ ID NO.: 13. 37 amino acids, and the length is 9-107 amino acids.
  • Z2 has the structure shown in formula III from the N end to the C end:
  • A is the N-terminal SH2 domain of SHP2 or SHP1 or its active fragment
  • L is no or connecting element
  • B is the C-terminal SH2 domain of SHP2 or SHP1 or its active fragment
  • element A is the N-terminal SH2 domain of SHP2, which has positions 1-99 or 9-51 in SEQ ID NO.: 2, and preferably contains No. 9 in SEQ ID No.: 2. , 28, 49 and 51, and the length is 43-99 amino acids.
  • element B is the C-terminal SH2 domain of SHP2, has the sequence shown in SEQ ID NO.: 3, and contains positions 27-39 or 29-37 in SEQ ID NO.: 3 It preferably contains the 29th, 31st, 36th and 37th positions in SEQ ID NO.: 3, and the length is 9-108 or 13-108 amino acids.
  • element A is the N-terminal SH2 domain of SHP1, which has amino acids 9-51 in SEQ ID NO.: 12, and preferably contains the 9th, 28th, and 49th amino acids in SEQ ID NO.: 12 , 51 amino acids, and the length is 43-99 amino acids.
  • element B has the C-terminal SH2 domain of SHP1, has amino acids 29-37 in SEQ ID NO.: 13, and preferably contains 29, 31, and 36 in SEQ ID NO.: 13 , 37 amino acids, and the length is 9-107 amino acids.
  • the length of the L'is 1-20aa preferably, 3-15aa, more preferably, 5-10aa.
  • amino acid sequence of L' is selected from the following group:
  • Z2 has the sequence shown in SEQ ID NO.: 11 or 14.
  • sequence of Z2 is shown in SEQ ID NO.: 11 or 14.
  • the length of the peptide linker is 0-10 amino acids, preferably 0-5 amino acids.
  • the fusion protein is selected from the following group:
  • (B) It has ⁇ 80% homology (preferably ⁇ 90% homology; preferably ⁇ 95% homology) to the amino acid sequence shown in any one of SEQ ID NO: 4-8; most preferably, ⁇ 97% homology, such as 98% or more, 99% or more), and the polypeptide has tumor suppressor activity;
  • (C) A derivative polypeptide formed by substituting, deleting or adding 1-5 amino acid residues to the amino acid sequence shown in any one of SEQ ID NOs: 4-8 and retaining tumor suppressor activity.
  • amino acid sequence of the fusion protein is shown in any one of SEQ ID NO.: 4-8.
  • the second aspect of the present invention provides an isolated polynucleotide which encodes the fusion protein of the first aspect of the present invention.
  • the polynucleotide additionally contains an auxiliary element selected from the group consisting of signal peptide, secretory peptide, tag sequence (such as 6His), or flank of the ORF of the mutein or fusion protein. Its combination.
  • the polynucleotide is selected from the following group: DNA sequence, RNA sequence, or a combination thereof.
  • the third aspect of the present invention provides a vector containing the polynucleotide according to the second aspect of the present invention.
  • the vector includes one or more promoters, which are operably linked to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, and selectable marker. , Nucleic acid restriction sites, and/or homologous recombination sites.
  • the vectors include plasmids and viral vectors.
  • the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.
  • AAV adeno-associated virus
  • adenovirus adenovirus
  • lentivirus lentivirus
  • retrovirus lentivirus
  • herpes virus SV40
  • poxvirus poxvirus
  • the vector includes an expression vector, a shuttle vector, and an integration vector.
  • the fourth aspect of the present invention provides a host cell that contains the vector of the third aspect of the present invention, or the polynucleotide of the second aspect of the present invention is integrated into its genome.
  • the host cell is a eukaryotic cell, such as a yeast cell, a plant cell or a mammalian cell (including human and non-human mammals).
  • the host cell is a prokaryotic cell, such as Escherichia coli.
  • the yeast cells are selected from one or more sources of yeast from the following group: Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cells include:
  • the yeast Luveyi is more preferably Kluyveromyces marxianus, and/or Kluyveromyces lactis.
  • the host cell is selected from the group consisting of Escherichia coli, wheat germ cells, insect cells, SF9, Hela, HEK293, CHO, yeast cells, or a combination thereof.
  • the fifth aspect of the present invention provides a method for producing the fusion protein of the first aspect of the present invention, and the method includes the steps:
  • the host cell according to the fourth aspect of the present invention is cultured to express the fusion protein; and/or the fusion protein is isolated.
  • the sixth aspect of the present invention provides a pharmaceutical composition, which contains the fusion protein described in the first aspect of the present invention and a pharmaceutically acceptable carrier thereof.
  • the pharmaceutical composition further includes other drugs for inhibiting tumor activity.
  • other drugs used to inhibit tumor activity are selected from the following group: PD-1 antibody, PD-L1 antibody, HER2 monoclonal antibody, BTLA antibody, CTLA-4 antibody, CD47 antibody, NKG2A antibody, NKTR- 214, GDF-15 antibody, LILRB4 antibody, LAIR1 antibody, Tim-3 antibody, Lag-3 antibody, Tight antibody, CD160 antibody, KLRG-1 antibody, GP49B antibody, CD31 antibody, CD38 antibody, Lair-1 antibody, CD200/ CD200R antibody, Catumaxomab, Blinatumomab, EGFR monoclonal antibody, CD20 monoclonal antibody, VEGF/VEGFR monoclonal antibody, Licartin, Zevalin, Bexxar, Mylotarg, Kadcyla, Abxi Generic, Conbercept, Apalutamide (ARN-509), Rova-T, TNF ⁇ , IFN ⁇ , IL-2, T ⁇ 4, small molecule inhibitors targeting EGFR, pac
  • the PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, teriplizumab, and Dilimumab, carrelizumab, tislelizumab, or a combination thereof.
  • the PD-L1 antibody is selected from the group consisting of atezolizumab, durvalumab, avelumab, or a combination thereof.
  • the HER2 monoclonal antibody is selected from the group consisting of trastuzumab, pertuzumab, T-DM1, or a combination thereof.
  • the CTLA-4 antibody includes Ipilimumab.
  • the EGFR monoclonal antibody includes Necitumumab, Panitumumab, Nimotuzumab, and Cetuximab.
  • the CD20 monoclonal antibody is selected from the group consisting of rituximab, ibrituximab, tositumomab, ofatumumab, orelizumab, attuzumab Monoclonal antibodies, or combinations thereof.
  • the VEGF/VEGFR monoclonal antibody is selected from the group consisting of bevacizumab, ramucirumab, ranibizumab, or a combination thereof.
  • the EGFR-targeting small molecule inhibitor is selected from the group consisting of erlotinib, gefitinib, icotinib, afatinib (including afatinib maleate) Tinib, dacomitinib, ossitinib, toartinib (Nazartinib), lenatinib (including lenatinib maleate), sorafenib (Sorafenib), apatinib (as Ai Tan, apatinib mesylate tablets), imatinib, sunitinib, dasatinib, lapatinib, pezopanib, crizotinib, vandetanib, regorafi Ni, Axitinib, Pranatinib, Lenatinib, Zebutinib, Anlotinib, Ceritinib, Fruquintinib, Pirrotinib, Lenvati
  • the seventh aspect of the present invention provides the fusion protein according to the first aspect of the present invention, the polynucleotide according to the second aspect of the present invention, the vector according to the third aspect of the present invention, and the host cell according to the fourth aspect of the present invention.
  • Use, for preparing a composition or preparation the composition or preparation is used to treat or prevent tumors.
  • composition or preparation is also used for one or more purposes selected from the following group:
  • the composition is a pharmaceutical composition.
  • the tumor is selected from the group consisting of breast cancer, colon cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, ovarian cancer, prostate cancer, kidney cancer, liver cancer, brain cancer, and melanoma , Multiple myeloma, leukemia, lymphoma, head and neck tumor, thyroid cancer, or a combination thereof.
  • the tumor cells are derived from one or more tumors selected from the group consisting of breast cancer, colon cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, ovarian cancer, prostate cancer, Kidney cancer, liver cancer, brain cancer, melanoma, multiple myeloma, leukemia, lymphoma, head and neck tumor, thyroid cancer, or a combination thereof.
  • the eighth aspect of the present invention provides a non-therapeutic in vitro method for inhibiting tumor growth, including the step of culturing tumor cells in the presence of the fusion protein of the first aspect of the present invention, thereby inhibiting tumor growth.
  • the tumor cells are derived from one or more tumors selected from the group consisting of breast cancer, colon cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, ovarian cancer, prostate cancer, Kidney cancer, liver cancer, brain cancer, melanoma, multiple myeloma, leukemia, lymphoma, head and neck tumor, thyroid cancer, or a combination thereof.
  • the tumor cell is a cell cultured in vitro.
  • the ninth aspect of the present invention provides a method for treating tumors, including the step of administering the fusion protein according to the first aspect of the present invention to a subject in need.
  • the fusion protein is administered in the form of monomer and/or dimer.
  • the subject is a human.
  • Figure 1 shows the results of pET-22b(+)TAT-SHP2–N-SH2 double restriction digestion.
  • Figure 2 shows the sequencing results of the recombinant plasmid pET-22b(+)TAT-SHP2-N-SH2.
  • FIG. 3 shows the expression of the target protein detected by SDS-PAGE
  • Figure 4 shows the SDS-PAGE to detect the expression of the target protein
  • Figure 5 shows the separation of target protein and impurity protein by cation exchange chromatography
  • SP1-SP4 are the elution peaks, and SP4 is the peak of the target protein.
  • FIG. 6 shows the purification effect of TAT-SHP2-N-SH2 detected by SDS-PAGE
  • FIG. 7 shows the Western-blot identification of SHP2-N-SH2 target protein
  • Figure 8 shows the HPLC purification chromatographic analysis results of the fusion protein.
  • Figure 9 shows the HPLC purification chromatographic analysis results of peptides
  • Figure 10 shows the molecular weight of polypeptides detected by mass spectrometry; A, L1 polypeptide; B, L2 polypeptide; C, D1 polypeptide; D, D2 polypeptide.
  • Figure 11 shows the laser confocal detection of the localization of fusion proteins and polypeptides in T cells
  • a Observe the ability of the control peptide without TAT transmembrane sequence (20 ⁇ g/mL) to enter the cell under a confocal microscope; b. SHP2-C-SH2 with TAT sequence; c. SHP2-N-SH2 with TAT sequence ; The action time is 0, 0.5, 4, 24, 72, 96, 120 and 144h (25 ⁇ m ( ⁇ 60).
  • Figure 12 shows the effect of recombinant TAT-N-SH2 fusion protein and L1 polypeptide on the proliferation of T lymphocytes
  • the CFSE method detects the effects of different treatment groups on the proliferation of T lymphocytes. d According to the results of flow cytometry, the proportion of T cell proliferation was counted. Compared with the control group, p>0.05 was not statistically significant.
  • Figure 13 shows the effect of recombinant TAT-N-SH2 fusion protein and L1 polypeptide on T lymphocyte apoptosis
  • ad Annexin V-FITC detects the effect of polypeptide on T lymphocyte apoptosis, and resuspended cells are added to different groups to treat the non-administered group SHP2-NC, SHP2-N-SH2 (80 ⁇ g/mL), TAT-SHP2-C-SH2 polypeptide (80 ⁇ g/mL), continue on the culture plate stimulated by EASY-T (add 50ng PD-L1 antibody 3h before adding the cell mixture to coat the 24-well plate at 37°C) for 3 days, collect the cells, stain with FITC and PI and pass Flow cytometry for detection; e According to the flow cytometry results, count the proportion of apoptotic cells, compared with the control group, *p ⁇ 0.05 or **p ⁇ 0.01.
  • Figure 14 shows the effect of fusion proteins and polypeptides on the phosphorylation level of downstream signal molecules mediated by T cells
  • TAT-SHP2-N-SH2 fusion protein treatment group a. TAT-SHP2-N-SH2 fusion protein treatment group; c. TAT-SHP2C-SH2 polypeptide treatment group; b, d.
  • ⁇ -actin is used as an internal reference to treat JNK mediated by T lymphocytes , AKT, ERK protein phosphorylation expression for grayscale analysis. Unpaired t-test, compared with the negative control, **p ⁇ 0.01, there is no statistical difference in NS.
  • Figure 15 shows the detection of the cytokine IL-2 secreted by T cells
  • Figure 16 shows the results of the direct tumor killing effect test.
  • Figure 17 shows the killing effect of TAT-SHP2-N-SH2 fusion protein on different tumor cell lines stimulated by T cells. Compared with the non-administered group, *p ⁇ 0.05 or **p ⁇ 0.01 or ***p ⁇ 0.001, p>0.05 was not statistically significant.
  • Figure 18 shows the killing effect of TAT-SHP2-C-SH2 polypeptide stimulated T cells on different tumor cell lines. Compared with the non-administered group, *p ⁇ 0.05 or **p ⁇ 0.01 or ***p ⁇ 0.001, p>0.05 was not statistically significant.
