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WO2022048574A1 - Molécule d'acide nucléique codant pour un mutant de gène kras - Google Patents

Molécule d'acide nucléique codant pour un mutant de gène kras Download PDF

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Publication number
WO2022048574A1
WO2022048574A1 PCT/CN2021/116075 CN2021116075W WO2022048574A1 WO 2022048574 A1 WO2022048574 A1 WO 2022048574A1 CN 2021116075 W CN2021116075 W CN 2021116075W WO 2022048574 A1 WO2022048574 A1 WO 2022048574A1
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kras
mutant
gene encoding
seq
nucleic acid
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Chinese (zh)
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张骅
华坚
陈晓庆
刘园园
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Sph Biotherapeutics Hk Ltd
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Sph Biotherapeutics Hk Ltd
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Priority to CN202180051207.XA priority Critical patent/CN116323947A/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • 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
    • 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
    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors

Definitions

  • the present application relates to the field of biomedicine, in particular to a nucleic acid molecule encoding a Kras gene mutant, and the application of an oncolytic herpes simplex virus (oHSV) vector containing the nucleic acid molecule in the preparation of antitumor drugs.
  • oHSV oncolytic herpes simplex virus
  • Oncolytic virus is a kind of natural or artificially modified virus that can specifically replicate in a large amount in tumor cells and eventually eliminate tumor cells, but has no killing effect on normal tissue cells.
  • viruses used in oncolytic therapy including herpes simplex virus, adenovirus, reovirus, measles virus, etc.
  • HSV herpes simplex virus
  • Herpes simplex virus is a commonly used virus in genetic engineering, which is divided into type 1 and type 2. With the development of virology and genetic engineering technology, people can modify viral genes and apply them in the treatment of tumors. As early as 1991, Martuza et al. genetically modified herpes simplex virus type 1 (HSV-1) and established an oncolytic virus strain that can inhibit the proliferation of tumor cells and has replication activity for malignant brain tumors. Treatment.
  • Kras gene is a proto-oncogene, about 35kb long, located on chromosome 12, is one of the members of RAS gene family, encoding Kras protein.
  • Kras protein is a membrane-bound protein that is located on the inner side of the cell membrane and is also located in the EGFR (epidermal growth factor receptor) signaling pathway, which plays an important role in the occurrence and development of tumors.
  • the growth, proliferation, angiogenesis and other processes of tumor cells require intracellular proteins for signal transduction, and the Kras gene is the determinant of the transduction protein.
  • the Kras mutant encodes an abnormal protein, which stimulates the growth and spread of malignant tumor cells, and is not affected by it. Signaling effects of upstream EGFR.
  • oncolytic viruses still have some problems in tumor treatment. For example, patients who have been attacked by viruses have residual virus antibodies in their bodies, and tumor cells cannot be completely eliminated. Therefore, it is necessary to develop novel oncolytic viruses and evaluate their antitumor activity.
  • the application provides an isolated nucleic acid molecule comprising one or more genes each independently selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V Mutant, Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T Mutant, Kras A146T mutant, Kras Y64H mutant and Kras A18D mutant.
  • genes each independently selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V Mutant, Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T Mutant, Kras A146T mutant
  • the application provides an isolated nucleic acid molecule that does not comprise a polynucleotide encoding a 6x His-tagged protein, and that comprises one or more genes each independently encoding a Kras mutant selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T mutant, Kras A146T mutant, Kras Y64H mutant and Kras A18D mutant.
  • a Kras mutant selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras
  • the isolated nucleic acid molecule comprises a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant, and KrasG12C mutant.
  • the isolated nucleic acid molecule comprises a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant, Kras Y64H mutant and Kras G12C mutant.
  • the 3' end of the gene encoding the Kras G12D mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras A146T mutant.
  • the 3' end of the gene encoding the Kras A46T mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras G12V mutant.
  • the 3' end of the gene encoding the Kras G12V mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras A59T mutant.
  • the 3' end of the gene encoding the Kras A59T mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras G13D mutant.
  • the 3' end of the gene encoding the Kras G13D mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras Y64H mutant.
  • the 3' end of the gene encoding the Kras Y64H mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras G12C mutant.
  • each of the genes encoding the Kras mutants are arranged in tandem in the isolated nucleic acid molecule.
  • each of the genes encoding the Kras mutants is a minigene Minigene.
  • each of the genes encoding the Kras mutants is arranged in tandem to yield a tandem minigene.
  • each of the Kras mutants comprises at least 20 amino acids.
  • each of the Kras mutants comprises at least 9 amino acids immediately N-terminal to the mutation site and at least 10 amino acids C-terminal to the mutation site.
  • the Kras G12D mutant comprises the amino acid sequence set forth in SEQ ID NO:1.
  • the Kras G13D mutant comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the Kras G12V mutant comprises the amino acid sequence set forth in SEQ ID NO:7.
  • the Kras G13C mutant comprises the amino acid sequence set forth in SEQ ID NO:8.
  • the Kras G12C mutant comprises the amino acid sequence set forth in SEQ ID NO:9.
  • the Kras G13A mutant comprises the amino acid sequence set forth in SEQ ID NO:10.
  • the Kras G12A mutant comprises the amino acid sequence set forth in SEQ ID NO:11.
  • the Kras Q61L mutant comprises the amino acid sequence set forth in SEQ ID NO:12.
  • the Kras G12R mutant comprises the amino acid sequence set forth in SEQ ID NO:15.
  • the Kras Q61H mutant comprises the amino acid sequence set forth in SEQ ID NO:16.
  • the Kras G12S mutant comprises the amino acid sequence set forth in SEQ ID NO:17.
  • the Kras Q61R mutant comprises the amino acid sequence set forth in SEQ ID NO:18.
  • the Kras A59T mutant comprises the amino acid sequence set forth in SEQ ID NO:54.
  • the Kras A146T mutant comprises the amino acid sequence set forth in SEQ ID NO:56.
  • the Kras Y64H mutant comprises the amino acid sequence set forth in SEQ ID NO:58.
  • the Kras A18D mutant comprises the amino acid sequence set forth in SEQ ID NO:60.
  • the gene encoding the Kras G12D mutant comprises the nucleotide sequence set forth in SEQ ID NO:19.
  • the gene encoding the Kras G13D mutant comprises the nucleotide sequence set forth in SEQ ID NO:22.
  • the gene encoding the Kras G12V mutant comprises the nucleotide sequence set forth in SEQ ID NO:25.
  • the gene encoding the Kras G13C mutant comprises the nucleotide sequence set forth in SEQ ID NO:26.
  • the gene encoding the Kras G12C mutant comprises the nucleotide sequence set forth in SEQ ID NO:27.
  • the gene encoding the Kras G13A mutant comprises the nucleotide sequence set forth in SEQ ID NO:28.
  • the gene encoding the Kras G12A mutant comprises the nucleotide sequence set forth in SEQ ID NO:29.
  • the gene encoding the Kras Q61L mutant comprises the nucleotide sequence set forth in SEQ ID NO:30.
  • the gene encoding the Kras G12R mutant comprises the nucleotide sequence set forth in SEQ ID NO:33.
  • the gene encoding the Kras Q61H mutant comprises the nucleotide sequence set forth in SEQ ID NO:34.
  • the gene encoding the Kras G12S mutant comprises the nucleotide sequence set forth in SEQ ID NO:35.
  • the gene encoding the Kras Q61R mutant comprises the nucleotide sequence set forth in SEQ ID NO:36.
  • the gene encoding the Kras A59T mutant comprises the nucleotide sequence set forth in SEQ ID NO:55.
  • the gene encoding the Kras A146T mutant comprises the nucleotide sequence set forth in SEQ ID NO:57.
  • the gene encoding the Kras Y64H mutant comprises the nucleotide sequence set forth in SEQ ID NO:59.
  • the gene encoding the Kras A18D mutant comprises the nucleotide sequence set forth in SEQ ID NO:61.
  • the tandem minigene comprises, from 5' end to 3' end, a gene encoding a Kras G12D mutant, a gene encoding a Kras A59T mutant, a gene encoding a Kras G12V mutant, and a gene encoding a Kras A146T mutation gene encoding Kras G13D mutant, gene encoding Kras Y64H mutant, gene encoding Kras G12C mutant, gene encoding Kras Q61H mutant, gene encoding Kras A18D mutant, gene encoding Kras G12A mutant Gene, the gene encoding the Kras A146T mutant, and the gene encoding the Kras G12S mutant.
  • the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:65.
  • the tandem minigene comprises, from 5' end to 3' end, a gene encoding a Kras G12D mutant, a gene encoding a Kras A146T mutant, a gene encoding a Kras G12V mutant, and a gene encoding a Kras A59T mutation
  • the gene encoding the Kras G13D mutant, the gene encoding the Kras Y64H mutant, and the gene encoding the Kras G12C mutant is selected from 5' end to 3' end, a gene encoding a Kras G12D mutant, a gene encoding a Kras A146T mutant, a gene encoding a Kras G12V mutant, and a gene encoding a Kras A59T mutation.
  • the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:66.
  • the tandem minigene comprises, from the 5' end to the 3' end, the gene encoding the Kras G12D mutant, the gene encoding the Kras A59T mutant, the gene encoding the Kras A18D mutant, and the gene encoding the Kras G12V mutant
  • the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:67.
  • the isolated nucleic acid molecule further comprises a polynucleotide encoding a secreted peptide.
  • the polynucleotide encoding the secreted peptide is a polynucleotide encoding the secreted peptide of CD14 protein.
  • the polynucleotide encoding the CD14 protein secretory peptide is located 5' to the gene encoding the Kras mutant.
  • the polynucleotide encoding the CD14 protein secreted peptide comprises the nucleotide sequence set forth in any one of SEQ ID NO:37.
  • the isolated nucleic acid molecule further comprises a polynucleotide encoding a tag protein.
  • the isolated nucleic acid molecule comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 62-64.
  • the present application provides a vector comprising the nucleic acid molecule.
  • the vector comprises a viral vector.
  • the vector comprises an oncolytic herpes simplex virus oHSV vector.
  • the vector comprises a herpes simplex virus type I HSV-1 vector.
  • the HSV-1 vector is deficient in the neurotoxic factor gamma 34.5 gene.
  • the isolated nucleic acid molecule is located between the UL3 gene and the UL4 gene of the HSV-1 vector.
  • the vector includes a promoter.
  • the promoter comprises a CMV promoter.
  • the vector comprises the nucleotide sequence shown in GenBank No: GU734771.1 of the NCBI database.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the nucleic acid molecule, and/or the carrier, and optionally a pharmaceutically acceptable adjuvant.
  • the present application provides a composition comprising the isolated nucleic acid molecule described herein, the carrier described herein or the pharmaceutical composition described herein, and physiological saline.
  • the isolated nucleic acid molecule in the composition comprises one or more genes each independently encoding a Kras mutant selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T mutant, Kras A146T mutant, Kras Y64H mutant and Kras A18D mutant.
  • a Kras mutant selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T mutant, Kra
  • the isolated nucleic acid molecule in the composition comprises a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant and KrasG12C mutant.
