WO2023143023A1 - 溶瘤病毒及其应用 - Google Patents

溶瘤病毒及其应用 Download PDF

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Publication number
WO2023143023A1
WO2023143023A1 PCT/CN2023/071375 CN2023071375W WO2023143023A1 WO 2023143023 A1 WO2023143023 A1 WO 2023143023A1 CN 2023071375 W CN2023071375 W CN 2023071375W WO 2023143023 A1 WO2023143023 A1 WO 2023143023A1
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protein
amino acid
oncolytic virus
acid substitution
mutation
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English (en)
French (fr)
Chinese (zh)
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周国庆
张凡
马良
田婷
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Joint Biosciences SH Ltd
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Joint Biosciences SH Ltd
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Priority to KR1020247028113A priority Critical patent/KR20240137069A/ko
Priority to AU2023210771A priority patent/AU2023210771A1/en
Priority to CA3243385A priority patent/CA3243385A1/en
Priority to EP23745906.0A priority patent/EP4471132A4/en
Priority to JP2024544471A priority patent/JP2025503188A/ja
Publication of WO2023143023A1 publication Critical patent/WO2023143023A1/zh
Priority to US18/785,273 priority patent/US20240424040A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/766—Rhabdovirus, e.g. vesicular stomatitis virus
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20221—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20232—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20251—Methods of production or purification of viral material
    • C12N2760/20252—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This application relates to the technical field of biomedicine, more specifically, it relates to an oncolytic virus and its application.
  • Oncolytic virus is a kind of tumor-killing virus with replication ability, which has been accepted by the public as an important branch of tumor immunotherapy.
  • Oncolytic viruses can specifically target and infect tumor cells, such as using the inactivation or defect of tumor suppressor genes in tumor cells to selectively infect tumor cells; after oncolytic viruses infect tumor cells, they will replicate in large numbers in tumor cells And eventually destroy tumor cells, thereby killing tumor cells.
  • oncolytic viruses can also provide the immunostimulatory signals necessary to improve the host's own anti-cancer response, thereby attracting more immune cells to continue to kill residual tumor cells.
  • oncolytic viruses Although oncolytic viruses have good application prospects in tumor immunotherapy, wild-type oncolytic viruses often cause inflammation in the nervous system of the body and other problems. disease risk. Therefore, in order to further promote the clinical application of oncolytic viruses, it is necessary to modify wild-type oncolytic viruses to obtain attenuated oncolytic viruses.
  • the attenuated oncolytic virus is used for clinical application, thereby reducing the pathogenic risk of the oncolytic virus and improving the safety of the oncolytic virus.
  • the application provides an oncolytic virus and its application.
  • an oncolytic virus provided by the present application adopts the following technical scheme:
  • an oncolytic virus the oncolytic virus includes an M protein, and the M protein, compared with the amino acid sequence shown in SEQ ID NO 1, contains amino acid substitutions at one or more of the following sites: 32nd, 33rd bit, 49th, 54th, 133rd, 225th.
  • the amino acid substitution of the M protein comprises a mutation of asparagine at position 32 to serine (N32S); and/or, a mutation of methionine at position 33 to alanine ( M33A); and/or, mutation of asparagine at position 49 to aspartic acid (N49D); and/or, mutation of histidine at position 54 to tyrosine (H54Y); and/or, Alanine at position 133 is mutated to threonine (A133T); and/or, valine at position 225 is mutated to isoleucine (V225I).
  • the M protein also includes amino acid substitutions at one or more of the following positions: 21st, 51st, 111th, 221st and 226th.
  • the amino acid substitution of the M protein further comprises a mutation of glycine at position 21 to glutamic acid (G21E); and/or, the amino acid substitution of the M protein comprises a methyl group at position 51 Mutation of thionine to arginine (M51R); and/or, mutation of methionine at position 51 to alanine (M51A); and/or, mutation of leucine to alanine at position 111 and/or, mutation of valine at position 221 to phenylalanine (V221F); and/or mutation of serine at position 226 to arginine (S226R).
  • M51R Mutation of thionine to arginine
  • M51A methionine at position 51 to alanine
  • V221F valine at position 221 to phenylalanine
  • S226R mutation of serine at position 226 to arginine
  • the amino acid substitution of the M protein comprises a mutation of glycine at position 21 to glutamic acid (G21E).
  • the amino acid substitution of the M protein comprises a mutation of asparagine at position 32 to serine (N32S).
  • the amino acid substitution of the M protein comprises a mutation of methionine at position 33 to alanine (M33A).
  • the amino acid substitution of the M protein comprises a mutation of asparagine at position 49 to aspartic acid (N49D).
  • the amino acid substitution of the M protein comprises a mutation of histidine at position 54 to tyrosine (H54Y).
  • the amino acid substitution of the M protein comprises a mutation of leucine to alanine at position 111 (L111A).
  • the amino acid substitution of the M protein comprises the mutation of alanine at position 133 to threonine (A133T).