  • Figure 19 shows the killing effect of L1, L2, D1, D2 polypeptide on lung cancer cell H460 stimulated by T cells;
  • Figure 20 shows the anti-tumor effect of TAT-SHP2-N-SH2 fusion protein on colon cancer-bearing mice
  • mice in the different dose administration group a. After 30 days of dissection, the tumor weight of mice in the different dose administration group; b. The tumor size; Unpaired t-test, compared with the control group, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 and ** **p ⁇ 0.0001.
  • Figure 21 shows the anti-tumor effect of TAT-SHP2-C-SH2 polypeptide on colon cancer-bearing mice
  • mice in different dose groups a. After 30 days of dissection, the tumor weight of mice in different dose groups; b. tumor size; Unpaired t-test, compared with the control group, **p ⁇ 0.01, ***p ⁇ 0.001 and ****p ⁇ 0.0001 .
  • Figure 22 shows the anti-tumor effect of TAT-SHP2-N-SH2 fusion protein and TAT-SHP2-C-SH2 polypeptide on breast cancer tumor-bearing mice;
  • Figure 23 shows the anti-tumor effect of recombinant TAT-N-SH2 fusion protein and L1 polypeptide on colon cancer nude mice;
  • a Tumor weight of mice after 30 days of dissection; b. Tumor size; c. Tumor growth curve; Unpaired t-test, compared with the control group, NS: no statistical difference.
  • Figure 24 shows the anti-tumor effect of recombinant TAT-N-SH2 fusion protein and L1 polypeptide on breast cancer in nude mice;
  • Figure 25 shows the anti-tumor effect of recombinant TAT-N-SH2 fusion protein and L1 polypeptide on PD-L1 knockout breast cancer tumor-bearing mice;
  • Figure 26 shows the detection of the anti-tumor effect of L1, L2, D1, D2 polypeptide on mouse lung cancer
  • a Tumor weight of mice after 21d anatomy; b. Tumor size; c. Tumor growth curve; Unpaired t-test, compared with the control group, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • the inventors unexpectedly discovered a fusion protein that contains a CPP element, an optional connecting element, and the SH2 domain of SHP2 or SHP1 or active fragments thereof.
  • the fusion protein obtained by the present invention has extremely Excellent tumor killing activity, and the fusion protein of the present invention can also significantly enhance the killing effect of T cells on tumor cells.
  • the present invention has been completed on this basis.
  • CPP Cell Penetration Peptide
  • the cell membrane is the main barrier between the cell and the extracellular environment, and because of this natural barrier, it is difficult for many biological macromolecules to enter the cell through the cell membrane, which is a major obstacle to targeted drug delivery at this stage.
  • Cell Penetration Peptide is a type of polypeptide composed of 5-30 amino acid residues and has the ability to penetrate the cell membrane. It can carry biological macromolecules such as proteins and nucleic acids into cells. CPP-mediated entry of biological macromolecules into cells is safer and more efficient than traditional methods, and is currently the most successful delivery system that can directly transport biological macromolecules in vivo and in vitro.
  • TAT arginine-rich polypeptide
  • HAV-1 human immunodeficiency virus
  • TAT-PTD TAT-protein transduction domain
  • CPP can be divided into three categories according to its chemical properties: (1) Cationic, rich in arginine and lysine residues, and has a strong positive charge at physiological pH, mainly including TAT, R9, hLF, etc. (2) The positive charge of amphiphilic peptides is provided by lysine residues. This type of CPP contains hydrophobic and hydrophilic domains, and its amphiphilic characteristics are shared by its primary structure and secondary structure. The decision mainly includes MPG, CADY, pVEC, SAP, etc. (3) Hydrophobic CPPs have a relatively low net charge and are mainly composed of non-polar amino acids. Such as Kaposi fibroblast growth factor (K-FGF) peptide.
  • K-FGF Kaposi fibroblast growth factor
  • the CPP element of the present invention is a TAT penetrating peptide.
  • the CPP element of the present invention is selected from the group consisting of polyarginine (R5-9), MPG (from the fusion protein domain of the human autoimmune deficiency virus type 1 gp41 protein and the simian vacuole virus
  • the regional composition of the nuclear localization sequence of the large T antigen, the amino acid sequence is: GLAFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO.: 15)), CADY (a secondary amphipathic consisting of 20 amino acids containing tryptophan residues and arginine residues) Peptide, amino acid sequence: GLWRALWRLLRSLWRLLWKA (SEQ ID NO.16)-CyA), pVEC (derived from murine vascular endothelial cell cadherin, amino acid sequence: LLIILRRRIRKQAHAHSK (SEQ ID NO.: 17)), Penetratin (Drosophila)
  • the third alpha helix in the antenna homeotype transcription factor protein has
  • ITIM Immune receptor tyrosine inhibitory motif
  • ITIM Immunorector tyrosine-based inhibitor motifs
  • ITIM is a common structural domain that inhibits many immune checkpoints, and it is also a key target for the design of universal drugs for immune checkpoints.
  • SHP1 and SHP2 are highly homologous and are often recruited to the same binding site. The different roles of SHP1 and SHP2 phosphatases are currently unclear.
  • Protein tyrosine phosphatase-1 (SH2 domain-containing protein tyrosine phosphates, SHP1) is mainly expressed in various human hematopoietic cells and is an important negative regulator that controls the level of phosphorylated tyrosine in the signal transduction pathway between lymphocytes.
  • SHP1 contains two SH2 domains, N-SH2 (3-101) and C-SH2 (109-215), a catalytic (PTP) domain (272-514), a proline-rich group and Tyrosine phosphorylation site tail.
  • SHP2 is a protein tyrosine phosphatase encoded by the human non-receptor protein tyrosine phosphatase 11 (Protein Tyrosine Phosphatase, Non Receptor Type 11, PTPN11) gene. It contains two SH2 domains, namely N-SH2 ( 5 ⁇ 103) and C-SH2 (111 ⁇ 218), a catalytic (PTP) domain (276 ⁇ 523), a tail rich in proline groups and tyrosine phosphorylation sites.
  • the PTPN11 gene includes 16 exons and produces a widely expressed 7 kb transcript, which contains a 1.779 bp open reading frame encoding a protein of 593 amino acids.
  • human and mouse SHP2 gene sequence homology is 100%.
  • the N-SH2 domain of SHP2/SHP1 In the inactive state (inactive) the N-SH2 domain of SHP2/SHP1 is in extensive contact with the catalytic domain PTP through charge-charge interactions, and part of the SH2 domain (NXGDY/F motif) is inserted into the catalytic cleft, preventing the bottom The substance enters the active site and loses its catalytic activity.
  • the binding of the ligand containing phosphorylated tyrosine residues to the N-SH2 domain can cause the allosteric switch in the SH2 domain to change from the inactive state to the active state.
  • This conformational change of the N-SH2 domain of SHP2/SHP1 disrupts the interaction between the SH2 domain and the phosphatase domain, so that its inhibition is eliminated and the substrate is allowed to enter.
  • the present invention to block the binding of N-SH2 and C-SH2 domains of SHP2/SHP1 in immune cells to ITIM as the entry point, genetic engineering and polypeptide solid-phase synthesis technology are used to design and prepare the binding of ITIM.
  • the penetrating peptide TAT is fused at the N-terminus of the fusion protein and the C-terminus of the polypeptide.
  • the transactivator protein (TAT) of HIV1 can efficiently and quickly introduce polypeptides and proteins into the cell without affecting the normal structure and function of the cell.
  • the fusion protein and polypeptide After the fusion protein and polypeptide enter the cell, they can compete with the SHP2/SHP1 normally expressed in T lymphocytes to bind to the ITIM of the inhibitory receptor intracellular segment, so that the SHP2/SHP1 in the immune cell is always in an inactive state, thereby making immunity
  • the inhibitory effect of the inhibitory receptor is inactivated, which is the development idea of the general immune checkpoint inhibitor designed by the present invention with ITIM as the target.
  • fusion protein of the present invention or “polypeptide” refers to the fusion protein described in the first aspect of the present invention.
  • the structure of the fusion protein is as shown in Z1-L-Z2(I) or Z2-L-Z1(II), where B is the CCP protein; L is no or connecting element; and Z2 It is the SH2 domain of SHP2 or SHP1 or its active fragment.
  • the fusion protein has an amino acid sequence as shown in any one of SEQ ID NO.: 4-8.
  • fusion protein also includes variant forms of the fusion protein (such as the sequence shown in any one of SEQ ID NO.: 4-8) having the above-mentioned activities. These variant forms include (but are not limited to): 1-3 (usually 1-2, more preferably 1) amino acid deletion, insertion and/or substitution, and addition or addition at the C-terminus and/or N-terminus One or several (usually 3 or less, preferably 2 or less, more preferably 1 or less) amino acids are deleted. For example, in the field, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed.
  • adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein.
  • the term also includes the polypeptide of the present invention in monomeric and multimeric forms.
  • the term also includes linear and non-linear polypeptides (such as cyclic peptides).
  • the present invention also includes active fragments, derivatives and analogs of the above-mentioned fusion protein.
  • fragment refers to a polypeptide that substantially retains the function or activity of the fusion protein of the present invention.
  • polypeptide fragments, derivatives or analogues of the present invention can be (i) one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) in one or more A polypeptide with substitution groups in three amino acid residues, or (iii) a polypeptide formed by fusing an antigenic peptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide fused to this polypeptide sequence (a fusion protein fused with a leader sequence, a secretory sequence, or a tag sequence such as 6 ⁇ His). According to the teachings herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.
  • a preferred type of active derivative means that compared with the amino acid sequence of Formula I or Formula II, there are at most 3, preferably at most 2, and more preferably at most 1 amino acid replaced by an amino acid with similar or similar properties. Peptides. These conservative variant polypeptides are best produced according to Table A by performing amino acid substitutions.
  • the present invention also provides analogs of the fusion protein of the present invention.
  • the difference between these analogs and the polypeptide shown in any one of SEQ ID NO.: 4-8 may be a difference in amino acid sequence, a difference in modification form that does not affect the sequence, or both.
  • Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modified (usually not changing the primary structure) forms include: chemically derived forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides produced by glycosylation modifications during the synthesis and processing of the polypeptide or during further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes polypeptides that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.
  • the present invention also relates to a vector containing the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the fusion protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention through recombinant technology.
  • the polynucleotide sequence of the present invention can be used to express or produce a recombinant fusion protein. Generally speaking, there are the following steps:
  • polynucleotide (or variant) of the present invention encoding the fusion protein of the present invention, or use a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;
  • the polynucleotide sequence encoding the fusion protein can be inserted into the recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors well known in the art. Any plasmid and vector can be used as long as it can be replicated and stabilized in the host.
  • An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
  • Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the fusion protein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti Transcriptional virus LTRs and some other known promoters that can control gene expression in prokaryotic or eukaryotic cells or viruses.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.
  • the host cell can be a prokaryotic cell (such as Escherichia coli), or a lower eukaryotic cell, or a higher eukaryotic cell, such as yeast cells, plant cells or mammalian cells (including human and non-human mammals).
  • a prokaryotic cell such as Escherichia coli
  • yeast cells such as Pichia pastoris, Kluyveromyces, or a combination thereof
  • yeast cells include: Kluyveromyces, more preferably Maxsk Luwei and/or Kluyveromyces lactis) are host cells.
  • Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, acting on promoters to enhance gene transcription. Examples include the 100 to 270 base pair SV40 enhancer on the late side of the replication initiation point, the polyoma enhancer on the late side of the replication initiation point, and adenovirus enhancers.
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method.
  • the steps used are well known in the art.
  • Another method is to use MgCl 2 .
  • the transformation can also be carried out by electroporation.
  • the host is a eukaryote
  • the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to a suitable cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the present invention provides a fusion protein, which may optionally contain a peptide linker.
  • the size and complexity of the peptide linker may affect the activity of the protein.
  • the peptide linker should have sufficient length and flexibility to ensure that the two proteins connected have enough freedom in space to perform their functions. At the same time, the influence of the formation of ⁇ helix or ⁇ sheet in the peptide linker on the stability of fusion protein is avoided.
  • the length of the connecting peptide is generally 0-10 amino acids, preferably 0-5 amino acids.
  • the composition is a pharmaceutical composition, which contains the above-mentioned fusion protein, and a pharmaceutically acceptable carrier, diluent, stabilizer and/or thickener, and can be prepared as a lyophilized powder , Tablets, capsules, syrups, solutions or suspensions.
  • “Pharmaceutically acceptable carrier or excipient (excipient)” refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and sufficient Low toxicity. "Compatibility” here means that the components in the composition can be blended with the active ingredients of the present invention and between them without significantly reducing the efficacy of the active ingredients.
  • the composition may be liquid or solid, such as powder, gel or paste.
  • the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art.
  • Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, and solid lubricants (such as stearic acid). , Magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween) ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose
  • the composition may comprise physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition is used to (a) treat or prevent tumors; (b) enhance the killing effect of T cells on tumor cells; (c) inhibit tumor growth; (d) inhibit T cell apoptosis; (e) ) Increase the level of IL-2 secreted by T cells.