  • the isolated nucleic acid molecule in the composition comprises a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant, Kras Y64H mutant and Kras G12C mutant.
  • the 3' end of the gene encoding the Kras G12D mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras A146T mutant.
  • the 3' end of the gene encoding the Kras A46T mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras G12V mutant.
  • the 3' end of the gene encoding the Kras G12V mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras A59T mutant.
  • the 3' end of the gene encoding the Kras A59T mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras G13D mutant.
  • the 3' end of the gene encoding the Kras G13D mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras Y64H mutant.
  • the 3' end of the gene encoding the Kras Y64H mutant is directly or indirectly linked to the 5' end of the gene encoding the Kras G12C mutant.
  • each of the genes encoding the Kras mutants are arranged in tandem in the isolated nucleic acid molecule.
  • each of the genes encoding the Kras mutants is a minigene Minigene.
  • each of the genes encoding the Kras mutants is arranged in tandem to yield a tandem minigene.
  • each of the Kras mutants comprises at least 20 amino acids.
  • each of the Kras mutants comprises at least 9 amino acids immediately N-terminal to the mutation site and at least 10 amino acids C-terminal to the mutation site.
  • the Kras G12D mutant comprises the amino acid sequence set forth in SEQ ID NO:1.
  • the Kras G13D mutant comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the Kras G12V mutant comprises the amino acid sequence set forth in SEQ ID NO:7.
  • the Kras G13C mutant comprises the amino acid sequence set forth in SEQ ID NO:8.
  • the Kras G12C mutant comprises the amino acid sequence set forth in SEQ ID NO:9.
  • the Kras G13A mutant comprises the amino acid sequence set forth in SEQ ID NO:10.
  • the Kras G12A mutant comprises the amino acid sequence set forth in SEQ ID NO:11.
  • the Kras Q61L mutant comprises the amino acid sequence set forth in SEQ ID NO:12.
  • the Kras G12R mutant comprises the amino acid sequence set forth in SEQ ID NO:15.
  • the Kras Q61H mutant comprises the amino acid sequence set forth in SEQ ID NO:16.
  • the Kras G12S mutant comprises the amino acid sequence set forth in SEQ ID NO:17.
  • the Kras Q61R mutant comprises the amino acid sequence set forth in SEQ ID NO:18.
  • the Kras A59T mutant comprises the amino acid sequence set forth in SEQ ID NO:54.
  • the Kras A146T mutant comprises the amino acid sequence set forth in SEQ ID NO:56.
  • the Kras Y64H mutant comprises the amino acid sequence set forth in SEQ ID NO:58.
  • the Kras A18D mutant comprises the amino acid sequence set forth in SEQ ID NO:60.
  • the gene encoding the Kras G12D mutant comprises the nucleotide sequence set forth in SEQ ID NO:19.
  • the gene encoding the Kras G13D mutant comprises the nucleotide sequence set forth in SEQ ID NO:22.
  • the gene encoding the Kras G12V mutant comprises the nucleotide sequence set forth in SEQ ID NO:25.
  • the gene encoding the Kras G13C mutant comprises the nucleotide sequence set forth in SEQ ID NO:26.
  • the gene encoding the Kras G12C mutant comprises the nucleotide sequence set forth in SEQ ID NO:27.
  • the gene encoding the Kras G13A mutant comprises the nucleotide sequence set forth in SEQ ID NO:28.
  • the gene encoding the Kras G12A mutant comprises the nucleotide sequence set forth in SEQ ID NO:29.
  • the gene encoding the Kras Q61L mutant comprises the nucleotide sequence set forth in SEQ ID NO:30.
  • the gene encoding the Kras G12R mutant comprises the nucleotide sequence set forth in SEQ ID NO:33.
  • the gene encoding the Kras Q61H mutant comprises the nucleotide sequence set forth in SEQ ID NO:34.
  • the gene encoding the Kras G12S mutant comprises the nucleotide sequence set forth in SEQ ID NO:35.
  • the gene encoding the Kras Q61R mutant comprises the nucleotide sequence set forth in SEQ ID NO:36.
  • the gene encoding the Kras A59T mutant comprises the nucleotide sequence set forth in SEQ ID NO:55.
  • the gene encoding the Kras A146T mutant comprises the nucleotide sequence set forth in SEQ ID NO:57.
  • the gene encoding the Kras Y64H mutant comprises the nucleotide sequence set forth in SEQ ID NO:59.
  • the gene encoding the Kras A18D mutant comprises the nucleotide sequence set forth in SEQ ID NO:61.
  • the tandem minigene comprises, from the 5' end to the 3' end, the gene encoding the Kras G12D mutant, the gene encoding the Kras A59T mutant, the gene encoding the Kras G12V mutant, and the gene encoding the Kras A146T mutation gene encoding Kras G13D mutant, gene encoding Kras Y64H mutant, gene encoding Kras G12C mutant, gene encoding Kras Q61H mutant, gene encoding Kras A18D mutant, gene encoding Kras G12A mutant Gene, the gene encoding the Kras A146T mutant, and the gene encoding the Kras G12S mutant.
  • the isolated nucleic acid molecule in the composition comprises the nucleotide sequence set forth in SEQ ID NO:65.
  • the tandem minigene comprises, from the 5' end to the 3' end, the gene encoding the Kras G12D mutant, the gene encoding the Kras A146T mutant, the gene encoding the Kras G12V mutant, and the Kras A59T mutation
  • the isolated nucleic acid molecule in the composition comprises the nucleotide sequence set forth in SEQ ID NO:66.
  • the tandem minigene comprises, from the 5' end to the 3' end, the gene encoding the Kras G12D mutant, the gene encoding the Kras A59T mutant, the gene encoding the Kras A18D mutant, and the gene encoding the Kras G12V mutant
  • the isolated nucleic acid molecule in the composition comprises the nucleotide sequence set forth in SEQ ID NO:67.
  • the isolated nucleic acid molecule in the composition further comprises a polynucleotide encoding a secreted peptide.
  • the polynucleotide encoding the secreted peptide is a polynucleotide encoding the secreted peptide of CD14 protein.
  • the polynucleotide encoding the CD14 protein secretory peptide is located 5' to the gene encoding the Kras mutant.
  • the polynucleotide encoding the CD14 protein secreted peptide comprises the nucleotide sequence set forth in any one of SEQ ID NO:37.
  • the isolated nucleic acid molecule in the composition further comprises a polynucleotide encoding a tag protein.
  • the isolated nucleic acid molecule in the composition comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 62-64.
  • the composition comprises a vector described herein comprising an isolated nucleic acid molecule described herein.
  • the vector in the composition comprises a viral vector.
  • the vector in the composition comprises an oncolytic herpes simplex virus oHSV vector.
  • the vector in the composition comprises a herpes simplex virus type I HSV-1 vector.
  • the HSV-1 vector is deficient in the neurotoxic factor gamma 34.5 gene.
  • the nucleic acid molecule in the composition, in the vector, is located between the UL3 gene and the UL4 gene of the HSV-1 vector.
  • the vector in the composition includes a promoter.
  • the promoter comprises a CMV promoter.
  • the vector in the composition comprises the nucleotide sequence shown in GenBank No: GU734771.1 of the NCBI database.
  • the present application provides the nucleic acid molecule, the carrier, the pharmaceutical composition and/or the application of the composition in the preparation of a medicament for treating tumors.
  • the tumor comprises a solid tumor.
  • the tumor comprises non-small cell lung cancer.
  • the tumor comprises colorectal cancer.
  • the tumor comprises breast cancer.
  • the tumor comprises pancreatic cancer.
  • Figure 1 shows the structure of the Kras mutant tandem minigene described in the present application.
  • FIG. 2A shows the structural composition of wild-type HSV-1 (F) described in this application.
  • Figure 2B shows the structural composition of the modified HSV-1(F)-KR10 described in the present application.
  • Figure 2C shows the structural composition of the BAC plasmid containing the KR11 nucleotide sequence described in this application.
  • Figure 2D shows the structural composition of the BAC plasmid containing the KR12 nucleotide sequence described in this application.
  • Figure 3 shows the body weight change trend of the CT26.WT subcutaneously transplanted tumor mice described in the present application.
  • Figure 4A shows the changes of tumor volume in the CT26.WT subcutaneously transplanted tumor mice described in the present application.
  • Figure 4B shows the individual data of the tumor volume change in the CT26.WT subcutaneously transplanted tumor mice described in the present application.
  • Figure 5 shows the statistics of tumor weight in CT26.WT subcutaneously transplanted mice described in the present application.
  • Figures 6A-6B show tumor photographs after euthanasia of the CT26.WT model described herein.
  • Figure 7 shows the changes in tumor volume in animals described herein (#5203, #5204).
  • Figure 8 shows the photo of the end point of the tumor re-challenge model animal experiment described in the present application.
  • Figure 9 shows the body weight change trend of the A549 subcutaneously transplanted tumor mice described in the present application.
  • Figure 10 shows the changes of tumor volume in mice with A549 subcutaneously transplanted tumor described in the present application.
  • FIG. 11 shows the changes in tumor volume of each group of animals described in this application.
  • Figure 12 shows the tumor weight statistics of each group described in this application.
  • Figure 13 shows a photograph of the tumor after euthanasia of the A549 model described in this application.
  • nucleic acid molecule generally refers to polymers of nucleotides (eg, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)), including naturally occurring (adenine, guanine, cytosine, uridine) pyrimidine and thymine), non-naturally occurring and modified nucleic acids.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the nucleic acid molecule comprises the nucleotide sequence of the gene encoding each of the Kras mutants, and each of the Kras mutants comprises at least 10 amino acids N-terminal to the mutation site immediately adjacent to the site and all The C-terminal of the mutation site is immediately adjacent to at least 10 amino acids of the site; the nucleic acid molecule further comprises a polynucleotide of the CD14 protein secreted peptide located at the 5' end of the gene encoding the Kras mutant, and the Kras mutation The polynucleotide of 6x His at the 3' end of the gene of the body.
  • secretory peptide generally refers to short peptide chains that direct the transfer of newly synthesized proteins to the secretory pathway.
  • the N of the Kras mutant polypeptide carries the CD14 protein secretion peptide sequence to guide the extracellular secretion of the Kras polypeptide, so as to facilitate the replication and expression of the recombinant HSV-1 in the tumor cells at a later stage, and release the Kras mutant polypeptide by antigen-presenting cells (antigen- presenting cell, APC) is effectively presented after ingestion.
  • antigen-presenting cells antigen-presenting cell, APC
  • tagged protein generally refers to a protein that is fused and expressed together with the target protein for the convenience of detection of the target protein.
  • the tag protein used in this application is a 6xHis tag protein, also known as a polyhistidine tag protein, using the sequence of histidine residues to bind to several types of immobilized ions (such as nickel under specific buffer conditions) , copper and cobalt), but has little effect on the properties of the target protein itself, so as to achieve the purpose of easy detection and purification of His-tagged proteins.
  • the term "vector” generally refers to a tool capable of carrying an exogenous gene or DNA fragment of interest into a host cell for replication and expression. According to the source, it can be divided into plasmid vector, phage vector, viral vector and yeast artificial chromosome vector.