  • the amino acid substitution of the M protein comprises a mutation of valine at position 225 to isoleucine (V225I).
  • the amino acid substitution of the M protein comprises a mutation of methionine at position 51 to arginine (M51R).
  • the amino acid substitution of the M protein comprises a mutation of methionine at position 51 to alanine (M51A).
  • the amino acid substitution of the M protein comprises a mutation of valine at position 221 to phenylalanine (V221F).
  • the amino acid substitution of the M protein comprises a mutation of serine at position 226 to arginine (S226R).
  • the amino acid substitution of the M protein comprises a mutation of serine at position 226 to glycine (S226G).
  • said M protein has an amino acid substitution of G21E.
  • the M protein has amino acid substitutions of G21E, N32S.
  • said M protein has amino acid substitutions of G21E, N32S, M33A.
  • said M protein has amino acid substitutions of G21E, N32S, M33A, N49D.
  • said M protein has amino acid substitutions of G21E, N32S, M33A, N49D, H54Y.
  • said M protein has amino acid substitutions of G21E, N32S, M33A, N49D, H54Y, L111A.
  • said M protein has amino acid substitutions of G21E, N32S, M33A, N49D, H54Y, L111A, A133T.
  • the M protein has amino acid substitutions of G21E, N32S, M33A, N49D, H54Y, L111A, A133T, V225I.
  • said M protein has G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V225I amino acid substitutions.
  • the M protein has amino acid substitutions of G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I.
  • the M protein has amino acid substitutions of G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, S226R.
  • said M protein has amino acid substitutions of N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, S226R.
  • the M protein has amino acid substitutions of M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, S226R.
  • said M protein has amino acid substitutions of N49D, M51R, H54Y, L111A, A133T, V221F, V225I, S226R.
  • the M protein has amino acid substitutions of M51R, H54Y, L111A, A133T, V221F, V225I, S226R.
  • said M protein has amino acid substitutions of H54Y, L111A, A133T, V221F, V225I, S226R.
  • said M protein has amino acid substitutions of L111A, A133T, V221F, V225I, S226R.
  • said M protein has amino acid substitutions of A133T, V221F, V225I, S226R.
  • said M protein has amino acid substitutions of V221F, V225I, S226R.
  • the M protein has amino acid substitutions of V225I, S226R.
  • said M protein has an amino acid substitution of S226R.
  • said M protein has amino acid substitutions of N32S, N49D, H54Y, V225I.
  • said M protein has amino acid substitutions of N32S, N49D, H54Y, V225I, S226G.
  • said M protein has amino acid substitutions of N32S, N49D, M51R, H54Y, V221F, V225I, S226R.
  • said M protein has amino acid substitutions of N32S, M33A, N49D, M51R, H54Y, V221F, V225I, S226R.
  • said M protein has amino acid substitutions of N32S, N49D, M51R, H54Y, A133T, V221F, V225I, S226R.
  • said M protein has amino acid substitutions of N32S, M33A, N49D, M51R, H54Y, A133T, V221F, V225I, S226R.
  • said M protein has amino acid substitutions of G21E, N32S, N49D, M51A, H54Y, L111A, V225I, S226R.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 2.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 3.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 4.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 5.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 6.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 7.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 8.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 9.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 10.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 11.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 12.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 13.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 14.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 15.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 16.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 17.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 18.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 19.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 20.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 21.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 22.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 23.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 24.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 25.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 26.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 27.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 28.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 29.
  • the M protein comprises the amino acid sequence shown in SEQ ID NO 30.
  • An oncolytic virus comprising the above-mentioned M protein; the oncolytic virus also includes a G protein; compared with the amino acid sequence shown in SEQ ID NO 31, the G protein comprises amino acid substitutions at one or more of the following sites: 438th, 453rd, 471st and 487th.
  • the G protein further comprises amino acid substitutions at one or more of the following positions: 53rd, 141st, 172nd, 217th, 232nd, 331st, 371st, 436th.
  • the amino acid substitution of the G protein comprises the mutation of valine at position 53 to isoleucine (V53I); and/or, the mutation of alanine at position 141 to valine acid (A141V); and/or, mutation of aspartic acid at position 172 to tyrosine (D172Y); and/or, mutation of lysine at position 217 to glutamic acid (K217E); and/or Or, aspartic acid at position 232 is mutated to glycine (D232G); and/or, valine at position 331 is mutated to alanine (V331A); and/or, valine at position 371 is mutated acid mutation to glutamic acid (V371E); and/or, glycine at position 436 to aspartic acid (G436D); and/or threonine at position 438 to serine (T438S); and /or, phenylalanine at position 453 is mutated to leucine (V53I); and
  • the amino acid substitution of the G protein comprises a mutation of valine at position 53 to isoleucine (V53I).
  • the amino acid substitution of the G protein comprises the mutation of alanine at position 141 to valine (A141V).
  • the amino acid substitution of the G protein comprises a mutation of aspartic acid at position 172 to tyrosine (D172Y).