  • the present invention finds a fusion protein for the first time.
  • the fusion protein contains CCP protein, optional connecting elements, and SH2 domain of SHP2 or SHP1 or active fragments thereof.
  • the fusion protein obtained in the present invention has extremely excellent tumor resistance. The killing activity.
  • the fusion protein of the present invention can also significantly enhance the killing effect of T cells on tumor cells; and/or inhibit tumor growth; and/or inhibit T cell apoptosis; and/or increase IL-2 secreted by T cells s level.
  • the fusion protein of the present invention can also enhance the killing effect of NK cells on tumor cells; increase the expression level of CD107a of NK cells; increase the secretion of perforin and granzyme of NK cells; increase the secretion of IFN- ⁇ and TNF- ⁇ of NK cells.
  • the fusion protein of the present invention can also enhance the phagocytic function of macrophages; enhance the phagocytosis and killing effect of macrophages on tumor cells; increase the secretion of NO, TNF- ⁇ and IL-1 ⁇ of macrophages.
  • the fusion protein and polypeptide After the fusion protein and polypeptide enter the cell, they can compete with SHP2 or SHP1 normally expressed in T lymphocytes/NK cells/macrophages to bind to the ITIM of the intracellular segment of the inhibitory receptor, so that the SHP2 or SHP1 in the immune cells is always in Inactive state, thereby inactivating the inhibitory effect of immunosuppressive receptors.
  • the fusion protein of the present invention has a broad-spectrum anti-tumor effect.
  • the recombinant TAT-N-SH2 fusion protein of the present invention connects the TAT penetrating peptide and N-SH2 through glycine and serine (TAT-N-SH2), and the connection mode is: TAT-Gly-Ser-N-SH2; Its amino acid sequence is shown in SEQ ID NO.: 4.
  • the nucleotide sequence of the recombinant TAT-N-SH2 fusion protein is the nucleotide sequence of the TAT penetrating peptide and the N-terminal SH2 domain of SHP2 connected by the BamHI restriction site, and the NdeI restriction site is added at the 5'end , Add the stop codon TGA and SalI restriction site to the 3'end, and the connection method is: NdeI restriction site-TAT gene-BamHI restriction site-N-SH2 gene-TGA-SalI restriction site, Its nucleotide sequence is shown in SEQ ID NO.:9.
  • Recombinant TAT-N-SH2 fusion protein nucleotides are constructed in expression vector or recombinant plasmid pET-22b(+)-TAT-N-SH2, said pET-22b(+)-TAT-N-SH2 is passed through NdeI enzyme
  • the cleavage site and the SalI restriction site connect the plasmid pET-22b(+) with the recombinant TAT-N-SH2 fusion protein described in claim 3 with nucleotide double restriction digestion.
  • the recombinant plasmid pET-22b(+)-TAT-N-SH2 was transformed into BL21 competent cells, cultured in a 37°C incubator for 12 hours, and then a single colony was picked and inoculated into LB culture medium containing 100 mg/L of ampicillin on a shaker 37 After culturing at °C, the bacteria were harvested and the plasmids were extracted. The plasmids were digested with NdeI and SalI, and identified by agarose gel electrophoresis. The expected 342bp insert fragment was obtained ( Figure 1). The DNA sequencing result ( Figure 2) showed that it was consistent with the synthesized sequence. The 22b(+)-TAT-N-SH2 recombinant plasmid was successfully constructed.
  • the recombinant plasmid pET-22b(+)TAT–SHP2–N-SH2 was double digested with Nde I and Sal I enzymes, and then subjected to agarose gel electrophoresis.
  • the red arrow is the target fragment.
  • the rabbit anti-human SHP2 polyclonal antibody can specifically bind to the target protein (as shown in Figure 7), which proves that the purified target protein is the target protein.
  • the purified fusion protein was detected by HPLC. A 20 ⁇ L sample was injected into the needle, the running time was 20 min, the flow rate was 0.8 mL/min, the detection wavelength was 280 nm, and the area of the main peak was used to determine the purity of the sample. The results showed that the purity of the fusion protein was 95.92% ( Figure 8).
  • Example 2 The fusion polypeptide of four TAT penetrating peptides and the ITIM motif binding region of the C-terminal SH2 domain of SHP2
  • L1 All L-type amino acids were used to send the company to synthesize the above-mentioned TAT-C-SH2 polypeptide (named L1). Taking the sequence of the polypeptide L1 as a reference, the reverse sequence of L1 was synthesized, the L1 sequence synthesized with D-type amino acid, and the L1 reverse sequence synthesized with D-type amino acid were respectively named L2, D1, D2. Its amino acid sequence is as follows:
  • Reverse L-shaped polypeptide (L2): LVFDGPHSQSERVPPRRRQRRKKRGY (SEQ ID NO.: 6)
  • Type D polypeptide (D1): YGRKKRRQRRRPPVRESQSHPGDFVL (SEQ ID NO.: 7)
  • Reverse D-type polypeptide (D1): LVFDGPHSQSERVPPRRRQRRKKRGY (SEQ ID NO.: 8)
  • the purity of the synthesized peptides was analyzed by HPLC. A 20 ⁇ L sample was injected into the needle, the running time was 20 min, the flow rate was 1.0 mL/min, the detection wavelength was 220 nm, and the area of the main peak was used to determine the purity of the sample. The results showed that the purity of L1 polypeptide was 96.74% (Figure 9A), the purity of L2 polypeptide was 98.1% (Figure 9B), the purity of D1 polypeptide was 99.27% ( Figure 9C), and the purity of D2 polypeptide was 96.66% (Figure 9D). .
  • Example 3 The penetrating peptide TAT can assist the recombinant fusion protein and polypeptide to enter T lymphocytes
  • TAT-N-SH2 fusion protein and polypeptide The ability of recombinant TAT-N-SH2 fusion protein and polypeptide to enter T lymphocytes is a prerequisite for binding to the ITIM of the inhibitory receptor on its cell membrane.
  • the penetrating peptide TAT is a short, positively charged peptide that can assist a variety of substances to pass through the cell membrane to perform their biological functions.
  • TAT-N-SH2 fusion protein, L1 polypeptide and control peptide with FITC, and observe their entry into T lymphocytes using a confocal microscope.
  • the action time is 0, 0.5, 4, 24, 72, 96, 120 and 144h ( Figure 11).
  • Example 4 The effect of recombinant fusion protein and polypeptide on T lymphocyte function
  • TAT-N-SH2 fusion protein and L1 TAT-SHP2-C-SH2
  • TAT-N-SH2 fusion protein and L1 TAT-SHP2-C-SH2
  • TAT-SHP2-C-SH2 polypeptide can increase the proliferation of T lymphocytes
  • the recombinant TAT-N-SH2 fusion protein group The proliferation rate of T lymphocytes was 61.05% (Figure 12b), and the proliferation rate of T lymphocytes in the L1 (TAT-SHP2-C-SH2) polypeptide group was 60.33% ( Figure 12c).
  • the statistical results showed that compared with the control group, the recombinant TAT-N-SH2 fusion protein and L1 polypeptide did not significantly increase the proliferation of T lymphocytes ( Figure 12d). This result indicates that the recombinant TAT-N-SH2 fusion protein and L1(TAT-SHP2- C-SH2) polypeptide has no effect on the proliferation of T lymphocytes.
  • TAT-N-SH2 fusion protein and L1 TAT-SHP2-C-SH2
  • FIG. 13a-d shows the effect of Annexin V-FITC detection polypeptide on T lymphocyte apoptosis.
  • Resuspended cells are added to different groups to treat non-administered groups SHP2-NC, SHP2-N-SH2 (80 ⁇ g/mL), TAT-SHP2 -C-SH2 polypeptide (80 ⁇ g/mL), then continue to culture on the EASY-T stimulated culture plate (add 50ng PD-L1 antibody to the 24-well plate at 37°C 3h before adding the cell mixture) for 3 days, collect the cells, FITC and After PI staining, it was detected by flow cytometry.
  • Figure 13e According to the results of flow cytometry, the proportion of apoptotic cells was counted. Compared with the control group, *p ⁇ 0.05 or **p ⁇ 0.01.
  • the apoptosis of T lymphocytes in the recombinant TAT-N-SH2 fusion protein and L1 polypeptide treatment group was significantly reduced.
  • TAT-N-SH2 fusion protein and L1 TAT-SHP2-C-SH2
  • T lymphocytes stimulated by the recombinant TAT-N-SH2 fusion protein and L1 polypeptide for 3 days were collected to detect the level of cytokine IL-2 secreted.
  • Example 6 Recombinant fusion proteins and polypeptides enhance the ability of T cells to kill tumor cells in vitro
  • TAT-SHP2-N-SH2 fusion protein Three protein concentrations (20 ⁇ g/mL, 40 ⁇ g/mL, 80 ⁇ g/mL) of TAT-SHP2-N-SH2 fusion protein were selected to stimulate human T lymphocytes and tested at 30:1 by CytoTox 96 non-radioactive cytotoxicity method Compare the killing effects on tumor cells MDA-MB-231 and SW480 respectively.
  • TAT-SHP2-N-SH2 fusion protein can improve the effect of T cells in killing tumor cells, and the effect of killing colon cancer is better than breast cancer cells.
  • TAT-SHP2-N-SH2 fusion protein stimulates the killing effect of T cells on different tumor cell lines
  • L1 TAT-SHP2-C-SH2
  • CytoTox 96 non-radioactive cytotoxicity method was used to detect TAT-SHP2-C-SH2 polypeptides (20 ⁇ g/mL, 40 ⁇ g/mL, 80 ⁇ g/mL) after stimulating human T lymphocytes for 4 days, respectively, to tumor cell lines MDA-MB-231 and SW480 The killing effect.
  • the effective target ratio is 30:1
  • the TAT-SHP2-C-SH2 polypeptide concentration is 40 ⁇ g/mL and 80 ⁇ g/mL respectively
  • the T lymphocytes stimulated by TAT-SHP2-C-SH2 polypeptide kill MDA-
  • the capabilities of MB-231 and SW480 are significantly improved ( Figure 18, Table 2).
  • TAT-SHP2-C-SH2 polypeptide stimulates the killing effect of T cells on different tumor cell lines
  • CytoTox 96 non-radioactive cytotoxicity method was used to detect the killing effect of L1, L2, D1, and D2 polypeptides (40 ⁇ g/mL) on human T lymphocytes after stimulating human T lymphocytes for 4 days.
  • the effective target ratio is 20:1
  • the L1, L2, D1, D2 polypeptide-stimulated T lymphocytes have significantly improved ability to kill lung cancer cells H460, and the D2 polypeptide has the strongest effect.
  • the MC38 colon cancer mouse subcutaneous xenograft model was established.
  • the dose of 50 ⁇ g/a PD-1 antibody was used as the positive control drug, and the selected concentration was 1.25 ⁇ g/head (N1.25), 2.5 ⁇ g/head (N2.5), 5 ⁇ g/head (N5) and 10 ⁇ g/head (N10) TAT-SHP2-N-SH2 fusion protein were administered by intraperitoneal injection. After 8 administrations, the tumor-bearing mice were dissected and weighed.
  • TAT-SHP2-C-SH2 polypeptide The effect of TAT-SHP2-C-SH2 polypeptide on the tumor growth of mice was evaluated through the established MC38 colon cancer mouse subcutaneous tumor model.
  • the effects of TAT-SHP2-C-SH2 polypeptide on mouse tumor growth were evaluated at 1.25 ⁇ g/mouse (C1.25) and 2.5 ⁇ g/mouse (C2.5). ), 5 ⁇ g/piece (C5) and 10 ⁇ g/piece (C10) TAT-SHP2-C-SH2 polypeptides were administered intraperitoneally, and at the same time, 50 ⁇ g/piece of ⁇ PD-1 antibody ( ⁇ PD-1) was administered as a positive control drug.
  • ⁇ PD-1 antibody ⁇ PD-1 antibody
  • the TAT-SHP2-N-SH2 (5 ⁇ g/head) group and the combination group were added, and it was found that compared with the control group, TAT- The tumor weight of the SHP2-C-SH2 polypeptide group, ⁇ PD-1 antibody group, TAT-SHP2-N-SH2 fusion protein group and the combination group was significantly reduced.
  • the combination group has the best tumor suppression effect, followed by the N5 group.
  • the TAT-SHP2-C-SH2 polypeptide has the slowest growth rate at a dose of 5 ⁇ g per tumor (Figure 21, ab); the combination group has the best tumor suppression rate.
  • N5 group Table 5
  • TAT-SHP2SHP2-N-SH2 fusion protein and TAT-SHP2SHP2-C-SH2 polypeptide were evaluated the effects of TAT-SHP2SHP2-N-SH2 fusion protein and TAT-SHP2SHP2-C-SH2 polypeptide on mouse tumor growth through the established EMT breast cancer mouse subcutaneous tumor model.