  • the vector may be an HSV-1 vector capable of being linked to the nucleic acid molecule and replicated and expressed in a host cell.
  • HSV-1 generally refers to a neurotropic, enveloped, double-stranded DNA virus belonging to the alphavirus subfamily of the family Herpesviridae, with a genome length of 152 kb consisting of two interconnected long The segment UL and the short segment US are composed. Because of its advantages of large gene capacity, short replication cycle, high infection efficiency, and the ability to insert multiple therapeutic genes, it has become the first choice for anti-tumor drug research in genetic engineering.
  • UL3 gene refers to the gene encoding protein_id: ADD59983.1 in human HSV [GU734771.1] and the term “UL4 gene” refers to the gene encoding protein_id: ADD60023.1 in human HSV [GU734771.1], the UL3 gene Neither the UL4 nor the UL4 gene is necessary to maintain the survival and replication of the HSV virus.
  • ⁇ 34.5 gene generally refers to the apoptosis suppressor gene in the HSV-1 genome, which is also an important neurotoxic gene, GeneBank No: GU734771.1. Knockout of the ⁇ 34.5 gene is also the most common attenuation strategy for oncolytic viruses based on HSV as the backbone. HSV-1 with defective ⁇ 34.5 gene function not only greatly reduces the toxicity, but also enables the virus to selectively replicate in tumor cells by exploiting the defect of IFN-PKR signaling in tumor cells.
  • HSV-1 ⁇ 34.5 can resist the inhibitory effect of PKR, enabling the virus to replicate in normal cells, while many tumor cells are defective in the PKR system, resulting in
  • the HSV virus with knockout of the ⁇ 34.5 gene can effectively replicate and kill tumors in tumor cells but cannot replicate in normal cells, which provides conditions for HSV virus engineering to specifically kill tumors.
  • promoter usually refers to a deoxyribonucleic acid (DNA) sequence located upstream of the 5' end of the transcription initiation site of the target gene, which can enable the transcription of the target gene, which can be recognized by RNA polymerase , and begin to transcribe synthetic RNA.
  • promoter refers to a DNA sequence located upstream of the 5' end of the transcription initiation site of the gene encoding the Kras mutant and controls its transcription.
  • pharmaceutical composition generally refers to a composition suitable for administration to a patient, comprising one or more pharmaceutically effective carriers, stabilizers, excipients, diluents, solubilizers, surfactants , emulsifiers, preservatives and/or adjuvants suitable formulations.
  • the pharmaceutical composition can be administered intravenously, intraperitoneally, subcutaneously, intramuscularly, topically or intradermally.
  • the pharmaceutical composition comprises a nucleic acid molecule encoding a Kras gene mutant, the oncolytic herpes simplex virus vector, and optionally a pharmaceutically acceptable adjuvant.
  • physiological saline can encompass any pharmaceutically acceptable concentration range.
  • the compositions described herein may include a physiological saline component.
  • Solid tumor generally refers to an abnormal tissue growth or mass that typically does not contain cysts or fluid areas. Solid tumors can be benign (noncancerous) or malignant (cancerous). About 90% of clinical cancer cases are currently solid tumors. Different types of solid tumors are named after the cell type that forms them.
  • gastrointestinal cancer pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocytoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, Salivary gland cancer, prostate cancer, vaginal cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct cancer, and head and neck cancer, etc.
  • pancreatic tumor generally includes pancreatic sarcoma, pancreatic cystadenoma, pancreatic cystadenocarcinoma.
  • Pancreatic tumor is one of the most common malignant tumors in the digestive tract, and it is the most common malignant tumor, which mostly occurs in the head of the pancreas.
  • surgical resection is usually the first choice for the treatment of pancreatic tumors.
  • the application provides an isolated nucleic acid molecule comprising one or more genes each independently selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V Mutant, Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T Mutant, Kras A146T mutant, Kras Y64H mutant and Kras A18D mutant.
  • genes each independently selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V Mutant, Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T Mutant, Kras A146T mutant
  • the application provides an isolated nucleic acid molecule that does not comprise a polynucleotide encoding a 6x His-tagged protein, and that comprises one or more genes each independently encoding a Kras mutant selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T mutant, Kras A146T mutant, Kras Y64H mutant and Kras A18D mutant.
  • a Kras mutant selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras
  • the isolated nucleic acid molecule may comprise one or more genes encoding each Kras mutant, respectively.
  • the isolated nucleic acid molecule may comprise non-repetitive one or more genes encoding each Kras mutant, respectively.
  • the isolated nucleic acid molecule may comprise duplicates of one or more genes encoding each Kras mutant, respectively.
  • the Kras mutant genes are independently selected from: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant, Kras G12C mutant , Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T mutant, Kras A146T mutant, Kras Y64H mutant and the Kras A18D mutant.
  • the isolated nucleic acid molecule may comprise a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant and KrasG12C mutant body.
  • the isolated nucleic acid molecule may comprise a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant, Kras Y64H Mutants and Kras G12C mutants.
  • the 3' end of the gene encoding the Kras G12D mutant may be directly or indirectly linked to the 5' end of the gene encoding the Kras A146T mutant.
  • the 3' end of the gene encoding the Kras A46T mutant may be directly or indirectly linked to the 5' end of the gene encoding the Kras G12V mutant.
  • the 3' end of the gene encoding the Kras G12V mutant may be directly or indirectly linked to the 5' end of the gene encoding the Kras A59T mutant.
  • the 3' end of the gene encoding the Kras A59T mutant may be directly or indirectly linked to the 5' end of the gene encoding the Kras G13D mutant.
  • the 3' end of the gene encoding the Kras G13D mutant may be directly or indirectly linked to the 5' end of the gene encoding the Kras Y64H mutant.
  • the 3' end of the gene encoding the Kras Y64H mutant may be directly or indirectly linked to the 5' end of the gene encoding the Kras G12C mutant.
  • each of the genes encoding the Kras mutants may be arranged in tandem in the isolated nucleic acid molecules.
  • each of the genes encoding Kras mutants may be Minigene.
  • tandem mini-genes can be obtained by arranging the genes encoding Kras mutants in tandem.
  • each of the Kras mutants may comprise at least 20 amino acids.
  • each of the Kras mutants may comprise at least 9 amino acids immediately N-terminal to the mutation site and at least 10 amino acids C-terminal to the mutation site.
  • the order of the Kras mutants from the 5' end to the 3' end is the gene encoding the Kras G12D mutant, the gene encoding the Kras A59T mutant, the gene encoding the Kras G12V mutant, Gene encoding Kras A146T mutant, gene encoding Kras G13D mutant, gene encoding Kras Y64H mutant, gene encoding Kras G12C mutant, gene encoding Kras Q61H mutant, gene encoding Kras A18D mutant, gene encoding Kras Gene of G12A mutant, gene encoding Kras A146T mutant, and gene encoding Kras G12S mutant.
  • the isolated nucleic acid molecule can comprise the amino acid sequence set forth in SEQ ID NO:65.
  • the order of the Kras mutants from the 5' end to the 3' end is the gene encoding the Kras G12D mutant, the gene encoding the Kras A146T mutant, the gene encoding the Kras G12V mutant, The gene encoding the Kras A59T mutant, the gene encoding the Kras G13D mutant, the gene encoding the Kras Y64H mutant, and the gene encoding the Kras G12C mutant.
  • the isolated nucleic acid molecule can comprise the amino acid sequence set forth in SEQ ID NO:66.
  • the order of the Kras mutants from the 5' end to the 3' end is the gene encoding the Kras G12D mutant, the gene encoding the Kras A59T mutant, the gene encoding the Kras A18D mutant, Gene encoding Kras G12V mutant, gene encoding Kras A146T mutant, gene encoding Kras Q61H mutant, gene encoding Kras G13D mutant, gene encoding Kras A59T mutant, gene encoding Kras A146T mutant and gene encoding Kras Gene of the G12C mutant.
  • the isolated nucleic acid molecule can comprise the amino acid sequence set forth in SEQ ID NO:67.
  • the nucleotide sequence encoding the wild-type Kras gene may comprise the nucleotide sequence shown in SEQ ID NO:39, and the amino acid sequence encoded by the wild-type Kras gene may comprise the amino acid sequence shown in SEQ ID NO:38.
  • the Kras G12D mutant refers to a segment comprising the amino acid G at position 12 cut from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at position 12 in the cut-out sequence is replaced by D, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26.
  • the Kras G12D mutant may comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site G12D and at least 10 (e.g., at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site G12D. at least 12, at least 13, at least 14) amino acids.
  • the amino acid sequence adjacent to the N-terminus of the mutation site G12D is the X extending from the 11th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, X can be 9, 10 or 11. It may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the amino acid sequence of the C-terminus adjacent to the mutation site G12D is the Y extending from the 13th (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the amino acid sequence of the Kras G12D mutant can sequentially comprise the amino acid sequence shown in SEQ ID NO:2, the Kras G12D site, and the amino acid sequence shown in SEQ ID NO:3 from the N-terminus to the C-terminus.
  • the Kras G12D mutant may comprise the amino acid sequence set forth in SEQ ID NO:1.
  • the gene encoding the Kras G12D mutant may comprise a nucleotide sequence encoding an amino acid sequence immediately adjacent to the N-terminal of the mutation site G12D, a nucleotide sequence encoding the mutation site G12D, a nucleotide sequence encoding the immediately adjacent mutation site The nucleotide sequence of the amino acid sequence of the C-terminus of G12D.
  • the gene encoding the Kras G12D mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20, a nucleotide sequence encoding the mutation site G12D, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20. : the nucleotide sequence of the amino acid sequence shown in 21.
  • the gene encoding the Kras G12D mutant may comprise the nucleotide sequence shown in SEQ ID NO:19.
  • the Kras G13D mutant refers to a sequence comprising the thirteenth amino acid G cut from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at the thirteenth position in the cut-out sequence is replaced by D, the said The sequence comprises at least 21 amino acids, such as 22, such as 23, such as 24, such as 25, such as 26, such as 27.
  • the Kras G13D mutant may comprise at least 10 (e.g., at least 11, at least 12) amino acids immediately adjacent to the N-terminus of the mutation site G13D and at least 10 (e.g., at least 11, at least 12) amino acids immediately adjacent to the C-terminus of the mutation site G13D. at least 12, at least 13, at least 14) amino acids.
  • the amino acid sequence adjacent to the N-terminus of the mutation site G13D is the X extending from the 12th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, X can be 10, 11 or 12. It may comprise the amino acid sequence shown in SEQ ID NO:5.
  • the amino acid sequence adjacent to the C-terminus of the mutation site G13D is the Y extending from the 14th (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:6.
  • the amino acid sequence of the Kras G13D mutant may comprise the amino acid sequence shown in SEQ ID NO: 5, the Kras G13D site, and the amino acid sequence shown in SEQ ID NO: 6 in order from the N-terminus to the C-terminus.
  • the Kras G13D mutant may comprise the amino acid sequence set forth in SEQ ID NO:4.
  • the gene encoding the Kras G13D mutant may comprise a nucleotide sequence encoding the amino acid sequence immediately adjacent to the N-terminal of the mutation site G13D, a nucleotide sequence encoding the mutation site G13D, a nucleotide sequence encoding the immediately adjacent mutation site The nucleotide sequence of the amino acid sequence of the C-terminus of G13D.