  • the amino acid substitution of the G protein comprises a mutation of lysine at position 217 to glutamic acid (K217E).
  • the amino acid substitution of the G protein comprises a mutation of aspartic acid at position 232 to glycine (D232G).
  • the amino acid substitution of the G protein comprises a mutation of valine at position 331 to alanine (V331A).
  • the amino acid substitution of the G protein comprises a mutation of valine at position 371 to glutamic acid (V371E).
  • the amino acid substitution of the G protein comprises the mutation of glycine at position 436 to aspartic acid (G436D).
  • the amino acid substitution of the G protein comprises the mutation of threonine at position 438 to serine (T438S).
  • the amino acid substitution of the G protein comprises a mutation of phenylalanine to leucine at position 453 (F453L).
  • the amino acid substitution of the G protein comprises the mutation of threonine at position 471 to isoleucine (T471I).
  • the amino acid substitution of the G protein comprises a mutation of tyrosine at position 487 to histidine (Y487H).
  • said G protein has a V53I amino acid substitution.
  • the G protein has amino acid substitutions of V53I and A141V.
  • the G protein has amino acid substitutions of V53I, A141V, D172Y.
  • the G protein has amino acid substitutions of V53I, A141V, D172Y, K217E.
  • the G protein has amino acid substitutions of V53I, A141V, D172Y, K217E, D232G.
  • the G protein has amino acid substitutions of V53I, A141V, D172Y, K217E, D232G, V331A.
  • the G protein has amino acid substitutions of V53I, A141V, D172Y, K217E, D232G, V331A, V371E.
  • the G protein has amino acid substitutions of V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D.
  • the G protein has amino acid substitutions of V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S.
  • the G protein has amino acid substitutions of V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L.
  • the G protein has amino acid substitutions of V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I.
  • the G protein has amino acid substitutions of V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, Y487H.
  • the G protein has amino acid substitutions of A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, Y487H.
  • the G protein has amino acid substitutions of D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, Y487H.
  • the G protein has amino acid substitutions of K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, Y487H.
  • the G protein has amino acid substitutions of D232G, V331A, V371E, G436D, T438S, F453L, T471I, Y487H.
  • the G protein has amino acid substitutions of V331A, V371E, G436D, T438S, F453L, T471I, Y487H.
  • the G protein has amino acid substitutions of V371E, G436D, T438S, F453L, T471I, Y487H.
  • the G protein has amino acid substitutions of G436D, T438S, F453L, T471I, Y487H.
  • the G protein has amino acid substitutions of T438S, F453L, T471I, Y487H.
  • the G protein has amino acid substitutions of F453L, T471I, Y487H.
  • the G protein has amino acid substitutions of T471I, Y487H.
  • said protein G has an amino acid substitution of Y487H.
  • the G protein has the amino acid sequence shown in SEQ ID NO 32.
  • the G protein has the amino acid sequence shown in SEQ ID NO 33.
  • the G protein has the amino acid sequence shown in SEQ ID NO 34.
  • the G protein has the amino acid sequence shown in SEQ ID NO 35.
  • the G protein has the amino acid sequence shown in SEQ ID NO 36.
  • the G protein has the amino acid sequence shown in SEQ ID NO 37.
  • the G protein has the amino acid sequence shown in SEQ ID NO 38.
  • the G protein has the amino acid sequence shown in SEQ ID NO 39.
  • the G protein has the amino acid sequence shown in SEQ ID NO 40.
  • the G protein has the amino acid sequence shown in SEQ ID NO 41.
  • the G protein has the amino acid sequence shown in SEQ ID NO 42.
  • the G protein has the amino acid sequence shown in SEQ ID NO 43.
  • the G protein has the amino acid sequence shown in SEQ ID NO 44.
  • the G protein has the amino acid sequence shown in SEQ ID NO 45.
  • the G protein has the amino acid sequence shown in SEQ ID NO 46.
  • the G protein has the amino acid sequence shown in SEQ ID NO 47.
  • the G protein has the amino acid sequence shown in SEQ ID NO 48.
  • the G protein has the amino acid sequence shown in SEQ ID NO 49.
  • the G protein has the amino acid sequence shown in SEQ ID NO 50.
  • the G protein has the amino acid sequence shown in SEQ ID NO 51.
  • the G protein has the amino acid sequence shown in SEQ ID NO 52.
  • the G protein has the amino acid sequence shown in SEQ ID NO 53.
  • the G protein has the amino acid sequence shown in SEQ ID NO 54.
  • An oncolytic virus comprising the above-mentioned M protein, or M protein and G protein; the oncolytic virus also includes an N protein; compared with the amino acid sequence shown in SEQ ID NO 55, the N protein contains one or more of the following Amino acid substitutions at positions: 14th, 155th, and 353rd.
  • the amino acid substitution of the N protein comprises the mutation of isoleucine at position 14 to valine (I14V); and/or, the mutation of arginine at position 155 to lysine acid (R155K); and/or, mutation of serine at position 353 to asparagine (S353N).