  • 10 ⁇ g/piece (C10), 15 ⁇ g/piece (C15) of TAT-SHP2SHP2-C-SH2 polypeptide and 2 ⁇ g/piece (N2), 5 ⁇ g/piece (N5), 10 ⁇ g/piece (N10), 15 ⁇ g/piece ( N15) TAT-SHP2SHP2-N-SH2 fusion protein was administered by intraperitoneal injection, and at the same time 50 ⁇ g/only ⁇ PD-1 antibody ( ⁇ PD-1) was administered as a positive control drug.
  • TAT-SHP2SHP2-C-SH2 polypeptide The tumor volume of each treatment group was significantly smaller than that of the control group, ⁇ PD-1 antibody group, and TAT-SHP2SHP2-N-SH2 fusion protein group; TAT-SHP2SHP2-N-SH2 fusion protein (5 ⁇ g/head) and TAT-SHP2SHP2 or SHP1-
  • the C-SH2 polypeptide (5 ⁇ g/head) group had the smallest tumor weight and the best tumor inhibition rate ( Figure 22, ab, Table 6).
  • the results indicate that the different dose groups have an inhibitory effect on the growth of mouse tumors, and the fusion protein and polypeptide doses are both 5 ⁇ g/mouse. The effect is the best, which is consistent with the tumor suppression results of colon cancer.
  • mice were randomly divided into three groups, each with 6 mice.
  • the tumor size was measured on the 4th day and the growth curve was drawn for 8 times.
  • the mice were sacrificed on the 21st day and their body weight was measured.
  • TAT-N-SH2 fusion protein and L1 TAT-SHP2-C-SH2
  • TAT-N-SH2 fusion protein and L1 TAT-SHP2-C-SH2
  • TAT-SHP2-C-SH2 L1
  • Cancer tumors are growing ( Figure 24, ac, Table 8).
  • TAT-N-SH2 fusion protein and L1 (TAT-SHP2-C-SH2) polypeptide are aimed at the ITIM motif of the inhibitory receptor, so it is better than PD- which only blocks PD-1, an inhibitory receptor.
  • the treatment range of monoclonal antibodies is wider, and theoretically, it can treat tumors that are ineffective in PD-1 antibody treatment.
  • PD-L1 stable knockout breast cancer EMT-6 cell line PD-L1-KO-EMT6
  • TAT-N- SH2 fusion protein (N5) and TAT-SHP2-C-SH2 polypeptide (C5) were administered by intraperitoneal injection, and at the same time, the dose of 50 ⁇ g/only ⁇ PD-1 antibody ( ⁇ PD-1) was used as the control drug.
  • the results show that: N5 and C5 can significantly inhibit the growth of PD-L1-KO-EMT6 tumors, but PD-1 antibody treatment is ineffective ( Figure 25, a-c, Table 9).

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Abstract

提供了一种抗肿瘤融合蛋白及其制法和应用。具体地，融合蛋白含有CPP元件、任选的连接元件和SHP2或SHP1的SH2结构域或其活性片段，所得到的融合蛋白具有极其优异的抗肿瘤作用。

Description

一种抗肿瘤融合蛋白及其制法和应用 技术领域
本发明涉及生物和药物领域，更具体地涉及一种抗肿瘤融合蛋白及其制法和应用。
背景技术
免疫检查点阻断剂已成为肿瘤免疫疗法的重要靶点，以PD-1/PD-L1为靶点的免疫检查点阻断疗法是利用人体自身的免疫系统抵御和抗击恶性肿瘤，实质性改善患者总生存期的新一代抗癌免疫疗法。然而，多项研究报道，仅有20％-30％的少数患者对该阻断剂有应答，且一些患者在接受药物治疗后出现耐药。在单药用药反应率低的情况下，新型免疫检查点阻断药物的开发以及针对不同免疫检查点药物的联用策略成为目前提高患者反应率的新途径。但仍然不能完全解决临床低反应率和耐药问题，并且多种疗法的联合应用存在联合时序、剂量优化、药物经济学等问题。因此，设计针对不同免疫检查点共同靶位的通用免疫检查点抑制剂(Universal immune check point blockade，UICB)可能是上述问题的关键突破口。
因此本领域迫切需要开发更有效的抗肿瘤的融合蛋白。
发明内容
本发明的目的就是提供一种更有效的抗肿瘤的融合蛋白。
在本发明的第一方面，提供了一种融合蛋白，所述融合蛋白具有从N端至C端的式I或II所示的结构：
Z1-L-Z2  (I)
Z2-L-Z1  (II)
式中，
Z1为CPP元件；
L为无或连接元件；
Z2为SHP2和/或SHP1的SH2结构域或其活性片段；
“-”表示连接上述元件的肽键或肽接头。
在另一优选例中，所述Z1、Z2以头-头、头-尾、尾-头、或尾-尾方式相连。
在另一优选例中，所述的“头部”指多肽或其片段的N端，尤其是野生型多肽的或其片段的N端。
在另一优选例中，所述的“尾部”指多肽或其片段的C端，尤其是野生型多肽的或其片段的C端。
在另一优选例中，所述Z1、Z2为D型或L型氨基酸。
在另一优选例中，当所述Z1、Z2为头-头方式相连时，所述Z1、Z2为L型或D型氨基酸。
在另一优选例中，当所述Z1、Z2为尾－尾方式相连时，所述Z1、Z2为L型或D型氨基酸。
在另一优选例中，所述的CPP元件来源于人或非人哺乳动物。
在另一优选例中，所述的CPP元件包括野生型和突变型。
在另一优选例中，所述的CPP元件包括全长的、成熟形式的CPP，或其活性片段，优选长度为5-30个氨基酸。
在另一优选例中，所述CPP元件为TAT穿膜肽。
在另一优选例中，所述CPP元件选自下组：聚精氨酸(R5-9)、MPG、CADY、pVEC、Penetratin、卡波西成纤维细胞生长因子肽、VP22、或其组合。
在另一优选例中，所述CPP元件的序列如SEQ ID NO.:1、15-21中任一所示。
在另一优选例中，L由甘氨酸和丝氨酸组成。
在另一优选例中，L具有如下结构：-(Gly-Ser)n-；其中，n为1-5，较佳地，1-3。
在另一优选例中，L具有如下结构：-(Gly-Ser-Ser-Ser-Ser)n-；其中，n为1-5，较佳地，1-3。
在另一优选例中，L包含1-6个脯氨酸，较佳地，1-3个脯氨酸。
在另一优选例中，所述的Z2来源于人或非人哺乳动物。
在另一优选例中，所述的Z2包括野生型和突变型。
在另一优选例中，所述的Z2包括全长的、成熟形式的SHP2或SHP1的SH2结构域，或其活性片段。
在另一优选例中，Z2选自下组：N端SH2结构域、C端SH2结构域、或其组合。
在另一优选例中，Z2还包括C端SH2结构域的ITIM基序结合区。
在另一优选例中，Z2为SHP2的N端SH2结构域，具有SEQ ID NO.:2中的第1-99位或9-51位氨基酸，优选含有SEQ ID No.:2中的第9、28、49和51位氨 基酸，并且长度为43-99个氨基酸。
在另一优选例中，Z2为SHP2的C端SH2结构域，具有SEQ ID NO.:3中的第27-39或第29-37位氨基酸，优选含有SEQ ID NO.:3中第29、31、36和37位氨基酸，并且长度为9-108或13－108个氨基酸。
在另一优选例中，Z2为SHP1的N端SH2结构域，具有SEQ ID NO.:12中的第9-51位氨基酸，优选含有SEQ ID NO.:12中的第9、28、49、51位氨基酸，并且长度为43-99个氨基酸。
在另一优选例中，Z2为SHP1的C端SH2结构域，具有SEQ ID NO.:13中的第29-37位氨基酸，优选含有SEQ ID NO.:13中的第29、31、36、37位氨基酸，并且长度为9-107个氨基酸。
在另一优选例中，Z2具有从N端－C端的式III所示的结构：
A-L’-B  (III)
其中，
A为SHP2或SHP1的N端SH2结构域或其活性片段；
L为无或连接元件；
B为SHP2或SHP1的C端SH2结构域或其活性片段；
“-”表示连接上述元件的肽键或肽接头。
在另一优选例中，元件A为SHP2的N端SH2结构域，具有SEQ ID NO.:2中第1-99位或第9-51位，优选含有SEQ ID No.:2中的第9、28、49和51位，并且长度为43-99个氨基酸。
在另一优选例中，元件B为SHP2的C端SH2结构域，具有SEQ ID NO.:3所示的序列，且含有SEQ ID NO.:3中第27-39位或第29-37位，优选含有SEQ ID NO.:3中第29、31、36和37位，并且长度为9-108或13-108个氨基酸。
在另一优选例中，元件A为SHP1的N端SH2结构域，具有SEQ ID NO.:12中的第9-51位氨基酸，优选含有SEQ ID NO.:12中的第9、28、49、51位氨基酸，并且长度为43-99个氨基酸。
在另一优选例中，元件B具有SHP1的C端SH2结构域，具有SEQ ID NO.:13中的第29-37位氨基酸，优选含有SEQ ID NO.:13中的第29、31、36、37位氨基酸，并且长度为9-107个氨基酸。
在另一优选例中，所述L’的长度为1-20aa，较佳地，3-15aa，更佳地，5-10aa。
在另一优选例中，所述L’的氨基酸序列选自下组：
(1)氨基酸序列如SEQ ID NO.:10所示的多肽；
(2)将SEQ ID NO.:10所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-8个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加而形成的，具有(1)所述多肽功能的由SEQ ID NO.:10所示氨基酸序列的多肽衍生的多肽。
在另一优选例中，Z2具有SEQ ID NO.:11或14所示的序列。
在另一优选例中，Z2的序列如SEQ ID NO.:11或14所示。
在另一优选例中，所述的肽接头的长度为0-10氨基酸，较佳地为0-5个氨基酸。
在另一优选例中，所述融合蛋白选自下组：
(A)具有SEQ ID NO:4－8任一所示氨基酸序列的多肽；
(B)具有与SEQ ID NO:4－8任一所示氨基酸序列≥80％同源性(优选地，≥90％的同源性；优选地≥95％的同源性；最优选地，≥97％的同源性，如98％以上，99％以上)的多肽，且所述多肽具有肿瘤抑制活性；
(C)将SEQ ID NO:4-8中任一所示氨基酸序列经过1-5个氨基酸残基的取代、缺失或添加而形成的，且保留肿瘤抑制活性的衍生多肽。
在另一优选例中，所述的融合蛋白的氨基酸序列如SEQ ID NO.:4－8任一所示。
本发明第二方面提供了一种分离的多核苷酸，所述的多核苷酸编码本发明第一方面所述的融合蛋白。
在另一优选例中，所述的多核苷酸在所述突变蛋白或融合蛋白的ORF的侧翼还额外含有选自下组的辅助元件：信号肽、分泌肽、标签序列(如6His)、或其组合。