  • the gene encoding the Kras G13D mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 23, a nucleotide sequence encoding the mutation site G13D, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 23. : the nucleotide sequence of the amino acid sequence shown in 24.
  • the gene encoding the Kras G13D mutant may comprise the nucleotide sequence shown in SEQ ID NO:22.
  • the Kras G12V mutant refers to a segment comprising the amino acid G at position 12 cut from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at position 12 in the cut-out sequence is replaced by V, and the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26.
  • the Kras G12V mutant may comprise at least 9 (e.g. at least 10, at least 11) amino acids next to the N-terminus of the mutation site G12V and at least 10 (e.g. at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site G12V. at least 12, at least 13, at least 14) amino acids.
  • the amino acid sequence adjacent to the N-terminus of the mutation site G12V is the X extending from the 11th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, X can be 9, 10 or 11. It may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the amino acid sequence of the C-terminus adjacent to the mutation site G12V is the Y extending from the 13th (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the amino acid sequence of the Kras G12V mutant may comprise the amino acid sequence shown in SEQ ID NO: 2, the Kras G12V site, and the amino acid sequence shown in SEQ ID NO: 3 in order from the N-terminus to the C-terminus.
  • the Kras G12V mutant may comprise the amino acid sequence set forth in SEQ ID NO:7.
  • the gene encoding the Kras G12V mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminus of the mutation site G12V, a nucleotide sequence encoding the mutation site G12V, and a nucleotide sequence encoding the immediately adjacent mutation site.
  • the gene encoding the Kras G12V mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20, a nucleotide sequence encoding the mutation site G12V, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20. : the nucleotide sequence of the amino acid sequence shown in 21.
  • the gene encoding the Kras G12V mutant may comprise the nucleotide sequence shown in SEQ ID NO:25.
  • the Kras G13C mutant refers to a segment comprising the amino acid G at position 13 cut from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at position 13 in the cut-out sequence is replaced by C, the The sequence comprises at least 21 amino acids, such as 22, such as 23, such as 24, such as 25, such as 26, such as 27.
  • the Kras G13C mutant may comprise at least 10 (e.g., at least 11, at least 12) amino acids immediately adjacent to the N-terminus of the mutation site G13C and at least 10 (e.g., at least 11, at least 12) amino acids immediately adjacent to the C-terminus of the mutation site G13C. at least 12, at least 13, at least 14) amino acids.
  • the amino acid sequence adjacent to the N-terminus of the mutation site G13C is the X extending from the 12th position (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, X can be 10, 11 or 12. It may comprise the amino acid sequence shown in SEQ ID NO:5.
  • the amino acid sequence of the C-terminus adjacent to the mutation site G13C is the Y extending from the 14th (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:6.
  • the amino acid sequence of the Kras G13C mutant may comprise the amino acid sequence shown in SEQ ID NO: 5, the Kras G13C site, and the amino acid sequence shown in SEQ ID NO: 6 in order from the N-terminus to the C-terminus.
  • the Kras G13C mutant may comprise the amino acid sequence shown in SEQ ID NO:8.
  • the gene encoding the Kras G13C mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminal of the mutation site G13C, a nucleotide sequence encoding the mutation site G13C, and a nucleotide sequence encoding the immediately adjacent mutation site.
  • the gene encoding the Kras G13C mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 23, a nucleotide sequence encoding the mutation site G13C, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 23. : the nucleotide sequence of the amino acid sequence shown in 24.
  • the gene encoding the Kras G13C mutant may comprise the nucleotide sequence shown in SEQ ID NO:26.
  • the Kras G12C mutant refers to a sequence comprising the twelfth amino acid G cut out from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at the twelfth position in the cut-out sequence is replaced by C, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26.
  • the Kras G12C mutant may comprise at least 9 (eg, at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site G12C and at least 10 (eg, at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site G12C. at least 12, at least 13, at least 14) amino acids.
  • the amino acid sequence adjacent to the N-terminus of the mutation site G12C is the X extending from the 11th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, X can be 9, 10 or 11. It may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the amino acid sequence of the C-terminus adjacent to the mutation site G12C is the Y extending from the 13th (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the amino acid sequence of the Kras G12C mutant can sequentially comprise the amino acid sequence shown in SEQ ID NO:2, the Kras G12C site, and the amino acid sequence shown in SEQ ID NO:3 from the N-terminus to the C-terminus.
  • the Kras G12C mutant may comprise the amino acid sequence set forth in SEQ ID NO:9.
  • the gene encoding the Kras G12C mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminus of the mutation site G12C, a nucleotide sequence encoding the mutation site G12C, and a nucleotide sequence encoding the immediately adjacent mutation site.
  • the gene encoding the Kras G12C mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20, a nucleotide sequence encoding the mutation site G12C, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20. : the nucleotide sequence of the amino acid sequence shown in 21.
  • the gene encoding the Kras G12C mutant may comprise the nucleotide sequence shown in SEQ ID NO:27.
  • the Kras G13A mutant refers to a sequence comprising the thirteenth amino acid G cut from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at the thirteenth position in the cut-out sequence is replaced by A, the The sequence comprises at least 21 amino acids, such as 22, such as 23, such as 24, such as 25, such as 26, such as 27.
  • the Kras G13A mutant may comprise at least 10 (e.g., at least 11, at least 12) amino acids immediately adjacent to the N-terminus of the mutation site G13A and at least 10 (e.g., at least 11, at least 12) amino acids immediately adjacent to the C-terminus of the mutation site G13A. at least 12, at least 13, at least 14) amino acids.
  • the amino acid sequence adjacent to the N-terminus of the mutation site G13A is the X extending from the 12th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, X can be 10, 11 or 12. It may comprise the amino acid sequence shown in SEQ ID NO:5.
  • the amino acid sequence of the C-terminus adjacent to the mutation site G13A is the Y extending from the 14th position (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:6.
  • the amino acid sequence of the Kras G13A mutant may comprise the amino acid sequence shown in SEQ ID NO: 5, the Kras G13A site, and the amino acid sequence shown in SEQ ID NO: 6 in order from the N-terminus to the C-terminus.
  • the Kras G13A mutant may comprise the amino acid sequence set forth in SEQ ID NO:10.
  • the gene encoding the Kras G13A mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminus of the mutation site G13A, a nucleotide sequence encoding the mutation site G13A, encoding the immediately adjacent mutation site The nucleotide sequence of the amino acid sequence of the C-terminus of G13A.
  • the gene encoding the Kras G13A mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 23, a nucleotide sequence encoding the mutation site G13A, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 23. : the nucleotide sequence of the amino acid sequence shown in 24.
  • the gene encoding the Kras G13A mutant may comprise the nucleotide sequence shown in SEQ ID NO:28.
  • the Kras G12A mutant refers to a sequence comprising the twelfth amino acid G cut out from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at the twelfth position in the cut-out sequence is replaced by A, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26.
  • the Kras G12A mutant may comprise at least 9 (eg, at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site G12A and at least 10 (eg, at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site G12A. at least 12, at least 13, at least 14) amino acids.
  • the amino acid sequence adjacent to the N-terminus of the mutation site G12A is the X extending from the 11th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, X can be 9, 10 or 11. It may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the amino acid sequence of the C-terminus adjacent to the mutation site G12A is the Y extending from the 13th position (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the amino acid sequence of the Kras G12A mutant may comprise the amino acid sequence shown in SEQ ID NO: 2, the Kras G12A site, and the amino acid sequence shown in SEQ ID NO: 3 in order from the N-terminus to the C-terminus.
  • the Kras G12A mutant may comprise the amino acid sequence set forth in SEQ ID NO:11.
  • the gene encoding the Kras G12A mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminus of the mutation site G12A, a nucleotide sequence encoding the mutation site G12A, encoding the immediately adjacent mutation site The nucleotide sequence of the amino acid sequence of the C-terminus of G12A.
  • the gene encoding the Kras G12A mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20, a nucleotide sequence encoding the mutation site G12A, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20. : the nucleotide sequence of the amino acid sequence shown in 21.
  • the gene encoding the Kras G12A mutant may comprise the nucleotide sequence shown in SEQ ID NO:29.
  • the Kras Q61L mutant refers to a segment comprising the amino acid Q at position 61 cut out from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid Q at position 61 in the cut-out sequence is replaced by L, the The sequence comprises at least 21 amino acids, such as 22, such as 23, such as 24, such as 25, such as 26, such as 27, such as 28, such as 29.
  • the Kras Q61L mutant may comprise at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately N-terminal to the mutation site Q61L and at least 10 amino acids immediately C-terminal to the mutation site Q61L. 10 (eg, at least 11, at least 12, at least 13, at least 14) amino acids.
  • the amino acid sequence adjacent to the N-terminus of the mutation site Q61L is the X extending from the 60th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, X can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:13.
  • the amino acid sequence of the C-terminus adjacent to the mutation site Q61L is the Y extending from the 62nd (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:14.
  • the amino acid sequence of the Kras Q61L mutant may comprise the amino acid sequence shown in SEQ ID NO: 13, the Kras Q61L site, and the amino acid sequence shown in SEQ ID NO: 14 in order from the N-terminus to the C-terminus.
  • the Kras Q61L mutant may comprise the amino acid sequence set forth in SEQ ID NO:12.
  • the gene encoding the Kras Q61L mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminus of the mutation site Q61L, a nucleotide sequence encoding the mutation site Q61L, encoding the immediately adjacent mutation site The nucleotide sequence of the amino acid sequence of the C-terminal of Q61L.
  • the gene encoding the Kras Q61L mutant may in turn comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 31, a nucleotide sequence encoding the mutation site Q61L, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 31. : the nucleotide sequence of the amino acid sequence shown in 32.
  • the gene encoding the Kras Q61L mutant may comprise the nucleotide sequence shown in SEQ ID NO:30.
  • the Kras G12R mutant refers to a segment comprising the amino acid G at position 12 cut from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at position 12 in the cut-out sequence is replaced by R, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26.
  • the Kras G12R mutant may comprise at least 9 (eg, at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site G12R and at least 10 (eg, at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site G12R. at least 12, at least 13, at least 14) amino acids.
  • the amino acid sequence adjacent to the N-terminus of the mutation site G12R is the X extending from the 11th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, X can be 9, 10 or 11. It may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the amino acid sequence of the C-terminus adjacent to the mutation site G12R is the Y extending from the 13th (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the amino acid sequence of the Kras G12R mutant may comprise the amino acid sequence shown in SEQ ID NO: 2, the Kras G12R site, and the amino acid sequence shown in SEQ ID NO: 3 in order from the N-terminus to the C-terminus.
  • the Kras G12R mutant may comprise the amino acid sequence set forth in SEQ ID NO:15.
  • the gene encoding the Kras G12R mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminal of the mutation site G12R, a nucleotide sequence encoding the mutation site G12R, and a nucleotide sequence encoding the immediately adjacent mutation site.
  • the gene encoding the Kras G12R mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20, a nucleotide sequence encoding the mutation site G12R, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20. : the nucleotide sequence of the amino acid sequence shown in 21.