  • the amino acid substitution of the N protein comprises a mutation of isoleucine to valine at position 14 (I14V).
  • the amino acid substitution of the N protein comprises arginine at position 155 to lysine (R155K).
  • the amino acid substitution of the N protein comprises a mutation of serine at position 353 to asparagine (S353N).
  • said N protein has an amino acid substitution of I14V.
  • the N protein has amino acid substitutions of I14V, R155K.
  • said N protein has amino acid substitutions of I14V, R155K, S353N.
  • the N protein has amino acid substitutions of R155K, S353N.
  • said N protein has an amino acid substitution of S353N.
  • the N protein comprises the amino acid sequence shown in SEQ ID NO 56.
  • the N protein comprises the amino acid sequence shown in SEQ ID NO 57.
  • the N protein comprises the amino acid sequence shown in SEQ ID NO 58.
  • the N protein comprises the amino acid sequence shown in SEQ ID NO 59.
  • the N protein comprises the amino acid sequence shown in SEQ ID NO 60.
  • An oncolytic virus comprising the above-mentioned M protein, or M protein and G protein, or M protein, G protein and N protein, or M protein and N protein; the oncolytic virus also includes P protein; the P protein and Compared with the amino acid sequence shown in SEQ ID NO 61, amino acid substitutions at one or more of the following positions are included: 50th, 76th, 99th, 126th, 140th, 151st, 168th bit, 170th, 189th, 237th.
  • the amino acid substitution of the P protein comprises the mutation of arginine at position 50 to lysine (R50K); and/or, the mutation of valine at position 76 to alanine (V76A); and/or, mutation of asparagine at position 99 to glutamic acid (D99E); and/or mutation of leucine at position 126 to serine (L126S); and/or, mutation of Leucine at position 140 is mutated to serine (L140S); and/or, histidine at position 151 is mutated to tyrosine (H151Y); and/or isoleucine at position 168 is mutated to formazan Thionine (I168M); and/or, lysine at position 170 to glutamic acid (K170E); and/or, tyrosine at position 189 to serine (Y189S); and/or , the asparagine at position 237 was mutated to aspartic acid (N237D).
  • the amino acid substitution of the P protein comprises the mutation of arginine at position 50 to lysine (R50K).
  • the amino acid substitution of the P protein comprises a mutation of valine at position 76 to alanine (V76A).
  • the amino acid substitution of the P protein comprises a mutation of asparagine at position 99 to glutamic acid (D99E).
  • the amino acid substitution of the P protein comprises a mutation of leucine to serine at position 126 (L126S).
  • the amino acid substitution of the P protein comprises a mutation of leucine to serine at position 140 (L140S).
  • the amino acid substitution of the P protein comprises a mutation of histidine at position 151 to tyrosine (H151Y).
  • the amino acid substitution of the P protein comprises a mutation of isoleucine at position 168 to methionine (I168M).
  • the amino acid substitution of the P protein comprises a mutation of lysine at position 170 to glutamic acid (K170E).
  • the amino acid substitution of the P protein comprises a mutation of tyrosine at position 189 to serine (Y189S).
  • the amino acid substitution of the P protein comprises a mutation of asparagine at position 237 to aspartic acid (N237D).
  • said P protein has an amino acid substitution of R50K.
  • the P protein has amino acid substitutions of R50K and V76A.
  • the P protein has amino acid substitutions of R50K, V76A, D99E.
  • the P protein has amino acid substitutions of R50K, V76A, D99E, L126S.
  • the P protein has amino acid substitutions of R50K, V76A, D99E, L126S, L140S.
  • the P protein has amino acid substitutions of R50K, V76A, D99E, L126S, L140S, H151Y.
  • the P protein has amino acid substitutions of R50K, V76A, D99E, L126S, L140S, H151Y, I168M.
  • the P protein has amino acid substitutions of R50K, V76A, D99E, L126S, L140S, H151Y, I168M, K170E.
  • the P protein has amino acid substitutions of R50K, V76A, D99E, L126S, L140S, H151Y, I168M, K170E, Y189S.
  • the P protein has amino acid substitutions of R50K, V76A, D99E, L126S, L140S, H151Y, I168M, K170E, Y189S, N237D.
  • the P protein has amino acid substitutions of V76A, D99E, L126S, L140S, H151Y, I168M, K170E, Y189S, N237D.
  • the P protein has amino acid substitutions of D99E, L126S, L140S, H151Y, I168M, K170E, Y189S, N237D.
  • said P protein has amino acid substitutions of L126S, L140S, H151Y, I168M, K170E, Y189S, N237D.
  • said P protein has amino acid substitutions of L140S, H151Y, I168M, K170E, Y189S, N237D.
  • the P protein has amino acid substitutions of H151Y, I168M, K170E, Y189S, N237D.
  • said P protein has amino acid substitutions of I168M, K170E, Y189S, N237D.
  • the P protein has amino acid substitutions of K170E, Y189S, N237D.
  • the P protein has amino acid substitutions of Y189S, N237D.