在另一优选例中，所述的多核苷酸选自下组：DNA序列、RNA序列、或其组合。
本发明第三方面提供了一种载体，它含有本发明第二方面所述的多核苷酸。
在另一优选例中，所述载体包含一个或多个启动子，所述启动子可操作地与所述核酸序列、增强子、转录终止信号、多腺苷酸化序列、复制起点、选择性标记、核酸限制性位点、和/或同源重组位点连接。
在另一优选例中，所述载体包括质粒、病毒载体。
在另一优选例中，所述的病毒载体选自下组：腺相关病毒(AAV)、腺病毒、慢病毒、逆转录病毒、疱疹病毒、SV40、痘病毒、或其组合。
在另一优选例中，所述载体包括表达载体、穿梭载体、整合载体。
本发明第四方面提供了一种宿主细胞，所述所述宿主细胞含有本发明第三方面所述的载体，或其基因组中整合有本发明第二方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞为真核细胞，如酵母细胞、植物细胞或哺乳动物细胞(包括人和非人哺乳动物)。
在另一优选例中，所述的宿主细胞为原核细胞，如大肠杆菌。
在另一优选例中，所述酵母细胞选自下组的一种或多种来源的酵母：毕氏酵母、克鲁维酵母、或其组合；较佳地，所述的酵母细胞包括：克鲁维酵母，更佳地为马克斯克鲁维酵母、和/或乳酸克鲁维酵母。
在另一优选例中，所述宿主细胞选自下组：大肠杆菌、麦胚细胞，昆虫细胞，SF9、Hela、HEK293、CHO、酵母细胞、或其组合。
本发明第五方面提供了一种产生本发明第一方面所述的融合蛋白的方法，所述的方法包括步骤：
在适合表达的条件下，培养本发明第四方面所述的宿主细胞，从而表达融合蛋白；和/或分离所述融合蛋白。
本发明第六方面提供了一种药物组合物，所述的药物组合物含有本发明第一方面所述的融合蛋白及其药物学上可接受的载体。
在另一优选例中，所述药物组合物还包括其他用于抑制肿瘤活性的药物。
在另一优选例中，其他用于抑制肿瘤活性的药物选自下组：PD-1抗体、PD-L1抗体、HER2单抗、BTLA抗体、CTLA-4抗体、CD47抗体、NKG2A抗体、NKTR-214,GDF-15抗体、LILRB4抗体、LAIR1抗体、Tim-3抗体、Lag-3抗体、Tight抗体、CD160抗体、KLRG-1抗体、GP49B抗体、CD31抗体、CD38抗体、Lair-1抗体、CD200/CD200R抗体、卡妥索单抗(Catumaxomab)、博纳吐单抗(Blinatumomab)、EGFR单抗、CD20单抗、VEGF/VEGFR单抗、利卡汀、Zevalin,Bexxar,Mylotarg,Kadcyla、阿柏西普、康柏西普、Apalutamide(ARN-509)、Rova-T、TNFα、IFNγ,IL-2,Tβ4、以EGFR为靶向的小分子抑制剂、紫杉醇(PTX)、多西他赛(TXT)、顺铂(DDP)、卡铂(CBP)、奥沙利铂、奈达铂、环磷酰胺(CTX)、异环磷酰(IFO)、多柔比星(ADM)、吡柔比星(THP)、表阿霉素(EPI)、氟尿嘧啶(5-Fu)、吉西他滨(GEM)、长春瑞滨(NVB)、培美曲塞(PEM)、伊立替康(CPT-11)、依托泊苷/足叶乙甙(VP-16)、卡培他滨(希罗达)、醋酸亮丙瑞林、醋酸戈舍瑞林、或其组合。
在另一优选例中，所述PD-1抗体选自下组：尼诺单抗(nivolumab)、派姆单抗(pembrolizumab)、塞米单抗(Cemiplimab)、特瑞普利单抗、信迪利单抗、卡瑞利珠单抗、替雷利珠单抗、或其组合。
在另一优选例中，所述PD-L1抗体选自下组：阿妥珠单抗(atezolizumab)、杜鲁伐单抗(durvalumab)、奥维单抗(avelumab)、或其组合。
在另一优选例中，所述HER2单抗选自下组：曲妥珠单抗、帕妥珠单抗、T－DM1、或其组合。
在另一优选例中，所述CTLA-4抗体包括伊匹单抗(Ipilimumab)。
在另一优选例中，所述EGFR单抗包括尼珠单抗(Necitumumab)、帕尼单抗(Panitumumab)、尼莫妥单抗(Nimotuzumab)、西妥昔单抗(Cetuximab)。
在另一优选例中，所述CD20单抗选自下组：利妥昔单抗、替伊莫单抗、托西莫单抗、奥法木单抗、奥瑞珠单抗、阿妥珠单抗、或其组合。
在另一优选例中，所述VEGF/VEGFR单抗选自下组：贝伐珠单抗、雷莫芦单抗、雷珠单抗、或其组合。
在另一优选例中，所述以EGFR为靶向的小分子抑制剂选自下组：厄洛替尼、吉非替尼、埃克替尼、阿法替尼(包括马来酸阿法替尼、达克替尼、奥西替尼、纳扎替尼(Nazartinib)、来那替尼(包括马来酸来那替尼)、索拉非尼(Sorafenib)、阿帕替尼(如艾坦、甲磺酸阿帕替尼片)、伊马替尼、舒尼替尼、达沙替尼、拉帕替尼、培唑帕尼、克唑替尼、凡德他尼、瑞戈非尼、阿西替尼、普纳替尼、来那替尼、泽布替尼、安罗替尼、色瑞替尼、呋喹替尼、吡咯替尼、仑伐替尼、或其组合。
本发明第七方面提供了本发明第一方面所述融合蛋白、本发明第二方面所述的多核苷酸、本发明第三方面所述的载体、本发明第四方面所述的宿主细胞的用途，用于制备组合物或制剂，所述组合物或制剂用于治疗或预防肿瘤。
在另一优选例中，所述组合物或制剂还用于选自下组的一种或多种用途：
(a)增强T细胞对肿瘤细胞的杀伤作用；
(b)抑制肿瘤的生长；
(c)抑制T细胞的凋亡；
(d)增加T细胞分泌的IL-2的水平；
(e)增强NK细胞对肿瘤细胞的杀伤作用；
(f)增加NK细胞CD107a表达水平；
(g)增加NK细胞穿孔素、颗粒酶分泌；
(h)增加NK细胞IFN-γ、TNF-α分泌；
(i)增强巨噬细胞的吞噬功能；
(j)增强巨噬细胞对肿瘤细胞的吞噬杀伤作用；
(k)增加巨噬细胞NO、TNF-α和IL-1β分泌。
在另一优选例中，所述的组合物为药物组合物。
在另一优选例中，所述肿瘤选自下组：乳腺癌、结肠癌、肺癌、大肠癌、胃癌、食道癌、胰腺癌、卵巢癌、前列腺癌、肾癌、肝癌、脑癌、黑色素瘤、多发性骨髓瘤、白血病、淋巴瘤、头颈部肿瘤、甲状腺癌、或其组合。
在另一优选例中，所述肿瘤细胞来自于选自下组的一种或多种肿瘤：乳腺癌、结肠癌、肺癌、大肠癌、胃癌、食道癌、胰腺癌、卵巢癌、前列腺癌、肾癌、肝癌、脑癌、黑色素瘤、多发性骨髓瘤、白血病、淋巴瘤、头颈部肿瘤、甲状腺癌、或其组合。
本发明第八方面提供了一种体外非治疗性的抑制肿瘤生长的方法，包括步骤：在本发明第一方面所述的融合蛋白存在下，培养肿瘤细胞，从而抑制肿瘤生长。
在另一优选例中，所述肿瘤细胞来自于选自下组的一种或多种肿瘤：乳腺癌、结肠癌、肺癌、大肠癌、胃癌、食道癌、胰腺癌、卵巢癌、前列腺癌、肾癌、肝癌、脑癌、黑色素瘤、多发性骨髓瘤、白血病、淋巴瘤、头颈部肿瘤、甲状腺癌、或其组合。
在另一优选例中，所述肿瘤细胞为体外培养的细胞。
本发明第九方面提供了一种治疗肿瘤的方法，包括步骤：给需要的对象施用本发明第一方面所述的融合蛋白。
在另一优选例中，所述的融合蛋白以单体和/或二聚体形式施用。
在另一优选例中，所述的对象是人。
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显示了pET-22b(+)TAT-SHP2–N-SH2双酶切鉴定结果。
图2显示了重组质粒pET-22b(+)TAT-SHP2-N-SH2测序结果。
图3显示了SDS-PAGE检测目的蛋白的表达情况；
1 Marker；2未诱导；3 IPTG诱导1；4 IPTG诱导2；红色箭头所指为目标蛋白。
图4显示了SDS-PAGE检测目的蛋白的表达情况；
注：1未诱导，2诱导，3裂菌上清，4裂解沉淀。
图5显示了阳离子交换层析分离目的蛋白和杂蛋白；
注：SP1-SP4为洗脱峰，其中SP4为目的蛋白所在峰。
图6显示了SDS-PAGE检测TAT-SHP2-N-SH2的纯化效果；
注：1穿过，2SP1，3SP2，4SP3，5SP4(目的蛋白峰)。
图7显示了Western-blot鉴定SHP2-N-SH2目的蛋白；
1 IPTG未诱导的重组菌；2 1mM IPTG诱导的重组菌。
图8显示了融合蛋白HPLC纯化色谱分析结果。
图9显示了多肽HPLC纯化色谱分析结果；
A,L1多肽；B,L2多肽；C,D1多肽；D,D2多肽。
图10显示了质谱检测多肽的分子量；A,L1多肽；B,L2多肽；C,D1多肽；D,D2多肽。
图11显示了激光共聚焦检测融合蛋白和多肽在T细胞中的定位；
a.共聚焦显微镜下观察未携带TAT穿膜序列对照肽(20μg/mL)进入细胞的能力；b.带有TAT序列的SHP2-C-SH2；c.带有TAT序列的SHP2-N-SH2；作用时间为0，0.5，4，24，72，96，120和144h(25μm(×60)。
图12显示了重组TAT-N-SH2融合蛋白和L1多肽对T淋巴细胞增殖的影响；
a-c CFSE法检测不同处理组对T淋巴细胞增殖的影响。d根据流式检测结果，统计T细胞增殖的比例，与对照组比较，p>0.05无统计学意义。
图13显示了重组TAT-N-SH2融合蛋白和L1多肽对T淋巴细胞凋亡的影响；
a-d Annexin V-FITC检测多肽对T淋巴细胞凋亡的影响，重悬细胞加入不同组处理未给药组SHP2-NC、SHP2-N-SH2(80μg/mL)、TAT-SHP2-C-SH2多肽(80 μg/mL)后继续在EASY-T刺激的培养板上(加入细胞混合液前3h加入50ng PD-L1抗体37℃包被24孔板)培养3d，收集细胞，FITC和PI染色后通过流式细胞仪进行检测；e根据流式检测结果，统计凋亡细胞的比例，与对照组比较，*p<0.05或**p<0.01。
图14显示了融合蛋白及多肽对T细胞介导下游信号分子磷酸化水平的影响；
a.TAT-SHP2-N-SH2融合蛋白处理组；c.TAT-SHP2C-SH2多肽处理组；b,d.根据Western-blot结果，以β-actin为内参，对T淋巴细胞介导的JNK，AKT，ERK蛋白磷酸化表达进行灰度分析。Unpaired t-test，与阴性对照比较，**p<0.01，NS无统计学差异。
图15显示了T细胞分泌的细胞因子IL-2的检测；
Unpaired t-test，与对照比较，*p<0.05和****p<0.0001。
图16显示了直接杀伤肿瘤作用检测的结果。
图17显示了TAT-SHP2-N-SH2融合蛋白刺激T细胞对不同肿瘤细胞株的杀伤作用。与未给药组比较，*p<0.05或**p<0.01或***p<0.001，p>0.05无统计学意义。
图18显示了TAT-SHP2-C-SH2多肽刺激T细胞对不同肿瘤细胞株的杀伤作用。与未给药组比较，*p<0.05或**p<0.01或***p<0.001，p>0.05无统计学意义。
图19显示了L1、L2、D1、D2多肽刺激T细胞对肺癌细胞H460的杀伤作用；
和对照组比， *P<0.05， **P<0.01， ***P<0.001， ****P<0.0001；
和D2组比， #P<0.05， ##P<0.01， ###P<0.001。
图20显示了TAT-SHP2-N-SH2融合蛋白对结肠癌荷瘤小鼠抑瘤作用；
a.30d解剖后不同剂量给药组小鼠瘤重；b.瘤大小；Unpaired t-test，与对照组比较，*p<0.05，**p<0.01，***p<0.001和****p<0.0001。
图21显示了TAT-SHP2-C-SH2多肽对结肠癌荷瘤小鼠抑瘤作用；
a.30d解剖后不同剂量给药组小鼠瘤重；b.瘤大小；Unpaired t-test，与对照组比较，**p<0.01，***p<0.001和****p<0.0001。
图22显示了TAT-SHP2-N-SH2融合蛋白和TAT-SHP2-C-SH2多肽对乳腺癌荷瘤小鼠抑瘤作用；
a.21d解剖后不同给药组小鼠瘤重；b.瘤大小；Unpaired t-test，与对照组比较，*p<0.05，**p<0.01，***p<0.001。
图23显示了重组TAT-N-SH2融合蛋白和L1多肽对结肠癌裸鼠的抑瘤作用；
a.30d解剖后小鼠瘤重；b.瘤大小；c.肿瘤生长曲线；Unpaired t-test，与对照组比较，NS：无统计学差异。
图24显示了重组TAT-N-SH2融合蛋白和L1多肽对裸鼠乳腺癌的抑瘤作用；
a.21d解剖后小鼠瘤重；b.瘤大小；c.肿瘤生长曲线；NS：无统计学差异。
图25显示了重组TAT-N-SH2融合蛋白和L1多肽对PD-L1敲除的乳腺癌荷瘤小鼠抑瘤作用；
a.23d解剖后小鼠瘤重；b.瘤大小；c.肿瘤生长曲线；Unpaired t-test，和对照组比，*P<0.05，**P<0.01，***P<0.001。
图26显示了L1、L2、D1、D2多肽对小鼠肺癌的抑瘤效果检测；
a.21d解剖后小鼠瘤重；b.瘤大小；c.肿瘤生长曲线；Unpaired t-test，和对照组比，*P<0.05，**P<0.01，***P<0.001。
具体实施方式
本发明人经过深入的研究，意外地发现一种融合蛋白，该融合蛋白含有CPP元件、任选的连接元件和SHP2或SHP1的SH2结构域或其活性片段，本发明所得到的融合蛋白具有极其优异的对肿瘤的杀伤活性，并且，本发明的融合蛋白还可显著增强T细胞对肿瘤细胞的杀伤作用。在此基础上完成了本发明。
CPP元件