  • the gene encoding the Kras G12R mutant may comprise the nucleotide sequence shown in SEQ ID NO:33. .
  • the Kras Q61H mutant refers to a sequence comprising the 61st amino acid Q cut from the amino acid sequence of the wild-type Kras polypeptide, and the 61st amino acid Q in the cut-out sequence is replaced by H, the The sequence comprises at least 21 amino acids, such as 22, such as 23, such as 24, such as 25, such as 26, such as 27, such as 28, such as 29.
  • the Kras Q61H mutant may comprise at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately adjacent to the N-terminus of the mutation site Q61H and at least 10 amino acids immediately adjacent to the C-terminus of the mutation site Q61H. 10 (eg, at least 11, at least 12, at least 13, at least 14) amino acids.
  • the amino acid sequence adjacent to the N-terminus of the mutation site Q61H is the X extending from the 60th position (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, X can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:13.
  • the amino acid sequence of the C-terminus adjacent to the mutation site Q61H is the Y extending from the 62nd (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:14.
  • the amino acid sequence of the Kras Q61H mutant may comprise the amino acid sequence shown in SEQ ID NO: 13, the Kras Q61H site, and the amino acid sequence shown in SEQ ID NO: 14 in order from the N-terminus to the C-terminus.
  • the Kras Q61H mutant may comprise the amino acid sequence set forth in SEQ ID NO:16.
  • the gene encoding the Kras Q61H mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminal of the mutation site Q61H, a nucleotide sequence encoding the mutation site Q61H, encoding the immediately adjacent mutation site The nucleotide sequence of the amino acid sequence of the C-terminal of Q61H.
  • the gene encoding the Kras Q61H mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 31, a nucleotide sequence encoding the mutation site Q61H, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 31. : the nucleotide sequence of the amino acid sequence shown in 32.
  • the gene encoding the Kras Q61H mutant may comprise the nucleotide sequence shown in SEQ ID NO:34.
  • the Kras G12S mutant refers to a segment comprising the amino acid G at position 12 cut from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid G at position 12 in the cut-out sequence is replaced by S, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26.
  • the Kras G12S mutant may comprise at least 9 (eg, at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site G12S and at least 10 (eg, at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site G12S. at least 12, at least 13, at least 14) amino acids.
  • the amino acid sequence adjacent to the N-terminus of the mutation site G12S is the X extending from the 11th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, X can be 9, 10 or 11. It may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the amino acid sequence of the C-terminus adjacent to the mutation site G12S is the Y extending from the 13th (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the amino acid sequence of the Kras G12S mutant can sequentially comprise the amino acid sequence shown in SEQ ID NO: 2, the Kras G12S site, and the amino acid sequence shown in SEQ ID NO: 3 from the N-terminus to the C-terminus.
  • the Kras G12S mutant may comprise the amino acid sequence set forth in SEQ ID NO:17.
  • the gene encoding the Kras G12S mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminus of the immediately adjacent mutation site G12S, a nucleotide sequence encoding the mutation site G12S, and a nucleotide sequence encoding the immediately adjacent mutation site.
  • the gene encoding the Kras G12S mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20, a nucleotide sequence encoding the mutation site G12S, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20. : the nucleotide sequence of the amino acid sequence shown in 21.
  • the gene encoding the Kras G12S mutant may comprise the nucleotide sequence shown in SEQ ID NO:35.
  • the Kras Q61R mutant refers to a sequence comprising the 61st amino acid Q cut out from the amino acid sequence of the wild-type Kras polypeptide, and the 61st amino acid Q in the cut-out sequence is replaced by R, the The sequence comprises at least 21 amino acids, such as 22, such as 23, such as 24, such as 25, such as 26, such as 27, such as 28, such as 29.
  • the Kras Q61R mutant may comprise at least 10 (e.g., at least 11, at least 12, at least 13, at least 14) amino acids immediately N-terminal to the mutation site Q61R and at least 10 amino acids immediately C-terminal to the mutation site Q61R. 10 (eg, at least 11, at least 12, at least 13, at least 14) amino acids.
  • the amino acid sequence adjacent to the N-terminus of the mutation site Q61R is the X extending from the 60th (the sequence from the N-terminus to the C-terminus) amino acid to the N-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, X can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:13.
  • the amino acid sequence of the C-terminus adjacent to the mutation site Q61R is the Y extending from the 62nd (the sequence from the N-terminus to the C-terminus) amino acid to the C-terminus in the wild-type Kras polypeptide sequence (as shown in SEQ ID NO: 38).
  • amino acids, Y can be 10, 11, 12, 13 or 14. It may comprise the amino acid sequence shown in SEQ ID NO:14.
  • the amino acid sequence of the Kras Q61R mutant may comprise the amino acid sequence shown in SEQ ID NO: 13, the Kras Q61R site, and the amino acid sequence shown in SEQ ID NO: 14 in order from the N-terminus to the C-terminus.
  • the Kras Q61R mutant may comprise the amino acid sequence set forth in SEQ ID NO:18.
  • the gene encoding the Kras Q61R mutant may comprise a nucleotide sequence encoding the amino acid sequence of the N-terminus of the mutation site Q61R, a nucleotide sequence encoding the mutation site Q61R, encoding the immediately adjacent mutation site The nucleotide sequence of the amino acid sequence of the C-terminal of Q61R.
  • the gene encoding the Kras Q61R mutant may sequentially comprise a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 31, a nucleotide sequence encoding the mutation site Q61R, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 31. : the nucleotide sequence of the amino acid sequence shown in 32.
  • the gene encoding the Kras Q61R mutant may comprise the nucleotide sequence shown in SEQ ID NO:36.
  • each of the genes encoding the Kras mutants is a Minigene.
  • the minigene usually refers to a short segment in a gene, which can be used for the study of the function and expression regulation mechanism of the gene and for the construction of a more complex minigene containing multiple exons and introns.
  • the minigene can be a fragment comprising the gene encoding each Kras mutant, and the encoding genes of each Kras mutant are arranged in series according to the sequence shown in FIG. 1 to form a Kras gene mutant. Tandem minigene (TMG).
  • the tandem mini-gene of the Kras gene mutant comprises, from the 5' end to the 3' end, the nucleotide sequence encoding the Kras G12D mutant, the nucleotide sequence encoding the Kras G12V mutant, the nucleotide sequence encoding the Kras G12V mutant, and the Kras G12V mutant.
  • tandem mini-gene of the Kras gene mutant may comprise the amino acid sequence encoding SEQ ID NO:43.
  • the tandem mini-gene of the Kras gene mutant comprises the nucleotide sequence shown in SEQ ID NO: 19, the nucleotide sequence shown in SEQ ID NO: 22, the nucleotide sequence shown in SEQ ID NO: 22 from the 5' end to the 3' end in order
  • the Kras A59T mutant refers to a sequence comprising amino acid A at position 59 cut from the amino acid sequence of a wild-type Kras polypeptide, and the amino acid A at position 59 in the cut-out sequence is replaced by T, and the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26.
  • the Kras A59T mutant may comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site A59T and at least 10 (e.g., at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site A59T. at least 12, at least 13, at least 14) amino acids.
  • the Kras A59T mutant may comprise the amino acid sequence set forth in SEQ ID NO:54.
  • the gene encoding the Kras A59T mutant may comprise the nucleotide sequence shown in SEQ ID NO:55.
  • the Kras A146T mutant refers to a sequence comprising the amino acid A at position 146 cut from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid A at position 146 in the cut-out sequence is replaced by T, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26.
  • the Kras A146T mutant may comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site A146T and at least 10 (e.g., at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site A146T. at least 12, at least 13, at least 14) amino acids.
  • the Kras A146T mutant may comprise the amino acid sequence set forth in SEQ ID NO:56.
  • the gene encoding the Kras A146T mutant may comprise the nucleotide sequence shown in SEQ ID NO:57.
  • Said Kras Y64H mutant refers to a segment comprising the amino acid Y at position 64 cut out from the amino acid sequence of the wild-type Kras polypeptide, and the amino acid Y at position 64 in the cut-out sequence is replaced by H, the said The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26.
  • the Kras Y64H mutant may comprise at least 9 (e.g. at least 10, at least 11) amino acids next to the N-terminus of the mutation site Y64H and at least 10 (e.g. at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site Y64H. at least 12, at least 13, at least 14) amino acids.
  • the Kras Y64H mutant may comprise the amino acid sequence set forth in SEQ ID NO:58.
  • the gene encoding the Kras Y64H mutant may comprise the nucleotide sequence shown in SEQ ID NO:59.
  • the Kras A18D mutant refers to a sequence comprising amino acid A at position 18 cut from the amino acid sequence of a wild-type Kras polypeptide, and the amino acid A at position 18 in the cut-out sequence is replaced by D, the The sequence comprises at least 20 amino acids, such as 21, such as 22, such as 23, such as 24, such as 25, such as 26.
  • the Kras A18D mutant may comprise at least 9 (e.g., at least 10, at least 11) amino acids immediately adjacent to the N-terminus of the mutation site A18D and at least 10 (e.g., at least 11, at least 11) amino acids immediately adjacent to the C-terminus of the mutation site A18D. at least 12, at least 13, at least 14) amino acids.
  • the Kras A18D mutant may comprise the amino acid sequence set forth in SEQ ID NO:60.
  • the gene encoding the Kras A18D mutant may comprise the nucleotide sequence shown in SEQ ID NO:61.
  • tandem minigene of the Kras gene mutant may comprise the nucleotide sequence shown in SEQ ID NO:44.
  • the nucleic acid molecule may also comprise mutants of other tumor antigens.
  • the nucleic acid molecule may also comprise mutants of tumor-associated driver genes.
  • the other tumor antigen can be TP53.
  • the other tumor antigen can be the Braf gene.
  • the nucleic acid molecule may also comprise a polynucleotide encoding a secreted peptide located at the 5' end of the tandem minigene of the Kras gene mutant.
  • the secretory peptide can guide the secretion of Kras polypeptide outside the tumor cells, and after being taken up by antigen-presenting cells (APC), it can be effectively presented to T cells, thereby exerting an immune effect.
  • the APC cells refer to a class of adjuvant cells that can present antigenic substances to T cells in the process of immune response, and the major histocompatibility complex (MHC) on the cell surface can interact with antigens, That is, the Kras mutant polypeptide is combined, and the combined complex of the two can be recognized by T cells.
  • the secreted peptide may include Hmm38 secreted peptide, CD14 protein secreted peptide.
  • the polynucleotide encoding the CD14 protein secretory peptide may comprise the nucleotide sequence shown in SEQ ID NO:37.
  • the isolated nucleic acid molecule comprising a polynucleotide encoding a CD14 protein secreted peptide can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 62-64.
  • the isolated nucleic acid molecule can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 62-64.
  • the nucleic acid molecule may also comprise a polynucleotide encoding a tag protein located at the 3' end of the gene encoding the Kras mutant.
  • the tag protein is used for detection of Kras mutant polypeptide.
  • the tagged proteins may include FLAG-tagged proteins, HA-tagged proteins, C-Myc-tagged proteins, 6x His-tagged proteins.