  • said P protein has an amino acid substitution of N237D.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 62.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 63.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 64.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 65.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 66.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 67.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 68.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 69.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 70.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 71.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 72.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 73.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 74.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 75.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 76.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 77.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 78.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 79.
  • the P protein comprises the amino acid sequence shown in SEQ ID NO 80.
  • An oncolytic virus comprising the above-mentioned M protein, or M protein and G protein, or M protein, G protein and N protein, or M protein, G protein, N protein and P protein, or M protein and N protein, or M protein protein and P protein, or M protein, G protein and P protein, or M protein, N protein and P protein; the oncolytic virus also includes L protein; the L protein is identical to the amino acid sequence shown in SEQ ID NO 81 ratio, including amino acid substitutions at one or more of the following positions: 87th, 487th.
  • the amino acid substitution of the L protein comprises a mutation of serine at position 87 to proline (S87P); and/or, a mutation of isoleucine at position 487 to threonine ( I487T).
  • the amino acid substitution of the L protein comprises a mutation of serine at position 87 to proline (S87P).
  • the amino acid substitution of the L protein comprises a mutation of isoleucine at position 487 to threonine (I487T).
  • said L protein has an amino acid substitution of S87P.
  • the L protein has amino acid substitutions of S87P, I487T.
  • said L protein has an amino acid substitution of I487T.
  • the L protein comprises the amino acid sequence shown in SEQ ID NO 82.
  • the L protein comprises the amino acid sequence shown in SEQ ID NO 83.
  • the L protein comprises the amino acid sequence shown in SEQ ID NO 84.
  • the oncolytic virus is obtained by site-directed mutation based on a baculovirus.
  • the oncolytic virus is obtained after site-directed mutation based on Vesicular Stomatitis Virus ("VSV”) virus.
  • VSV Vesicular Stomatitis Virus
  • the oncolytic virus is obtained after performing site-directed mutation based on the Indiana MuddSummer subtype of VSV virus.
  • the oncolytic virus comprises or expresses an exogenous protein of interest.
  • the oncolytic virus comprises a nucleic acid molecule comprising a nucleic acid sequence encoding the M protein with amino acid substitutions, and/or a nucleic acid sequence encoding the G protein with amino acid substitutions , and/or the nucleic acid sequence encoding the N protein with amino acid substitutions, and/or the nucleic acid sequence encoding the P protein with amino acid substitutions, and/or the nucleic acid sequence encoding the L protein with amino acid substitutions.
  • the nucleic acid molecule comprises a nucleic acid sequence encoding the exogenous protein of interest.
  • the nucleic acid sequence encoding the exogenous target protein in the nucleic acid molecule is located in the nucleic acid sequence encoding the M protein with amino acid substitutions, and/or the nucleic acid sequence encoding the G protein with amino acid substitutions Nucleic acid sequence, and/or the nucleic acid sequence encoding the N protein with amino acid substitution, and/or the nucleic acid sequence encoding the P protein with amino acid substitution, and/or the nucleic acid sequence encoding the L protein with amino acid substitution between.
  • the present application provides an oncolytic virus expression vector, which adopts the following technical scheme:
  • An oncolytic virus expression vector capable of producing any one of the oncolytic viruses described in this application.
  • the present application provides a virus production cell, adopting the following technical scheme:
  • a virus-producing cell capable of producing any one of the oncolytic viruses described in the application.
  • the present application provides a pharmaceutical composition, adopting the following technical scheme:
  • a pharmaceutical composition which includes any oncolytic virus described in this application, and optionally a pharmaceutically acceptable carrier.
  • the present application provides methods for preparing the above-mentioned oncolytic virus, oncolytic virus expression vector, virus production cell and/or pharmaceutical composition.
  • the present application provides the use of the above-mentioned oncolytic virus, oncolytic virus expression vector, virus production cell and/or pharmaceutical composition in the preparation of medicines for preventing and/or treating diseases and/or disorders.
  • the oncolytic virus, oncolytic virus expression vector, virus producing cell and/or pharmaceutical composition are used in a method for slow and sustained killing of dysplastic cells.
  • the diseases and/or conditions include: abnormal proliferative cells selected from tumor cells or related cells of tumor tissues; preferably, the tumor cells are cancer cells; more preferably, the The cancer cells are metastatic cancer cells.
  • the tumor comprises solid tumors and/or hematological tumors.
  • the oncolytic viruses provided by this application all have good infective ability and in vitro killing ability to abnormally proliferative (tumor) LLC cells, 4T1 cells, MC38 cells, and Hela cells, and are effective in LLC cells, 4T1 cells, MC38 cells, and Hela cells. are not easily cleared from the cells. Moreover, all the oncolytic viruses provided in the present application have poor infective ability to normal MEF cells, and poor killing ability in vitro, and the oncolytic viruses provided in the present application are all easy to clear in normal MEF cells.