细胞膜是细胞与细胞外环境的主要屏障，也正因为存在这一天然屏障，许多生物大分子难以透过细胞膜进入细胞，是现阶段靶向给药的一大障碍。细胞穿膜肽(cell penetration peptide，CPP)是一类由5～30个氨基酸残基组成，具有细胞膜穿透能力的多肽，可携带蛋白质、核酸等生物大分子进入细胞。CPP介导的生物大分子的入胞因其相较于传统方式更加安全和高效，是目前最为成功的可在体内、外直接转运生物大分子的递送系统。最早的CPP研究就是 发现人免疫缺陷病毒(HIV-1)的反式激活转录蛋白中的一个富含精氨酸序列的多肽(TAT)可以有效穿过细胞膜并激活相应病毒启动子的转录。TAT能够高效地将药物传递进入细胞。TAT的穿膜功能被精确定位于11个氨基酸的核心区段(aa47-aa57)，该区段被命名为TAT-蛋白转导域(protein transduction domain，TAT-PTD)，并被成功应用于不同外源蛋白的胞内转运。
CPP根据其化学性质的不同，可以分为三类:(1)阳离子型，富含精氨酸和赖氨酸残基，在生理pH下具有强正电荷，主要包括TAT、R9、hLF等。(2)两亲型肽类的正电荷由赖氨酸残基提供，这类CPP含疏水性和亲水性结构域，并且其两亲性的特征是由其一级结构和二级结构共同决定的，主要包括MPG、CADY、pVEC、SAP等。(3)疏水型CPPs具有较低的净电荷，主要由非极性氨基酸组成。比如卡波西成纤维细胞生长因子(K-FGF)肽。
在一优选实施方式中，本发明的CPP元件为TAT穿膜肽。
在一优选实施方式中，本发明的CPP元件选自下组：聚精氨酸(R5-9)、MPG(由人自身免疫缺陷病毒1型的gp41蛋白的融合蛋白结构域和猿猴空泡病毒大T抗原的核定位序列区域组成，氨基酸序列为：GLAFLGFLGAAGSTMGAWSQPKKKRKV(SEQ ID NO.:15))、CADY(含有色氨酸残基和精氨酸残基的20个氨基酸构成的次级两亲性肽，氨基酸序列为：GLWRALWRLLRSLWRLLWKA(SEQ ID NO.16)-CyA)、pVEC(来源于鼠源血管内皮细胞钙黏蛋白，氨基酸序列为：LLIILRRRIRKQAHAHSK(SEQ ID NO.:17))、Penetratin(果蝇触角同源异型转录因子蛋白中第三个α螺旋中具有较多正电荷的序列43－58，氨基酸序列为：RQIKIWFQNRRMKWKK(SEQ ID NO.:18))、卡波西成纤维细胞生长因子肽(K-FGF,氨基酸序列为：AAVLLPVLLAAP(SEQ ID NO.:19))、VP22(1型单纯疱疹病毒间层蛋白，氨基酸序列为：DAATATRGRSAASRPTERPRAPARSASRPRRVD(SEQ ID NO.:20))、或其组合。
免疫受体酪氨酸抑制性基序(ITIM)
目前已知多数免疫检查点抑制性受体(PD-1，BTLA，KIR，CD31和SIRPα等)，尽管它们的胞外区不同，但在其胞内段均含有一个或多个免疫受体酪氨酸抑制基序(Immunorector tyrosine based inhibitor motifs，ITIM)，当ITIM中的酪氨酸发生磷酸化后，可募集信号分子蛋白酪氨酸磷酸酶1或2(SH2domain-containing inositol phosphatase-1，SHP1；SH2 domain-containing  inositol phosphatase-2，SHP2)，引发抑制信号的产生，致使效应性T细胞失能和/或凋亡。因此，ITIM是许多免疫检查点发挥抑制作用的共同结构域，也是针对免疫检查点设计通用性药物的关键靶位。SHP1和SHP2高度同源，且经常被招募到相同的结合位点，SHP1和SHP2磷酸酶的不同作用目前尚不清楚。
SHP1
蛋白酪氨酸磷酸酶-1(SH2 domain-containing protein tyrosine phosphates,SHP1)主要表达于人体各种造血细胞,是控制淋巴细胞间信号传导通路中磷酸化酪氨酸水平的重要负调节因子。SHP1包含两个SH2结构域，分别为N-SH2(3～101)和C-SH2(109～215)，一个催化(PTP)结构域(272～514)，一个富含脯氨酸基团和酪氨酸磷酸化位点的尾巴。
SHP2
SHP2是由人非受体型蛋白酪氨酸磷酸酶11(Protein Tyrosine Phosphatase，Non Receptor Type 11，PTPN11)基因编码的蛋白酪氨酸磷酸酶，包含两个SH2结构域，分别为N-SH2(5～103)和C-SH2(111～218)，一个催化(PTP)结构域(276～523)，一个富含脯氨酸基团和酪氨酸磷酸化位点的尾巴。PTPN11基因包括16个外显子并产生广泛表达的7kb转录物，其含有编码593个氨基酸蛋白的1.779bp开放阅读框。此外，人和小鼠的SHP2基因序列同源性为100％。
在非活性状态(inactive)SHP2/SHP1的N-SH2结构域通过电荷-电荷相互作用与催化结构域PTP广泛接触，并且部分SH2结构域(NXGDY/F基序)插入催化裂缝中，阻止了底物进入活性位点而失去催化活性。当含有磷酸化酪氨酸残基的配体与N-SH2结构域的结合即可导致该SH2结构域中的变构开关从inactive状态转变为active状态。这种SHP2/SHP1的N-SH2结构域的构象变化破坏了SH2结构域和磷酸酶结构域之间的相互作用，使自身的抑制消除，从而允许底物进入。
在本发明中，以阻断免疫细胞内SHP2/SHP1的N-SH2和C-SH2结构域与ITIM的结合为切入点，应用基因工程和多肽固相合成技术分别设计和制备能够与ITIM结合的SHP2/SHP1的N-SH2和C-SH2结构域的融合蛋白和模拟多肽。为了使融合蛋白和多肽能够进入细胞，在融合蛋白的N-端和多肽的C-端融合了穿膜肽TAT。HIV1的反式激活蛋白(transactivator protein，TAT)能够将多肽和蛋白质高效快速地导入细胞内，而不影响细胞的正常结构和功能。融合蛋 白和多肽进入细胞后，能够与T淋巴细胞内正常表达的SHP2/SHP1竞争性结合抑制性受体胞内段的ITIM，使免疫细胞内的SHP2/SHP1一直处于非活性状态，从而使免疫抑制性受体的抑制作用失活，这就是本发明设计的以ITIM为靶点的通用免疫检查点抑制剂的研制思路。
融合蛋白
如本文所用，“本发明的融合蛋白”、或“多肽”均指本发明第一方面所述的融合蛋白。
在另一优选例中，所述融合蛋白的结构如Z1-L-Z2(I)或Z2-L-Z1(II)所示，式中B为CCP蛋白；L为无或连接元件；以及Z2为SHP2或SHP1的SH2结构域或其活性片段。
在另一优选例中，所述融合蛋白具有如SEQ ID NO.:4-8任一所示的氨基酸序列。
如本文所用，术语“融合蛋白”还包括具有上述活性的融合蛋白(如SEQ ID NO.:4-8任一所示的序列)的变异形式。这些变异形式包括(但并不限于)：1-3个(通常为1-2个，更佳地1个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为3个以内，较佳地为2个以内，更佳地为1个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。此外，所述术语还包括单体和多聚体形式的本发明多肽。该术语还包括线性以及非线性的多肽(如环肽)。
本发明还包括上述融合蛋白的活性片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明融合蛋白的功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或几个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)抗原肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6×His等标签序列融合而形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与式I或式II的氨基酸序列相比，有至多3个，较佳地至多2个，更佳地至多1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供本发明融合蛋白的类似物。这些类似物与SEQ ID NO.:4-8任一所示的多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
表达载体和宿主细胞
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发明融合蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组的融合蛋白。一般来说有以下步骤：
(1).用本发明的编码本发明融合蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明中，编码融合蛋白的多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含本发明融合蛋白编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞(如大肠杆菌)，或是低等真核细胞，或是高等真核细胞，如酵母细胞、植物细胞或哺乳动物细胞(包括人和非人哺乳动物)。代表性例子有：大肠杆菌、麦胚细胞，昆虫细胞，SF9、Hela、HEK293、CHO、酵母细胞等。在本发明的一个优选实施方式中，选择酵母细胞(如毕氏酵母、克鲁维酵母、或其组合；较佳地，所述的酵母细胞包括：克鲁维酵母，更佳地为马克斯克鲁维酵母、和/或乳酸克鲁维酵母)为宿主细胞。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序 列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
肽接头
本发明提供了一种融合蛋白，它可任选地含有肽接头。肽接头大小和复杂性可能会影响蛋白的活性。通常，肽接头应当具有足够的长度和柔韧性，以保证连接的两个蛋白在空间上有足够的自由度以发挥其功能。同时避免肽接头中形成α螺旋或β折叠等对融合蛋白的稳定性的影响。
连接肽的长度一般为0-10个氨基酸，较佳地0-5个氨基酸。
药物组合物
本发明还提供了一种药物组合物。在优选例中，所述的组合物是药物组合物，它含有上述的融合蛋白，以及药学上可接受的载体、稀释剂、稳定剂和/或增稠剂，并可制备成如冻干粉、片剂、胶囊、糖浆、溶液或悬浮液的药剂类型。
“药学上可接受的载体或赋形剂(excipient)”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的活性成分以及它们之间相互掺和，而不明显降低活性成分的药效。
组合物可以是液体或固体，例如粉末、凝胶或糊剂。优选地，组合物是液体，优选地可注射液体。合适的赋形剂将是本领域技术人员己知的。
药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温
Figure PCTCN2021071335-appb-000001
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：腹膜内、静脉内、或局部给药。所述药物组合物用于(a)用于治疗或预防肿瘤；(b)增强T细胞对肿瘤细胞的杀伤作用；(c)抑制肿瘤的生长；(d)抑制T细胞的凋亡；(e)增加T细胞分泌的IL-2的水平。(a)增强NK细胞对肿瘤细胞的杀伤作用；(b)增加NK细胞CD107a表达水平；(c)增加NK细胞穿孔素、颗粒酶分泌；(d)增加NK细胞IFN-γ、TNF-α分泌；(a)增强M细胞的吞噬功能；(b)增强M细胞对肿瘤细胞的吞噬杀伤作用；(c)增加M细胞NO、TNF-α和IL-1β分泌。
本发明的主要优点包括：
(a)本发明首次发现一种融合蛋白，该融合蛋白含有CCP蛋白、任选的连接元件和SHP2或SHP1的SH2结构域或其活性片段，本发明所得到的融合蛋白具有极其优异的对肿瘤的杀伤活性。
(b)本发明的融合蛋白还可显著增强T细胞对肿瘤细胞的杀伤作用；和/或抑制肿瘤的生长；和/或抑制T细胞的凋亡；和/或增加T细胞分泌的IL-2的 水平。
(c)本发明的融合蛋白还可增强NK细胞对肿瘤细胞的杀伤作用；增加NK细胞CD107a表达水平；增加NK细胞穿孔素、颗粒酶分泌；增加NK细胞IFN-γ、TNF-α分泌。
(d)本发明的融合蛋白还可增强巨噬细胞的吞噬功能；增强巨噬细胞对肿瘤细胞的吞噬杀伤作用；增加巨噬细胞NO、TNF-α和IL-1β分泌。
(e)本发明首次以阻断免疫细胞内SHP2或SHP1的N-SH2和C-SH2结构域与ITIM的结合为切入点，应用基因工程和多肽固相合成技术分别设计和制备能够与ITIM结合的SHP2或SHP1N-SH2和C-SH2结构域的融合蛋白和模拟多肽。为了使融合蛋白和多肽能够进入细胞，在融合蛋白的N-端和多肽的C-端融合了穿膜肽TAT。HIV1的反式激活蛋白(transactivator protein，TAT)能够将多肽和蛋白质高效快速地导入细胞内，而不影响细胞的正常结构和功能。融合蛋白和多肽进入细胞后，能够与T淋巴细胞/NK细胞/巨噬细胞内正常表达的SHP2或SHP1竞争性结合抑制性受体胞内段的ITIM，使免疫细胞内的SHP2或SHP1一直处于非活性状态，从而使免疫抑制性受体的抑制作用失活。