  • polynucleotide encoding the 6x His-tagged protein may comprise the nucleotide sequence shown in SEQ ID NO:40.
  • the present application provides a vector comprising the nucleic acid molecule.
  • the vector may be a viral vector.
  • the nucleic acid molecules described herein can be inserted into viral vectors.
  • the nucleotide sequence set forth in any one of SEQ ID NOs: 65-67 can be inserted into a viral vector.
  • the viral vector may comprise the nucleotide sequence shown in GenBank No: GU734771.1 of the NCBI database.
  • genes described herein that do not contain a 6x His-tagged protein can be inserted into a viral vector.
  • the vector may be an oncolytic herpes simplex virus (oHSV) vector.
  • oHSV oncolytic herpes simplex virus
  • the oncolytic herpes simplex virus (oHSV) vector can be a herpes simplex virus type I (HSV-1) vector.
  • the HSV-1 vector is a gene deletion type, and the deleted gene may be neurotoxic factor ⁇ 34.5.
  • the vector may be an oncolytic herpes simplex virus (oHSV) vector.
  • the oncolytic herpes simplex virus (oHSV) vector can be a herpes simplex virus type I (HSV-1) vector.
  • the HSV-1 vector is a gene deletion type, and the deleted gene may be neurotoxic factor ⁇ 34.5.
  • the HSV-1 vector lacks two copies of the neurotoxic factor ⁇ 34.5.
  • the vector also includes a promoter.
  • the promoter may include an elongation factor 1 ⁇ short (EFS) promoter, an elongation factor 1 ⁇ (EF-1 ⁇ ) promoter, a CMV promoter, a SV40 early or late promoter, an OPEFS promoter, or derivatives thereof.
  • the promoter may comprise a CMV promoter.
  • the promoter is located 5' upstream of the transcription initiation site of the gene encoding the Kras mutant and controls its transcription.
  • the promoter sequence can include the sequence shown in SEQ ID NO:41.
  • the tandem mini-gene of the Kras gene mutant may be located between the UL3 gene and the UL4 gene of the HSV-1 vector.
  • the viral vector may comprise the nucleotide sequence shown in GenBank No: GU734771.1 of the NCBI database.
  • the present application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the nucleic acid molecule, and/or the carrier, and optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition refers to a formulation in a form that allows the biological activity of the active ingredient contained therein to be effective and free of additional ingredients that would be unacceptably toxic to a subject to whom the composition would be administered.
  • the pharmaceutically acceptable adjuvant refers to any substance capable of assisting or improving the action of a drug.
  • the adjuvant may be a particulate adjuvant, eg, an aluminum hydroxide adjuvant.
  • the adjuvant may be a non-particulate adjuvant, eg, a cytokine.
  • the adjuvant may be derived from plants such as saponins and polysaccharide extracts.
  • the adjuvant may be derived from pathogenic microorganisms, eg, monophospholipids, cholera toxin, and the like.
  • the present application also provides a composition, which may comprise the isolated nucleic acid molecule described in the present application, the carrier described in the present application or the pharmaceutical composition described in the present application, and physiological saline.
  • composition may comprise any one or more of the isolated nucleic acid molecules described herein, and physiological saline.
  • the isolated nucleic acid molecule may comprise one or more genes each independently encoding a Kras mutant selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T mutant, Kras A146T mutant mutant, Kras Y64H mutant and Kras A18D mutant.
  • a Kras mutant selected from the group consisting of: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant Kras G12C mutant, Kras G13A mutant, Kras G12A mutant, Kras Q61L mutant, Kras G12R mutant, Kras Q61H mutant, Kras G12S mutant, Kras Q61R mutant, Kras A59T mutant, Kras A146
  • the isolated nucleic acid molecule may comprise a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D mutant, and KrasG12C mutant.
  • the isolated nucleic acid molecule in the composition comprises a gene encoding a Kras mutant selected from the group consisting of: Kras A59T mutant, Kras G12D mutant, Kras G12V mutant, KrasA146T mutant, Kras G13D Mutant, Kras Y64H mutant and Kras G12C mutant.
  • the 3' end of the gene encoding the Kras G12D mutant can be directly or indirectly with the 5' end of the gene encoding the Kras A146T mutant connected.
  • the 3' end of the gene encoding the Kras A46T mutant can be directly or indirectly with the 5' end of the gene encoding the Kras G12V mutant connected.
  • the 3' end of the gene encoding the Kras G12V mutant can be directly or indirectly with the 5' end of the gene encoding the Kras A59T mutant connected.
  • the 3' end of the gene encoding the Kras A59T mutant can be directly or indirectly with the 5' end of the gene encoding the Kras G13D mutant connected.
  • the 3' end of the gene encoding the Kras G13D mutant can be directly or indirectly with the 5' end of the gene encoding the Kras Y64H mutant connected.
  • the 3' end of the gene encoding the Kras Y64H mutant can be directly or indirectly with the 5' end of the gene encoding the Kras G12C mutant connected.
  • each of the genes encoding the Kras mutants may be arranged in tandem in the isolated nucleic acid molecules.
  • each of the genes encoding Kras mutants may be Minigene.
  • tandem mini-genes can be obtained by arranging the genes encoding Kras mutants in tandem.
  • each of the Kras mutants may comprise at least 20 amino acids.
  • each of the Kras mutants in the isolated nucleic acid molecule in the composition, may comprise at least 9 amino acids N-terminal to the mutation site immediately adjacent to the site and the mutation site At least 10 amino acids immediately C-terminal to this site.
  • the Kras G12D mutant may comprise the amino acid sequence shown in SEQ ID NO:1.
  • the Kras G13D mutant may comprise the amino acid sequence shown in SEQ ID NO:4.
  • the Kras G12V mutant may comprise the amino acid sequence shown in SEQ ID NO:7.
  • the Kras G13C mutant may comprise the amino acid sequence shown in SEQ ID NO:8.
  • the Kras G12C mutant may comprise the amino acid sequence shown in SEQ ID NO:9.
  • the Kras G13A mutant may comprise the amino acid sequence shown in SEQ ID NO:10.
  • the Kras G12A mutant may comprise the amino acid sequence shown in SEQ ID NO: 11.
  • the Kras G12A mutant may comprise the amino acid sequence shown in SEQ ID NO: 11.
  • the Kras Q61L mutant may comprise the amino acid sequence shown in SEQ ID NO:12.
  • the Kras G12R mutant may comprise the amino acid sequence shown in SEQ ID NO:15.
  • the Kras Q61H mutant may comprise the amino acid sequence shown in SEQ ID NO:16.
  • the Kras G12S mutant may comprise the amino acid sequence shown in SEQ ID NO:17.
  • the Kras Q61R mutant may comprise the amino acid sequence shown in SEQ ID NO:18.
  • the Kras A59T mutant may comprise the amino acid sequence shown in SEQ ID NO:54.
  • the Kras A146T mutant may comprise the amino acid sequence shown in SEQ ID NO:56.
  • the Kras Y64H mutant may comprise the amino acid sequence shown in SEQ ID NO:58.
  • the Kras A18D mutant may comprise the amino acid sequence shown in SEQ ID NO:60.
  • the tandem mini-gene may sequentially comprise a gene encoding a Kras G12D mutant, a gene encoding a Kras A59T mutant from the 5' end to the 3' end.
  • the tandem minigene may comprise the nucleotide sequence set forth in SEQ ID NO:65.
  • the tandem mini-gene in the isolated nucleic acid molecule in the composition, may sequentially comprise a gene encoding a Kras G12D mutant, a gene encoding a Kras A146T mutant from the 5' end to the 3' end.
  • the tandem minigene may comprise the nucleotide sequence set forth in SEQ ID NO:66.
  • the tandem mini-gene may sequentially comprise a gene encoding a Kras G12D mutant, a gene encoding a Kras A59T mutant from the 5' end to the 3' end.
  • the tandem minigene may comprise the nucleotide sequence set forth in SEQ ID NO:67.
  • the composition may comprise the nucleotide sequence shown in any one of SEQ ID NOs: 62-67, and physiological saline. In certain embodiments, the composition may comprise the nucleotide sequence set forth in SEQ ID NO: 63, and physiological saline.
  • composition may comprise any one or more of the carriers described in this application, and physiological saline.
  • the composition may comprise physiological saline, and a viral vector comprising the nucleotide sequence shown in any one of SEQ ID NOs: 62-67.
  • the composition may comprise physiological saline, and a viral vector comprising the nucleotide sequence set forth in any one of SEQ ID NO:63.
  • composition may comprise any one or more of the pharmaceutical compositions described in the present application, and physiological saline.
  • the present application also provides the nucleic acid molecule, the carrier, the pharmaceutical composition and/or the application of the composition in the preparation of a medicament for treating tumors.
  • the tumor can be a solid tumor.
  • the tumor can be non-small cell lung cancer.
  • the tumor can be colorectal cancer.
  • the tumor can be pancreatic cancer.
  • the tumor can be breast cancer.
  • the present application provides the nucleic acid molecule, the carrier, the pharmaceutical composition and/or the composition, which can treat tumors.
  • the present application provides a method of treating a tumor, which may include the steps of: administering to a subject an effective amount comprising the nucleic acid molecule, the carrier, the pharmaceutical composition, and/or the composition.
  • the effective amount generally refers to any drug amount that promotes disease regression when used alone or in combination with another therapeutic agent.
  • the isolated nucleic acid molecule, the carrier, the pharmaceutical composition and/or the composition described herein can be effectively used to inhibit the proliferation and/or growth of tumor cells.
  • the Kras mutant TMG-2, Kras mutant TMG-5, and Kras mutant TMG-7 constructed in the present application are expressed in oncolytic virus vectors and can exert good therapeutic effects.
  • An isolated nucleic acid molecule comprising genes encoding the following Kras mutants, respectively: Kras G12D mutant, Kras G13D mutant, Kras G12V mutant, Kras G13C mutant, Kras G12C mutant, Kras G13A mutant, Kras G12A Mutants, Kras Q61L mutants, Kras G12R mutants, Kras Q61H mutants, Kras G12S mutants and Kras Q61R mutants.
  • each of the Kras mutants comprises at least 20 amino acids.
  • each of the Kras mutants comprises at least 9 amino acids N-terminal to the mutant site immediately adjacent to the site and the mutant site The C-terminus of the site is immediately adjacent to at least 10 amino acids of the site.
  • the isolated nucleic acid molecule of any one of embodiments 1-20, wherein the gene encoding the Kras G13C mutant comprises the nucleotide sequence set forth in SEQ ID NO:26.
  • the isolated nucleic acid molecule of any one of embodiments 1-22, wherein the gene encoding the Kras G13A mutant comprises the nucleotide sequence set forth in SEQ ID NO:28.
  • the isolated nucleic acid molecule of any one of embodiments 1-23, wherein the gene encoding the Kras G12A mutant comprises the nucleotide sequence set forth in SEQ ID NO:29.
  • the isolated nucleic acid molecule of any one of embodiments 1-24, wherein the gene encoding the Kras Q61L mutant comprises the nucleotide sequence set forth in SEQ ID NO:30.