  • the oncolytic virus provided by this application can be better used for the infection and killing of cells such as tumors and cancers, and it is not easy to be cleared in cells such as tumors and cancers, which further improves the oncolytic virus’s effect on cells such as tumors and cancers. Cure rate; at the same time, the above-mentioned oncolytic virus will not damage normal cells, and is easier to remove when inside normal cells, further ensuring the safety of normal cells.
  • Fig. 1 is the detection result of the infectivity of the oncolytic virus prepared in the present application and the wild-type oncolytic virus to LLC cells.
  • Fig. 2 is the detection result of the infectivity of the oncolytic virus prepared in the present application and the wild-type oncolytic virus to 4T1 cells.
  • Fig. 3 is the detection result of the infectivity of the oncolytic virus prepared in the present application and the wild-type oncolytic virus to MC38 cells.
  • Fig. 4 is the detection result of the infectivity of the oncolytic virus prepared in the present application and the wild-type oncolytic virus to Hela cells.
  • Fig. 5 is the detection result of the infectivity of the oncolytic virus prepared in the present application and the wild-type oncolytic virus to MEF cells.
  • Fig. 6 is the detection result of the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on LLC cells.
  • Fig. 7 is the test result of the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on 4T1 cells.
  • Fig. 8 is the test result of the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on MC38 cells.
  • Fig. 9 is the detection result of the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on Hela cells.
  • Fig. 10 is the detection result of the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on MEF cells.
  • Figure 11 shows the expression of IFN- ⁇ induced by the oncolytic virus prepared in the present application and the wild-type oncolytic virus in LLC cells.
  • Figure 12 shows the expression of IFN- ⁇ induced by the oncolytic virus prepared in the present application and the wild-type oncolytic virus in 4T1 cells.
  • Figure 13 shows the expression of IFN- ⁇ induced by the oncolytic virus prepared in the present application and the wild-type oncolytic virus in MC38 cells.
  • Figure 14 shows the expression of IFN- ⁇ induced by the oncolytic virus prepared in the present application and the wild-type oncolytic virus in Hela cells.
  • Figure 15 shows the expression of IFN- ⁇ induced by the oncolytic virus prepared in the present application and the wild-type oncolytic virus in MEF cells.
  • the number 0 on the abscissa represents the wild-type oncolytic virus; the numbers 1-79 on the abscissa represent the oncolytic viruses prepared in Preparation Examples 1-79, respectively.
  • the ordinate Log 10 TCID50 represents the TCID50 value calculated by the Karber method. The larger the value of Log 10 TCID50, the better the ability of the oncolytic virus to infect the cell; the smaller the value of Log 10 TCID50, the better the oncolytic virus The poorer the ability to infect the cell;
  • the vertical axis OD 570 represents the OD value of the cell. The larger the value of OD 570 , the worse the killing ability of the oncolytic virus on the cell; the smaller the value of OD 570 , the better the killing ability of the oncolytic virus on the cell.
  • the IFN- ⁇ level on the ordinate indicates the expression of the IFN- ⁇ gene.
  • the term "oncolytic virus” generally refers to a virus capable of replicating in and killing tumor cells.
  • Oncolytic viruses include, but are not limited to: Vesicular Stomatitis Virus (VSV virus for short), poxvirus, herpes simplex virus, measles virus, Semliki Forest virus, poliovirus, reovirus virus, Seneca Valley virus, Echo enterovirus, Coxsackie virus, Newcastle disease virus, and Maraba virus.
  • VSV virus Vesicular Stomatitis Virus
  • poxvirus herpes simplex virus
  • measles virus Semliki Forest virus
  • poliovirus poliovirus
  • reovirus virus Seneca Valley virus
  • Echo enterovirus Coxsackie virus
  • Newcastle disease virus and Maraba virus.
  • the oncolytic virus is engineered to increase selectivity for tumor cells.
  • the oncolytic virus is engineered to reduce its immunogenicity.
  • the oncolytic virus described herein is a VSV virus.
  • the VSV virus is a mutant of the Indiana MuddSummer subtype strain of the VSV virus.
  • site-directed gene mutation can be performed on the M protein, and/or G protein, and/or N protein, and/or P protein, and/or L protein of VSV virus.
  • the oncolytic virus described in the present application may be an oncolytic virus that has undergone genetic modification, such as modification of one or more genes, so as to improve its tumor selectivity and/or enhance its tumor selectivity in dividing cells. Copy first.
  • the modification at the gene level can be the modification of genes involved in DNA replication, nucleic acid metabolism, host tropism, surface attachment, virulence, lysis and diffusion, or the modification of integrating foreign genes.
  • the exogenous genes may include exogenous immune regulation genes, exogenous screening genes, exogenous reporter genes and the like.
  • the modified oncolytic virus may also be an oncolytic virus modified at the amino acid level, such as insertion, deletion, or substitution of one or more amino acids.
  • M protein generally refers to the VSV viral matrix protein. M protein is an important virulence factor of VSV virus, and it is also a protein in VSV virus that is known to interfere with the natural immune response of mice. The term “M protein” also includes homologues, orthologs, variants, functionally active fragments, etc. thereof.