(d)本发明的融合蛋白具有广谱抗肿瘤作用。
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(New York:Cold Spring Harbor Laboratory Press,1989)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，否则百分比和份数是重量百分比和重量份数。
除非有特别说明，否则本发明实施例中的试剂和材料均为市售产品。
实施例1重组TAT-N-SH2融合蛋白及其制备方法
本发明的重组TAT-N-SH2融合蛋白，通过甘氨酸和丝氨酸将TAT穿膜肽和N-SH2连接起来(TAT-N-SH2)，其连接方式为：TAT-Gly-Ser-N-SH2；其氨基酸序列SEQ ID NO.:4所示。
该重组TAT-N-SH2融合蛋白的核苷酸序列是用BamHⅠ酶切位点连接TAT穿膜肽与SHP2的N端SH2结构域的核苷酸序列，在5’端加入NdeⅠ酶切位点,3’端加入入终止密码子TGA和SalⅠ酶切位点，其连接方式为：NdeⅠ酶切位点-TAT基因 -BamHⅠ酶切位点-N-SH2基因-TGA-SalⅠ酶切位点，其核苷酸序列如SEQ ID NO.:9所示。
重组TAT-N-SH2融合蛋白核苷酸构建于表达载体或重组质粒pET-22b(+)-TAT-N-SH2，所述的pET-22b(+)-TAT-N-SH2是通过NdeⅠ酶切位点和SalⅠ酶切位点将质粒pET-22b(+)与权利要求3所述的重组TAT-N-SH2融合蛋白核苷酸双酶切连接。
重组TAT-N-SH2融合蛋白的制备方法，按照以下步骤：
1、构建重组TAT-N-SH2融合蛋白基因表达载体或重组质粒pET-22b(+)-TAT-N-SH2
1)TAT-N-SH2的设计与合成
依据大肠杆菌偏爱的密码子，将TAT和N-SH2的氨基酸序列转化为基因序列，用BamHⅠ酶切位点连接TAT和N-SH2的基因序列，再在5’端加入NdeⅠ酶切位点,3’端加入终止密码子TGA和SalⅠ酶切位点，合成TAT-N-SH2基因，其核苷酸序列如下所示：
Figure PCTCN2021071335-appb-000002
送公司合成上述TAT-N-SH2基因。
2)表达载体pET-22b(+)-TAT-N-SH2的鉴定
将重组质粒pET-22b(+)-TAT-N-SH2转化BL21感受态细胞，37℃恒温箱培养12h后挑取单克隆菌落接种于含氨苄青霉素100mg/L的LB培养液中，摇床37℃培养后收菌提取质粒，NdeⅠ和SalⅠ双酶切，琼脂糖凝胶电泳鉴定，获得预期342bp大小的插入片段(图1)，DNA测序结果(图2)显示与所合成序列相符，pET-22b(+)-TAT-N-SH2重组质粒构建成功。
利用Nde I和Sal I酶对获得的重组质粒pET-22b(+)TAT–SHP2–N-SH2进行双酶切，然后进行琼脂糖凝胶电泳，红色箭头为目的片段。
2、重组TAT-N-SH2融合蛋白的表达
重组质粒pET-22b(+)-TAT-N-SH2转化大肠杆菌BL21感受态细胞，37℃恒温箱培养12h后挑取边缘整齐，生长状态良好的菌落，接种于含氨苄青霉素100mg/L的LB培养液中，摇床37℃培养过夜；次日以1∶100的比例接种于新鲜的含氨苄青霉素100mg/L的LB培养液中，37℃继续培养至细菌密度达到OD600＝0.4～0.6，加入0.01～0.1mM IPTG诱导4h后12000rpm离心20min收菌，-20℃保存。
3、重组TAT-N-SH2融合蛋白的纯化
1)称取菌液，按照1:7比例加入pH 6.5PB裂解缓冲液，置于4℃冰箱搅拌均匀至溶解；在功率为300W，工作时间5s，间隙时间10s，冰浴超声20min；4℃，12000rpm离心20min，收集上清；
2)取25mL裂菌上清装入事先准备好的透析袋中，以30倍体积的平衡A液(20mM pH 6.5，PB)为外液，4℃搅拌透析过夜，4℃ 12000rpm离心20min，收集上清过0.22μm的滤膜，记录体积；取事先装好的25mLSP-阳离子交换层析柱，用平衡A液平衡柱子至基线平稳，调零，以1mL/min的流速进行上样，收集穿过峰，待基线平稳后停止收峰；以流速1.5mL/min，使用平衡A液和洗脱B液(20mM pH(5.4，6.5，7.4)PB，1M NaCl)进行0～100％线性洗脱，并且收集洗脱峰4；以30倍体积PBS对目的蛋白的洗脱峰进行透析。透析后用0.22μm滤膜过滤除菌，即得到目的蛋白TAT-N-SH2融合蛋白。
4、重组TAT-N-SH2融合蛋白的诱导表达和纯化结果鉴定
1)用SDS-PAGE(聚丙烯酰胺凝胶电泳)分析目的蛋白的表达量(如图3所示)，与未诱导组比较，诱导组均有特异性蛋白条带，大小与理论分子量(12.870KD)相符。
超声裂菌后，分别收集上清和沉淀，SDS-PAGE检测目的蛋白是否为上清表达，结果如图4所示，裂菌上清和裂菌沉淀中均有目的蛋白，但是与裂菌上清相比，裂菌沉淀的量很少，可以忽略不计。因此，在后续的实验中我们均有裂菌上清为原料进行精细分离。
经过裂菌粗提的裂解上清，经透析、离心后，再利用SP-阳离子交换层析柱进行进一步分离，在不同的检测波长下收集峰液，最后发现在只有在215nm的波长下才能检测到目的蛋白，总共收到1个穿过峰和4个洗脱峰，分别为：穿过峰、SP峰1、SP峰2、SP峰3、SP峰4(如图5所示)；SDS-PAGE分析各峰，检测目的蛋白所在峰为SP4(如图6)。
5、重组TAT-N-SH2融合蛋白的特异性和纯度的鉴定
经过Western-blot定性检测目的蛋白，发现兔抗人SHP2多克隆抗体可以特异性的结合目的蛋白(如图7所示)，证明纯化得到的是目的蛋白。
对纯化所得的融合蛋白进行HPLC检测，取20μL样品进针，运行时间20min，流速0.8mL/min，检测波长280nm，主峰的面积判定样品的纯度。结果表明，该融合蛋白纯度为95.92％(图8)。
实施例2四种TAT穿膜肽与SHP2的C端SH2结构域的ITIM基序结合区域的融合多肽
1、一种TAT穿膜肽与SHP2的C端SH2结构域的ITIM基序结合区域的融合多肽，其特征在于，通过两个脯氨酸将TAT穿膜肽和C-SH2的ITIM基序结合区域连接起来(TAT-C-SH2)，其连接方式为：TAT-Phe-Phe-C-SH2；其氨基酸序列如下所示：
YGRKKRRQRRR-PP-VRESQSHPGDFVL(SEQ ID NO.:5)
全部用L型氨基酸，送公司合成上述TAT-C-SH2多肽(命名为L1)。以多肽L1的序列为参照，分别合成L1的反向序列，用D型氨基酸合成的L1序列，用D型氨基酸合成的L1反向序列，分别命名为L2、D1、D2。其氨基酸序列如下所示：
1)反向L型多肽(L2)：LVFDGPHSQSERVPPRRRQRRKKRGY(SEQ ID NO.:6)
2)D型多肽(D1)：YGRKKRRQRRRPPVRESQSHPGDFVL(SEQ ID NO.:7)
3)反向D型多肽(D1)：LVFDGPHSQSERVPPRRRQRRKKRGY(SEQ ID NO.:8)
全部送公司化学合成。
2、多肽的鉴定
利用HPLC分析合成多肽的纯度，取20μL样品进针，运行时间20min，流速1.0mL/min，检测波长220nm，主峰的面积判定样品的纯度。结果显示：L1多肽的纯度为96.74％(图9A)，L2多肽的纯度为98.1％(图9B)，D1多肽的纯度为99.27％(图9C)，D2多肽的纯度为96.66％(图9D)。
利用质谱检测合成多肽的分子量。结果显示：L1多肽的分子量是3206(图10A)，L2多肽的分子量是3206.67(图10B)，D1多肽的分子量是3206.63(图10C)，D2多肽的分子量是3206.79(图10D)，与理论分子量相一致。
结果表明，成功合成多肽，可用于进一步的体内外实验研究。
实施例3穿膜肽TAT能够协助重组融合蛋白和多肽进入T淋巴细胞
重组TAT-N-SH2融合蛋白和多肽能够进入T淋巴细胞内是与其细胞膜上抑制性受体胞内段ITIM结合发挥作用的前提条件。穿膜肽TAT是带正电荷的短肽，可以协助多种物质穿过细胞膜而发挥其生物学功能。为检测含TAT穿膜序列的融合蛋白和多肽能否进入T淋巴细胞，我们将TAT-N-SH2融合蛋白、L1多肽以及对照肽进行FITC标记，运用共聚焦显微镜观察其进入T淋巴细胞的情况，作用时间为0，0.5，4，24，72，96，120和144h(图11)。结果发现带有TAT穿膜序列的融合蛋白24h多数进入T细胞，直到120h逐渐减弱(图11c)。而带有TAT穿膜序列的L1多肽0.5h大多数已经进入T细胞，直到72h逐渐减弱(图11b)；无TAT穿膜序列的对照肽无法进入T细胞(图11a)。这一结果说明TAT穿膜序列可以协助融合蛋白和多肽进入目的细胞，且多肽与融合蛋白进入T细胞的时间不同，这可能与其分子量大小有关。
实施例4重组融合蛋白和多肽对T淋巴细胞功能的影响
1、重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽对T淋巴细胞增殖的影响
为了检测重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽是否能够提高T淋巴细胞的增殖，我们分别使用剂量为80μg/mLTAT-SHP2-N-SH2融合蛋白和TAT-SHP2-C-SH2多肽刺激T淋巴细胞3d后，通过CFSE检测T淋巴细胞的增殖，结果显示，对照组的T淋巴细胞增殖率为62.83％(图12a)，重组TAT-N-SH2融合蛋白组的T淋巴细胞增殖率为61.05％(图12b)，L1(TAT-SHP2-C-SH2)多肽组的T淋巴细胞增殖率为60.33％(图12c)。统计结果显示与对照组比较，重组TAT-N-SH2融合蛋白和L1多肽没有明显提高T淋巴细胞的增殖(图12d)，该结果说明重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽对T淋巴细胞增殖不产生影响。
2、重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽对T淋巴细胞凋亡的影响
使用重组TAT-N-SH2融合蛋白和L1多肽刺激3d后的T淋巴细胞，通过流式细胞术检测不同处理组T淋巴细胞凋亡的情况。图13a-d所示Annexin V-FITC检测多肽对T淋巴细胞凋亡的影响，重悬细胞加入不同组处理未给药组SHP2-NC、SHP2-N-SH2(80μg/mL)、TAT-SHP2-C-SH2多肽(80μg/mL)后继续在EASY-T 刺激的培养板上(加入细胞混合液前3h加入50ng PD-L1抗体37℃包被24孔板)培养3d，收集细胞，FITC和PI染色后通过流式细胞仪进行检测。图13e根据流式检测结果，统计凋亡细胞的比例，与对照组比较，*p<0.05或**p<0.01。综上所述，与对照组比较，重组TAT-N-SH2融合蛋白和L1多肽处理组的T淋巴细胞凋亡明显下降。
3、重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽对T淋巴细胞介导的下游信号分子磷酸化水平的影响
收集重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽刺激3d后的T淋巴细胞检测在ERK、AKT和JNK分子磷酸化水平的变化，结果显示，与未给药组比较，ERK、AKT和JNK的表达水平没有明显变化，但是TAT-SHP2-N-SH2融合蛋白刺激组p-ERK和p-AKT表达水平变化最明显，TAT-SHP2-C-SH2多肽刺激组p-AKT表达水平变化最明显(图14，a-d)。
4、重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽对T淋巴细胞分泌的细胞因子IL-2的影响
收集重组TAT-N-SH2融合蛋白和L1多肽刺激3d后的T淋巴细胞检测其分泌的细胞因子IL-2水平，结果显示，与对照组比较，TAT-SHP2-N-SH2融合蛋白和SHP2-C-SH多肽刺激T淋巴细胞分泌的IL-2水平显著升高(图15)。
实施例5重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽对肿瘤细胞的直接杀伤能力的研究
为了检测重组TAT-N-SH2融合蛋白和L1多肽对肿瘤细胞是否存在直接杀伤作用，我们将不同浓度(20μg/mL，40μg/mL，80μg/mL)重组TAT-N-SH2融合蛋白和L1多肽与肿瘤细胞MDA-MB-231和SW480共培养后，应用zenCell owl活细胞动态成像分析系统实时观察其对肿瘤细胞的影响。实验结果发现，与未加药组比较，重组TAT-N-SH2融合蛋白和L1多肽对肿瘤细胞MDA-MB-231和SW480无直接毒性作用，该结果说明重组TAT-N-SH2融合蛋白和L1多肽是通过作用于T细胞而发挥杀伤肿瘤细胞作用(图16)。
实施例6重组融合蛋白和多肽在体外增强T细胞对肿瘤细胞杀伤能力的研究
1、重组TAT-N-SH2融合蛋白对T细胞杀伤乳腺癌细胞MDA-MB-231和结肠癌细 胞SW480作用的影响