  • the isolated nucleic acid molecule of any one of embodiments 1-28, wherein the gene encoding the Kras Q61R mutant comprises the nucleotide sequence set forth in SEQ ID NO:36.
  • the isolated nucleic acid molecule of any one of embodiments 31-32, wherein the polynucleotide encoding the CD14 protein secreted peptide comprises the nucleotide sequence set forth in any one of SEQ ID NO:37.
  • nucleic acid molecule of any one of embodiments 34-36, wherein the polynucleotide encoding the tag protein comprises the nucleotide sequence set forth in SEQ ID NO:40.
  • a vector comprising the nucleic acid molecule of any one of embodiments 1-38.
  • the vector of embodiment 39 comprising a viral vector.
  • a pharmaceutical composition comprising the nucleic acid molecule of any one of embodiments 1-38, and/or the carrier of any one of embodiments 39-47, and optionally a pharmaceutically acceptable adjuvant. agent.
  • nucleic acid molecule according to any one of Embodiments 1-38 and/or the vector according to any one of Embodiments 39-47 in the preparation of a medicament for treating tumors.
  • the Kras G12D mutant (as shown in SEQ ID NO: 19), the Kras A59T mutant (as shown in SEQ ID NO: 55), and the Kras G12V mutant (as shown in SEQ ID NO: 25) were synthesized respectively , Kras A146T mutant (as shown in SEQ ID NO:57), Kras G13D mutant (as shown in SEQ ID NO:22), Kras Y64H mutant (as shown in SEQ ID NO:59), Kras G12C mutant (as shown in SEQ ID NO:27), Kras Q61H mutant (as shown in SEQ ID NO:34), Kras A18D mutant (as shown in SEQ ID NO:61), Kras G12A mutant (as shown in SEQ ID NO:61) : 29), the Kras A146T mutant (as shown in SEQ ID NO: 57) and the Kras G12S mutant (as shown in SEQ ID NO: 35), and will encode each Kras in
  • the Kras G12D mutant (as shown in SEQ ID NO: 19), the Kras A146T mutant (as shown in SEQ ID NO: 57), and the Kras G12V mutant (as shown in SEQ ID NO: 25) were synthesized respectively.
  • Kras A59T mutant (as shown in SEQ ID NO:55), Kras G13D mutant (as shown in SEQ ID NO:22), Kras Y64H mutant (as shown in SEQ ID NO:59) and Kras G12C mutant (as shown in SEQ ID NO: 27), and concatenate the nucleotide sequences encoding each Kras mutant according to the sequence to obtain a Kras mutant in the form of a tandem minigene (Tandem minigene, TMG), Called TMG-5.
  • TMG tandem minigene
  • Kras G12D mutant, Kras A59T mutant, Kras A18D mutant, Kras G12V mutant, Kras A146T mutant, Kras Q61H, Kras G13D mutant, Kras A59T mutant, Kras A146T mutant and Kras were synthesized respectively
  • the nucleotide sequence of the G12C mutant, and the nucleotide sequences encoding each Kras mutant were concatenated according to the stated sequence to obtain a Kras mutant in the form of a tandem minigene (TMG), named TMG-7.
  • TMG-7 tandem minigene
  • CD14 protein secretory peptide (synthesized in Aike Biotechnology, nucleotide sequence SEQ ID NO: 37) was introduced at the 5' end of the gene encoding the above-mentioned Kras mutant in the form of TMG, and a 6x His tag (synthesized at the 3' end) was introduced Yu Aike Biotechnology Company, nucleotide sequence SEQ ID NO: 40), obtain the nucleotide sequence encoding CD14 protein-Kras mutant (TMG)-6x His (as shown in SEQ ID NO: 47-49), The structural form of CD14 protein-Kras mutant (TMG)-6x His is shown in Figure 1.
  • an intermediate plasmid pKO5.1 for BAC recombination (gifted by Prof. Dr. Bernard Roizman, University of Chicago) was constructed, and the nucleotide sequence of UL3-CD14 protein-Kras-6xHis-UL4 described in Example 1.2 Inserted into pKO5.1, it was transformed into E. coli of HSV BAC with two copies of neurovirulence factor ⁇ 34.5 gene (shown in SEQ ID NO: 53) deleted by electroporation to obtain recombinant virus KR10 the BAC plasmid.
  • the nucleic acid sequence structure of KR10 is shown in Figure 2B.
  • a CD14 protein secretion peptide (Aike Biotechnology, SEQ ID NO: 37) was introduced into the N-terminus of the GFP protein, and a 6x His tag was introduced at the C-terminus (Aike Biotechnology, SEQ ID NO: 40) .
  • the negative control virus UL3-CD14 protein-GFP-6xHis-UL4 nucleotide sequence (SEQ ID NO:45) was synthesized with reference to the method of Example 1.2, and the BAC plasmid containing the KR11 nucleotide sequence was constructed with reference to the method of Example 1.3.
  • the nucleic acid sequence structure of KR11 is shown in Figure 2C.
  • Flu A Mp antigenic peptide shown in SEQ ID NO: 42 obtain its nucleotide sequence according to its amino acid sequence, and introduce the CD14 protein secreted peptide (Aike Biotechnology) at its N-terminus with reference to the method of Example 1.1. technology company, SEQ ID NO: 37), and a 6x His tag was introduced into the C-terminal (Aike Biotechnology, SEQ ID NO: 40).
  • the nucleotide sequence (SEQ ID NO: 46) of the positive control virus UL3-CD14 protein-FLU-6xHis-UL4 was synthesized with reference to the method of Example 1.2, and the BAC plasmid containing the nucleotide sequence of KR12 was constructed with reference to the method of Example 1.3 .
  • the nucleic acid sequence structure of KR12 is shown in Figure 2D.
  • the BAC plasmid containing the KR10 nucleotide sequence, the BAC plasmid containing the KR11 nucleotide sequence, and the BAC plasmid containing the KR12 nucleotide sequence constructed in the above Example 1-3 were respectively mixed with the pRB103 plasmid (containing the HSV-1F virus TK gene). ) were co-transfected into Vero cells, incubated in a 37°C, 5% CO 2 incubator for 4 hours, then the medium was changed, fresh complete growth medium (5% NBCS/DMEM) was added, and the culture was continued until the virus was packaged successfully. Plaques appear.
  • Vero- ⁇ TK cells Vero cells lacking TK gene
  • HAT was added for screening to pick out monoclonal viruses.
  • Vero- ⁇ TK cells were re-infected with monoclonal virus, and HAT was added to select the monoclonal virus.
  • the monoclonal virus picked after repeated screening by HAT infects Vero cells and amplifies the virus to obtain the recombinant virus with the final TK gene repaired.
  • the expressions of Kras polypeptide, GFP protein and Flu A MP antigen peptide were detected by western blotting method.
  • the antibody used in WB was HRP-labeled 6*His monoclonal antibody purchased from Proteintech. The results of WB detection showed the expression of Kras polypeptide, GFP protein and Flu A MP antigen peptide.
  • Tumor cell plates were laid, and the recombinant viruses KR10, KR11, and KR12 prepared in Example 4.1 were respectively infected with tumor cells at different titers. After 72 hours of virus exposure, CCK8 was used to detect the cell proliferation toxicity, so as to detect that the recombinant viruses KR10, KR11, and KR12 killed tumors. Ability. The results showed that KR10, KR11 and KR12 had the ability to kill tumor cells.
  • Thymidine kinase gene repair after the repaired virus is successfully repaired by protein detection, in vivo experiments in mice are carried out. The results showed that KR10 and KR11 had good tumor suppressing effect.
  • KR10 is a genetically engineered oncolytic virus.
  • KR10 herpes virus is based on the wild-type herpes virus HSV-1 (F strain), knocking out one copy of the ⁇ 34.5 gene in the IR region and TR region, resulting in the simultaneous knockout of two copies of the ⁇ 34.5 gene. The virus is less virulent.
  • KRAS mutant polypeptide is inserted into the virus, and the TMG form of the mutant polypeptide is G12D-A146T-G12V-A59T-G13D-Y64H-G12C.
  • KR11 is GFP inserted into the viral backbone of KR10.
  • mice The experimental animals used in this experiment were BALB/c mice, SPF grade, female, 80, 5-6 weeks old. Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd., animal certificate number: 20201214Abzz0619000226.
  • CT26 WT mouse colorectal cancer cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) supplemented with 100 U/mL penicillin and 100 ⁇ g/mL streptomycin. Incubate at 37°C with 5% CO2.
  • FBS fetal bovine serum
  • a subcutaneous xenograft model of mouse colon cancer (CT26.WT) in BALB/c mice was established.
  • the tumor cell suspension was subcutaneously inoculated in the right middle wing of BALB/c mice.
  • the day of group administration is D1
  • vehicle control group the vehicle is DPBS containing 10% (w/v) glycerol
  • KR11 low-dose group 1 ⁇ 10 6 PFU/only virus backbone control group
  • KR11 High-dose group 1 ⁇ 10 7 PFU/donor virus backbone control group
  • KR10 high-dose group (1 ⁇ 10 7 PFU/shot).
  • the experimental animal groups and dosing schedules are shown in Table 1.
  • the test product was administered by intratumoral injection, the administration volume was 50 ⁇ L/animal, and when the tumor volume was less than 80 mm, single - point injection (the syringe entered the lesion area through a single needle inlet, and the injection point was the middle of the tumor tissue);
  • the tumor volume is 80mm 3 to 140mm 3 , inject at 2 points (the syringe enters the lesion area through a single needle inlet, and the injection point is 1/3 and 2/3 of the long diameter of the tumor tissue);
  • the tumor volume is greater than 140mm 3 , Injection at 3 points (the syringe enters the lesion area through one needle inlet, the injection point is 1/3 and 2/3 of the long diameter of the tumor tissue, the second needle enters the lesion area from the other needle inlet, and the injection point is the tumor tissue
  • the middle is on the outside.
  • the animal state, animal death and clinical symptoms were observed every day, and the observation content included but not limited to: mental state, behavioral activities, tumor rupture, etc.
  • the tumor diameter was measured and the body weight of the animals was weighed twice a week.
  • the tumors of the surviving animals were stripped and weighed.
  • the relative tumor proliferation rate T/C%, tumor growth inhibition rate TGI% and tumor weight inhibition rate IR TW % were calculated.
  • Complete tumor remission CR tumor volume less than 50 mm 3 .
  • Tumor weight inhibition rate IR TW (%) (W control group- W administration group )/W control group ⁇ 100%
  • mice in the vehicle control group was 24.13 ⁇ 0.48g
  • the average weights of the KR11 low and high dose groups were 23.03 ⁇ 0.38g and 22.23 ⁇ 0.89g, respectively
  • the average weights of the KR10 low and high dose groups were 21.83 ⁇ 0.59g and 22.39 ⁇ 0.49g.
  • the weight statistics are shown in Table 3-1 and Table 3-2.
  • the individual data of body weight are shown in Table 4-1 and Table 4-2.
  • the body weight increase trend is shown in Figure 3 (the red triangle symbol indicates the time point of administration, and D1 is the day of the grouping).