  • the wild-type VSV virus Indiana MuddSummer subtype M protein can comprise the amino acid sequence shown in SEQ ID NO 1.
  • the M protein of the oncolytic virus may comprise the amino acid sequence shown in SEQ ID NO 2-29.
  • G protein generally refers to the glycoprotein of water VSV virus, also known as envelope protein.
  • G protein also includes homologues, orthologs, variants, functionally active fragments and the like thereof.
  • wild-type VSV virus Indiana MuddSummer subtype G protein can comprise the amino acid sequence shown in SEQ ID NO 31.
  • the G protein of the oncolytic virus may comprise the amino acid sequence shown in SEQ ID NO 32-54.
  • N protein generally refers to the nucleocapsid protein of VSV virus.
  • the term “N protein” also includes its homologues, orthologs, variants, functionally active fragments and the like.
  • the wild-type VSV virus Indiana MuddSummer subtype N protein can comprise the amino acid sequence shown in SEQ ID NO 55.
  • the N protein of the oncolytic virus may comprise the amino acid sequence shown in SEQ ID NO 56-60.
  • the term "P protein” generally refers to the phosphoprotein of VSV virus.
  • the term “P protein” also includes its homologues, orthologs, variants, functionally active fragments and the like.
  • the wild-type VSV virus Indiana MuddSummer subtype P protein can comprise the amino acid sequence shown in SEQ ID NO 61.
  • the P protein of the oncolytic virus may comprise the amino acid sequence shown in SEQ ID NO 62-80.
  • L protein generally refers to the VSV viral RNA polymerase protein.
  • the L gene of VSV virus encodes RNA poly E protein.
  • L protein also includes its homologues, orthologs, variants, functionally active fragments and the like.
  • the wild-type VSV virus Indiana MuddSummer subtype L protein can comprise the amino acid sequence shown in SEQ ID NO 81.
  • the L protein of the oncolytic virus may comprise the amino acid sequence shown in SEQ ID NO 82-84.
  • the protein mutation site is usually expressed by "amino acid + amino acid number + (mutated amino acid)".
  • the mutation may include, but not limited to, addition, substitution, deletion and/or deletion of amino acids.
  • M51R generally refers to a mutation of methionine M at position 51 to arginine R.
  • amino acid substitution generally refers to the replacement of an amino acid residue present in a parent sequence by another amino acid residue.
  • Amino acids in the parent sequence may be substituted, eg, via chemical peptide synthesis or by recombinant methods known in the art.
  • substitution at position xx generally refers to substitution of the amino acid present at position xx with an alternative amino acid residue.
  • the amino acid substitution may include amino acid mutation.
  • mutation generally refers to changing the nucleotide or amino acid sequence of a wild-type molecule.
  • Amino acid changes may include amino acid substitutions, deletions, deletions, insertions, additions, truncations, or processing or cleavage of proteins.
  • nucleic acid molecule generally refers to nucleotides of any length.
  • nucleic acid molecule may encode a protein comprised by said oncolytic virus.
  • the nucleic acid molecule may comprise DNA and/or RNA.
  • the RNA can comprise single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA), and single-stranded RNA can comprise sense RNA or antisense RNA (anti-sense RNA).
  • ssRNA single-stranded RNA
  • dsRNA double-stranded RNA
  • anti-sense RNA anti-sense RNA
  • the term "expression vector” generally refers to a nucleic acid vector. Under appropriate conditions, it is usually capable of expressing the gene and/or protein of interest.
  • the expression vector includes a nucleic acid molecule for expressing one or more components of a virus (eg, an oncolytic virus).
  • the expression vector can include at least one viral genomic element and can be packaged into a virus or as a virion.
  • virus producing cell generally refers to a cell, cell line or cell culture that can or already contains the nucleic acid molecule or expression vector described in the application, or can express the oncolytic virus described in the application.
  • the cells may include progeny of a single host cell.
  • the cells can be obtained by in vitro transfection using the expression vector described in this application.
  • the term "pharmaceutical composition” generally refers to a preparation in a form that allows the biological activity of the active ingredients to be effective, and which does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is to be administered .
  • these formulations may comprise the active ingredient of a drug together with a pharmaceutically acceptable carrier.
  • the drug product comprises a drug product for parenteral, transdermal, intracavity, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the pharmaceutical product can be administered in different ways, for example intravenously, intraperitoneally, subcutaneously, intramuscularly, topically or intradermally.
  • prevention generally refers to preventing the occurrence and onset, recurrence, and/or spread of a disease or one or more symptoms thereof by taking certain measures in advance.
  • treating generally refers to eliminating or ameliorating a disease, or one or more symptoms associated with a disease.
  • treatment generally refers to the administration of one or more drugs to a patient with the disease such that the disease is eliminated or remitted.
  • treatment may be administration of the drug combination and/or drug product in the presence or absence of other drugs after the onset of symptoms of a particular disease.