选择三种蛋白浓度(20μg/mL，40μg/mL，80μg/mL)的TAT-SHP2-N-SH2融合蛋白刺激人T淋巴细胞并通过CytoTox 96非放射性细胞毒性法检测在30:1的效靶比下分别对肿瘤细胞MDA-MB-231和SW480的杀伤作用。实验结果显示，靶细胞为乳腺癌细胞株MDA-MB-231时，40μg/mL和80μg/mL浓度的TAT-SHP2-N-SH2融合蛋白组的杀伤作均显著高于对照组高(p＝0.0379和p＝0.0367)。当靶细胞为结肠癌细胞株SW480时，三个浓度的蛋白组均明显高于未给药组(图17，表1)。说明TAT-SHP2-N-SH2融合蛋白能够提高T细胞杀伤肿瘤细胞的作用，且杀伤结肠癌的效果优于乳腺癌细胞。
表1.TAT-SHP2-N-SH2融合蛋白刺激T细胞对不同肿瘤细胞株的杀伤作用
Figure PCTCN2021071335-appb-000003
与未给药组比较，*p<0.05或**p<0.01或***p<0.001，p>0.05无统计学意义
2、L1(TAT-SHP2-C-SH2)多肽对T细胞杀伤乳腺癌细胞MDA-MB-231和结肠癌细胞SW480作用的影响
运用CytoTox 96非放射性细胞毒性法检测TAT-SHP2-C-SH2多肽(20μg/mL，40μg/mL，80μg/mL)刺激人的T淋巴细胞4d后分别对肿瘤细胞株MDA-MB-231和SW480的杀伤作用。在效靶比为30:1且TAT-SHP2-C-SH2多肽浓度分别为40μg/mL和80μg/mL时，与对照组比较，TAT-SHP2-C-SH2多肽刺激的T淋巴细胞杀伤MDA-MB-231和SW480的能力显著提高(图18，表2)。
表2.TAT-SHP2-C-SH2多肽刺激T细胞对不同肿瘤细胞株的杀伤作用
Figure PCTCN2021071335-appb-000004
Figure PCTCN2021071335-appb-000005
与未给药组比较，*p<0.05或**p<0.01或***p<0.001，p>0.05无统计学意义。
3、L1、L2、D1、D2多肽对T细胞杀伤肺癌细胞H460作用的影响
运用CytoTox 96非放射性细胞毒性法检测L1、L2、D1、D2多肽(40μg/mL)刺激人的T淋巴细胞4d后分别对肺癌细胞H460的杀伤作用，在效靶比为20:1时，与未给药组和对照肽比较，L1、L2、D1、D2多肽刺激的T淋巴细胞杀伤肺癌细胞H460的能力显著提高，且以D2多肽效果最强。(图19，表3)。
表3.L1、L2、D1、D2多肽刺激T细胞对肺癌细胞H460的杀伤作用
Figure PCTCN2021071335-appb-000006
和对照组比，*P<0.05，**P<0.01，***P<0.001，****P<0.0001；
和D2组比，#P<0.05，##P<0.01，###P<0.001
实施例7重组融合蛋白和多肽在体内抗肿瘤作用的研究
1、重组TAT-N-SH2融合蛋白对小鼠结肠癌的抑瘤效果检测
建立MC38结肠癌小鼠皮下移植瘤模型，以50μg/只αPD-1抗体给药剂量为阳性对照药物，选取浓度为1.25μg/只(N1.25)，2.5μg/只(N2.5)，5μg/只(N5)和10μg/只(N10)TAT-SHP2-N-SH2融合蛋白腹腔注射给药。待给药8次后，解剖荷瘤小鼠称瘤重，发现TAT-SHP2-N-SH2融合蛋白组瘤重明显小于对照组，且N5组瘤重小于αPD-1抗体组(图20，a-b)，N5组的抑瘤率最高(表4)，达到80.9％，该结果表明5μg/只的TAT-SHP2 1-N-SH2融合蛋白给药剂量抑制结肠癌肿瘤的效果最佳。
表4 TAT-SHP2-N-SH2融合蛋白对结肠癌荷瘤小鼠的抑瘤率
Figure PCTCN2021071335-appb-000007
和Control组比， *p<0.05， **p<0.01， ***p<0.001
2、L1(TAT-SHP2-C-SH2)多肽对小鼠结肠癌的抑瘤效果检测
通过建立的MC38结肠癌小鼠皮下荷瘤模型评价了TAT-SHP2-C-SH2多肽对小鼠肿瘤生长的影响，分别以1.25μg/只(C1.25)，2.5μg/只(C2.5)，5μg/只(C5)和10μg/只(C10)TAT-SHP2-C-SH2多肽腹腔注射给药，同时以50μg/只αPD-1抗体(αPD-1)给药剂量为阳性对照药物，并且加入TAT-SHP2-N-SH2(5μg/只)组和联合组(TAT-SHP2-N-SH2融合蛋白和TAT-SHP2C-SH2多肽各5μg/只)，发现相比于对照组、TAT-SHP2-C-SH2多肽组、αPD-1抗体组、TAT-SHP2-N-SH2融合蛋白组及联合组的肿瘤瘤重明显降低。另外，联合组抑瘤效果最佳，其次是N5组，TAT-SHP2-C-SH2多肽在给药剂量为5μg/只肿瘤生长最缓慢(图21，a-b)；联合组抑瘤率最佳，其次是N5组(表5)，该结果表明不同剂量组对小鼠肿瘤的生长均表现有抑制作用，其中联合组效果最佳。
表5.TAT-SHP2-C-SH2多肽对结肠癌荷瘤小鼠的抑瘤率
Figure PCTCN2021071335-appb-000008
和Control组比， ***p<0.001
3、重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽对小鼠乳腺癌的抑瘤效果检测
我们进一步通过建立的EMT乳腺癌小鼠皮下荷瘤模型评价了TAT-SHP2SHP2-N-SH2融合蛋白和TAT-SHP2SHP2-C-SH2多肽对小鼠肿瘤生长的影响，我们分别以5μg/只(C5)，10μg/只(C10)，15μg/只(C15)的TAT-SHP2SHP2-C-SH2多肽和2μg/只(N2)，5μg/只(N5)，10μg/只(N10)，15μg/只(N15)的TAT-SHP2SHP2-N-SH2融合蛋白腹腔注射给药，同时以50μg/只αPD-1抗体(αPD-1)给药剂量为阳性对照药物，结果表明：TAT-SHP2SHP2-C-SH2多肽组、αPD-1抗体组、TAT-SHP2SHP2-N-SH2融合蛋白组各治疗组的肿瘤体积明显小于对照组；TAT-SHP2SHP2-N-SH2融合蛋白(5μg/只)和TAT-SHP2SHP2或SHP1-C-SH2多肽(5μg/只)组的瘤重最小，抑瘤率最佳(图22，a-b，表6)。该结果说明不同剂量组对小鼠肿瘤的生长均表现有抑制作用，其中融合蛋白和多肽剂量均为5μg/只时效果最佳，与结肠癌抑瘤结果一致。
表6.TAT-SHP2-N-SH2融合蛋白和TAT-SHP2-C-SH2多肽对乳腺癌荷瘤小鼠的抑瘤率
Figure PCTCN2021071335-appb-000009
与对照组比较，*p<0.05，**p<0.01，***p<0.001
4、重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽对裸鼠移植瘤的抑瘤作用
1)重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽对MC38结肠癌裸鼠的抑瘤作用
为了排除重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽对肿瘤的直接抑制作用，我们选择免疫缺陷的裸鼠接种MC38结肠癌肿瘤，14d成瘤后开始给 药，4d测量一次瘤体积，待30d后处死小鼠进行解剖，测量瘤重，结果发现，重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽小鼠瘤体积和瘤重，抑瘤率与对照组无统计学差异(图23，a-c，表7)，该结果表明TAT-SHP2-N-SH2融合蛋白和TAT-SHP2-C-SH2多肽对结肠癌无直接抑制作用。
表7.重组TAT-N-SH2融合蛋白和L1多肽对结肠癌裸鼠的抑瘤作用
Figure PCTCN2021071335-appb-000010
和Control组比， ns p>0.05
2)重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽对EMT-6乳腺癌裸鼠的抑瘤作用
同时我们选择免疫缺陷的裸鼠小鼠接种EMT-6乳腺癌肿瘤，5d成瘤后随机分三组，每组6只，开始给药，给药剂量同上，隔一天给药1次，共给药8次，4d测量瘤体大小，绘制生长曲线，第21d处死小鼠测量体重，发现与对照组比较，重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽处理组瘤体积和瘤重，抑瘤率无明显差异，该结果说明重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽无法直接抑制肿瘤细胞，而是通过免疫细胞发挥抑制乳腺癌肿瘤生长的(图24，a-c，表8)。
表8.重组TAT-N-SH2融合蛋白和L1多肽对裸鼠乳腺癌的抑瘤率
Figure PCTCN2021071335-appb-000011
和Control组比， ns p>0.05
5、重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽对小鼠PD-L1敲除的乳腺癌移植瘤的抑瘤效果检测
重组TAT-N-SH2融合蛋白和L1(TAT-SHP2-C-SH2)多肽针对的是抑制性 受体的ITIM基序，所以比单一阻断PD-1这一种抑制性受体的PD-1单抗的治疗范围要更广，理论上可以治疗PD-1抗体治疗无效的肿瘤。为此，我们构建了PD-L1稳定敲除的乳腺癌EMT-6细胞系(PD-L1-KO-EMT6),接种到小鼠乳腺脂肪垫下，分别以5μg/只的重组TAT-N-SH2融合蛋白(N5)和TAT-SHP2-C-SH2多肽(C5)腹腔注射给药，同时以50μg/只αPD-1抗体(αPD-1)给药剂量为对照药物。结果表明：N5,C5均能显著抑制PD-L1-KO-EMT6肿瘤的生长，但是PD-1抗体治疗无效(图25，a-c，表9)。
表9.不同药物对BALB/c小鼠PD-L1敲除的EMT-6皮下移植瘤的抑瘤率
Figure PCTCN2021071335-appb-000012
和对照组比，**P<0.01，*P<0.05
6、L1、L2、D1、D2多肽对小鼠肺癌的抑瘤效果检测
我们进一步通过建立的小鼠Lewis肺癌移植瘤模型评价了L1、L2、D1、D2多肽对小鼠肿瘤生长的影响，我们分别以5μg/只的L1、L2、D1、D2多肽腹腔注射给药，同时以5μg/只对照肽(R肽)给药剂量为阴性对照药物，生理盐水为空白对照组(NC)，隔一天打一次。共打8次。结果表明：L1、L2、D1、D2多肽均能显著抑制肿瘤的生长，D2组的抑瘤效果最好、抑瘤率最强(P<0.001)，与体外杀伤实验结果一致(图26，a-c，表10)。
表10.L1、L2、D1、D2多肽对小鼠肺癌的抑瘤效果
Figure PCTCN2021071335-appb-000013
和对照组比，*P<0.05，**P<0.01，***P<0.001；
和D2组比，#P<0.05，##P<0.01，###P<0.001，####P<0.0001
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. 一种融合蛋白，其特征在于，所述融合蛋白具有从N端至C端的式I或II所示的结构：
    Z1-L-Z2  (I)
    Z2-L-Z1  (II)
    式中，
    Z1为CPP元件；
    L为无或连接元件；
    Z2为SHP2和/或SHP1的SH2结构域或其活性片段；
    “-”表示连接上述元件的肽键或肽接头。
  2. 如权利要求1所述的融合蛋白，其特征在于，所述Z1、Z2以头-头、头-尾、尾-头、或尾-尾方式相连。
  3. 如权利要求1所述的融合蛋白，其特征在于，所述Z1、Z2为D型或L型氨基酸。
  4. 一种分离的多核苷酸，其特征在于，所述的多核苷酸编码权利要求1所述的融合蛋白。
  5. 一种载体，其特征在于，它含有权利要求4所述的多核苷酸。
  6. 一种宿主细胞，其特征在于，所示所述宿主细胞含有权利要求5所述的载体，或其基因组中整合有权利要求4所述的多核苷酸。
  7. 一种产生权利要求1所述的融合蛋白的方法，其特征在于，所述的方法包括步骤：
    在适合表达的条件下，培养权利要求6所述的宿主细胞，从而表达融合蛋白；和/或分离所述融合蛋白。
  8. 一种药物组合物，其特征在于，所述的药物组合物含有权利要求1所述的融合蛋白及其药物学上可接受的载体。
  9. 权利要求1所述融合蛋白、权利要求4所述的多核苷酸、权利要求5所述的载体、权利要求6所述的宿主细胞的用途，其特征在于，用于制备组合物或制剂，所述组合物或制剂用于治疗或预防肿瘤。
  10. 一种体外非治疗性的抑制肿瘤生长的方法，其特征在于，包括步骤：在权利要求1所述的融合蛋白存在下，培养肿瘤细胞，从而抑制肿瘤生长。
PCT/CN2021/071335 2020-01-13 2021-01-12 一种抗肿瘤融合蛋白及其制法和应用 WO2021143695A1 (zh)

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