  • the average tumor volume of the mice in the vehicle control group was 3968.41 ⁇ 653.80mm 3
  • the average tumor volume of the KR11 low and high dose groups were 3093.03 ⁇ 886.41mm 3 and 2308.04 ⁇ 789.41mm 3
  • the KR10 low and high dose groups were The mean tumor volumes were 1701.34 ⁇ 512.96 mm 3 and 1309.76 ⁇ 628.21 mm 3 , respectively.
  • the average tumor volume in the KR11 low and high dose groups decreased, but there was no significant difference (P>0.05), and the average tumor volume in the KR10 low and high dose groups decreased significantly (P ⁇ 0.05);
  • the tumor volume in the high-dose group had a decreasing trend, but there was no statistical difference (P>0.05).
  • the relative tumor proliferation rates T/C% of KR11 low and high dose groups were 62.23% and 53.22%, respectively, and the relative tumor proliferation rates T/C% of KR10 low and high dose groups were 35.97% and 20.99%, respectively.
  • the tumor growth inhibition rates TGI% of KR11 low and high dose groups were 37.77% and 46.78%, respectively, and the tumor growth inhibition rates of KR10 low and high dose groups were 64.03% and 79.01%, respectively.
  • the inhibition rate of KR10 on tumor was higher than that of KR11.
  • a total of 2 animals with high dose of KR10 had complete tumor remission (tumor disappeared); in other groups, no tumor disappeared.
  • the statistical results of tumor volume are shown in Table 5.
  • the statistical data of tumor volume are shown in Table 6-1 and Table 6-2, the individual data are shown in Table 7-1 and Table 7-2, and the change trend of tumor volume is shown in Figure 4A-4B.
  • the average tumor weight of the vehicle control group was 3.914 ⁇ 0.617g
  • the average tumor weight of the KR11 low-dose and high-dose groups were 2.788 ⁇ 0.628g and 2.353 ⁇ 0.804g, respectively
  • the average tumor weight of the KR10 low-dose and high-dose groups was 1.615, respectively ⁇ 0.508g and 1.226 ⁇ 0.526g.
  • the average tumor weight of the KR11 low and high dose groups decreased, but there was no significant difference (P>0.05).
  • the mean tumor weight of KR10 low and high dose groups decreased significantly (P ⁇ 0.05).
  • the tumor weight inhibition rate IR TW (%) of KR11 low and high dose groups were 28.77% and 39.88%, respectively.
  • the tumor weight inhibition rate IR TW (%) of KR10 low-dose and high-dose groups was 58.74% and 68.68%, respectively.
  • the statistical results are shown in Table 8.
  • the tumor weight statistics are shown in Table 9. Individual data are shown in Table 10. Tumor weight statistics are shown in Figure 5. Euthanasia photographs are shown in Figures 6A-6B.
  • CT26.WT tumor was re-challenged in 2 animals in the KR10 high-dose group whose tumors had completely disappeared, that is, the same amount of CT26.WT tumor cells was re-inoculated on the contralateral side of the mice to observe the tumor growth. Thirty days after rechallenge, none of the animals developed tumors. It was proved that after administration of KR10, the long-term anti-CT26.WT tumor immune memory function was established in the above two animals. The changes in tumor volume after tumor re-challenge are shown in Figure 7. The individual data of tumor volume are shown in Table 7-3. Figure 8 shows the photos of the end point of the tumor re-challenge model animal experiment.
  • mice The experimental animals used in this experiment were BALB/C nude mice, SPF grade, female, 50, 5-6 weeks old. From Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd., animal certificate number: 20201214Abzz0619000711.
  • A549 human non-small cell lung cancer cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) supplemented with 100 U/mL penicillin and 100 ⁇ g/mL streptomycin. Incubate at 37°C with 5% CO 2 .
  • FBS fetal bovine serum
  • a BALB/c nude mouse subcutaneous xenograft model of human non-small cell lung cancer cells was established. Tumor cell suspensions were subcutaneously inoculated in the right middle wing of BALB/c nude mice. On the 11th day after subcutaneous inoculation, when the average tumor volume in the mice grew to about 75 mm 3 , 42 tumor-bearing mice were selected.
  • the tumor volume ranged from 49.94-99.10mm 3 , the weight of the animals was 17.8-21.5g, they were randomly divided into 5 groups according to the tumor volume, 10 in the vehicle group, 8 animals/group in each treatment group, respectively: the vehicle control group (the vehicle contains 10% ( w/v) glycerol in DPBS), KR11 low-dose control group (viral backbone) (1 ⁇ 10 5 PFU/piece, QW ⁇ 3), KR11 high-dose control group (1 ⁇ 10 6 PFU/piece, QW ⁇ 3) , KR10 low-dose group (1 ⁇ 10 5 PFU/only, QW ⁇ 3), KR10 high-dose group (1 ⁇ 10 6 PFU/only, QW ⁇ 3). 1 time a week, a total of 3 doses.
  • the vehicle control group the vehicle contains 10% ( w/v) glycerol in DPBS
  • KR11 low-dose control group viral backbone
  • KR11 high-dose control group (1 ⁇ 10 6 PFU/piece, QW ⁇ 3
  • the day of the first dose was defined as D1.
  • the experimental animal groups and dosing schedules are shown in Table 11.
  • the test product was administered by intratumoral injection, the administration volume was 50 ⁇ L/animal, and when the tumor volume was less than 80 mm, single - point injection (the syringe entered the lesion area through a single needle inlet, and the injection point was the middle of the tumor tissue);
  • the tumor volume is 80mm 3 to 140mm 3
  • inject at 2 points the syringe enters the lesion area through a single needle inlet, and the injection point is 1/3 and 2/3 of the long diameter of the tumor tissue
  • the tumor volume is greater than 140mm 3
  • Injection at 3 points the syringe enters the lesion area through one needle inlet, the injection point is 1/3 and 2/3 of the long diameter of the tumor tissue, the second needle enters the lesion area from the other needle inlet, and the injection point is the tumor tissue middle to the outside).
  • the animal state was observed every day, and the observation content included but not limited to: mental state, behavioral activities, tumor rupture, etc.
  • the tumor diameter was measured and the body weight of the animals was weighed twice a week.
  • the tumors of the surviving animals were stripped and weighed.
  • the relative tumor proliferation rate T/C%, tumor growth inhibition rate TGI% and tumor weight inhibition rate IR TW % were calculated.
  • the relative tumor proliferation rate T/C (%) and tumor growth inhibition rate TGI (%) were used as experimental evaluation indicators.
  • Tumor weight inhibition rate IR TW (%) (W control group - W administration group )/W control group ⁇ 100%
  • the observation time was 21 days, and the experiment was terminated.
  • the average body weight of the animals in the vehicle control group was 21.77 ⁇ 0.31g, and the average weights of the animals in the KR11 low and high dose groups, KR10 low and high dose groups were 22.05 ⁇ 0.56g, 22.05 ⁇ 0.36g, 21.58 ⁇ 0.45 g and 21.49 ⁇ 0.40 g.
  • Weight statistics are shown in Table 13.
  • Body weight individual data are shown in Table 14.
  • the weight gain trend is shown in Figure 9.
  • the red triangle symbol indicates the time point of administration, and D1 is the day of grouping.
  • the mean tumor volume of the animals in the vehicle control group was 506.16 ⁇ 51.47mm 3
  • the mean tumor volumes of the animals in the KR11 low and high dose groups, KR10 low and high dose groups were 242.17 ⁇ 42.89mm 3 , 138.75 ⁇ 39.51mm 3 , 161.33 ⁇ 47.50mm 3 , 66.12 ⁇ 26.42mm 3 .
  • There were significant differences in mean tumor volume between each treatment group and the vehicle control group (P ⁇ 0.001).
  • the relative tumor proliferation rates T/C% of each treatment group were 48.57%, 30.02%, 34.20% and 14.95%, respectively.
  • the tumor growth inhibition rate TGI% was 51.43%, 69.98%, 65.80% and 85.05%, respectively.
  • Tumor volume statistics are shown in Table 15. Individual data are shown in Table 16. The statistical table of tumor volume in each group is shown in Table 17. The trend of tumor volume change is shown in Figure 10. Individual data are shown in Figure 11.
  • the mean tumor weight of the vehicle control group was 0.47 ⁇ 0.05g
  • the mean tumor weights of the animals in the KR11 low and high dose groups, KR10 low and high dose groups were 0.27 ⁇ 0.04g, 0.19 ⁇ 0.04g, 0.23 ⁇ 0.05g and 0.13 ⁇ 0.13 ⁇ 0.05g, respectively. 0.04g, which was significantly lower than that of the vehicle control group (vs the vehicle control group, P ⁇ 0.01). There was no significant difference in the mean tumor weight of each group under the same administration level of KR10 and KR11 (P>0.05).
  • the tumor weight inhibition rates IR TW (%) were 43.38%, 58.87%, 50.32% and 72.76%, respectively.
  • the statistical results are shown in Table 18.
  • the tumor weight statistics are shown in Table 19, the individual data are shown in Table 20, and the tumor weight statistics of each group are shown in Figure 12. Photos of euthanized tumors are shown in Figure 13.

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Abstract

L'invention concerne une molécule d'acide nucléique codant pour un mutant du gène Kras et une application d'un vecteur du virus de l'herpès simplex oncolytique (oHSV) contenant ladite molécule d'acide nucléique dans la préparation d'un médicament antitumoral. Le mutant comprend un ou plusieurs des éléments suivants : un mutant Kras G12D, un mutant Kras G13D, un mutant Kras G12V, un mutant Kras G13C, un mutant Kras G12C, un mutant Kras G13A, un mutant Kras G12A, un mutant Kras Q61L, un mutant Kras G12R, un mutant Kras Q61H, un mutant Kras G12S, un mutant Kras Q61R, un mutant Kras A59T, un mutant Kras A146T, un mutant Kras Y64H, et un mutant Kras A18D.
PCT/CN2021/116075 2020-09-01 2021-09-01 Molécule d'acide nucléique codant pour un mutant de gène kras Ceased WO2022048574A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102108356A (zh) * 2009-12-29 2011-06-29 台北荣民总医院 新颖启动子及包含其之病毒载体
WO2016053339A1 (fr) * 2014-10-02 2016-04-07 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Méthodes d'isolation de cellules t présentant une spécificité antigénique pour une mutation spécifique d'un cancer
CN106659705A (zh) * 2014-09-10 2017-05-10 加利福尼亚大学董事会 通过prostratin靶向k‑ras介导的信号转导通路和恶性肿瘤

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102108356A (zh) * 2009-12-29 2011-06-29 台北荣民总医院 新颖启动子及包含其之病毒载体
CN106659705A (zh) * 2014-09-10 2017-05-10 加利福尼亚大学董事会 通过prostratin靶向k‑ras介导的信号转导通路和恶性肿瘤
WO2016053339A1 (fr) * 2014-10-02 2016-04-07 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Méthodes d'isolation de cellules t présentant une spécificité antigénique pour une mutation spécifique d'un cancer

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YONG-CHEN LU ET AL.: "An Efficient Single- Cell RNA-Seq Approach to Identify Neoantigen- Specific T Cell Receptors", MOL THER., vol. 26, no. 2, 7 February 2018 (2018-02-07), XP002781571 *

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