  • the use of the pharmaceutical combinations and/or pharmaceutical products described herein prevents the development, progression, recurrence and/or metastasis of tumors.
  • the term "neoplastic” generally refers to any new pathological growth of tissue. Tumors can be benign or malignant. In the present application, the tumor may be a solid tumor and/or a hematological tumor. For research, these tissues can be isolated from readily available sources by methods well known to those skilled in the art.
  • the application provides an oncolytic virus.
  • the oncolytic virus is based on the wild-type VSV virus, specifically on the basis of the Indiana strain of the VSV virus and the Indiana MuddSummer subtype strain of the VSV virus.
  • M protein, G protein Through its M protein, G protein, It is obtained by mutating the amino acid sequence of N protein, P protein and L protein.
  • the amino acid sequence of its M protein is shown in SEQ ID NO 1; the amino acid sequence of its G protein is shown in SEQ ID NO 31; the amino acid sequence of its N protein is shown in SEQ ID NO 55; the amino acid sequence of its P protein is shown in SEQ ID NO 55 Shown in ID NO 61;
  • the amino acid sequence of its L protein is shown in SEQ ID NO 81.
  • the M protein, G protein, N protein, P protein, and L protein can all be modified.
  • the present application makes the following modifications to the VSV virus to obtain an oncolytic virus.
  • an oncolytic virus the oncolytic virus includes an M protein, and the M protein, compared with the amino acid sequence shown in SEQ ID NO 1, contains amino acid substitutions at one or more of the following sites: 32nd, 33rd, 49th, 54th, 133rd, 225th.
  • the M protein also includes amino acid substitutions at one or more of the following positions: 21st, 51st, 111th, 221st and 226th.
  • the amino acid substitution of the M protein comprises a mutation of asparagine at position 32 to serine (N32S); and/or, a mutation of methionine at position 33 to alanine (M33A); and/or, at Asparagine at position 49 to aspartic acid (N49D); and/or, histidine at position 54 to tyrosine (H54Y); and/or, alanine at position 133 Mutation to threonine (A133T); and/or, mutation of valine at position 225 to isoleucine (V225I); the amino acid substitution of the M protein also includes the mutation of glycine at position 21 to glutamine acid (G21E); and/or, the amino acid substitution of the M protein comprises the mutation of methionine at position 51 to arginine (M51R); and/or, the mutation of methionine at position 51 to Alanine (M51A); and/or, comprising a mutation of leucine at position 111 to alanine (L111A
  • the M protein may contain an amino acid mutation at position 21.
  • the M protein may contain amino acid mutations at positions 21 and 32.
  • the M protein may comprise amino acid mutations at positions 21, 32 and 33.
  • the M protein may comprise amino acid mutations at positions 21, 32, 33 and 49.
  • the M protein may comprise amino acid mutations at positions 21, 32, 33, 49 and 54.
  • the M protein may comprise amino acid mutations at positions 21, 32, 33, 49, 54 and 111.
  • the M protein may comprise amino acid mutations at positions 21, 32, 33, 49, 54, 111 and 133.
  • the M protein may comprise amino acid mutations at positions 21, 32, 33, 49, 54, 111, 133 and 225.
  • the M protein may comprise amino acid mutations at positions 21, 32, 33, 49, 51, 54, 111, 133 and 225.
  • the M protein may comprise the 21st, 32nd, 33rd, 49th, 51st, 54th, 111th, 133rd, 221st and 225th amino acid mutations.
  • the M protein may comprise the 21st, 32nd, 33rd, 49th, 51st, 54th, 111th, 133rd, 221st, 225th and amino acid mutation at position 226.
  • the M protein may comprise the 32nd, 33rd, 49th, 51st, 54th, 111th, 133rd, 221st, 225th and 226th amino acid mutations.
  • the M protein may comprise amino acid mutations at positions 33, 49, 51, 54, 111, 133, 221, 225 and 226.
  • the M protein may comprise amino acid mutations at positions 49, 51, 54, 111, 133, 221, 225 and 226.
  • the M protein may comprise amino acid mutations at positions 51, 54, 111, 133, 221, 225 and 226.
  • the M protein may comprise amino acid mutations at positions 54, 111, 133, 221, 225 and 226.
  • the M protein may comprise amino acid mutations at positions 111, 133, 221, 225 and 226.
  • the M protein may comprise amino acid mutations at positions 133, 221, 225 and 226.
  • the M protein may comprise amino acid mutations at positions 221, 225 and 226.
  • the M protein may comprise amino acid mutations at positions 225 and 226.
  • the M protein may contain an amino acid mutation at position 226.
  • the M protein may comprise amino acid mutations at positions 32, 49, 54 and 225.
  • the M protein may comprise amino acid mutations at positions 32, 49, 54, 225 and 226.
  • the M protein may comprise amino acid mutations at positions 32, 49, 51, 54, 221, 225 and 226.
  • the M protein may comprise amino acid mutations at positions 32, 33, 49, 51, 54, 221, 225 and 226.
  • the M protein may comprise amino acid mutations at positions 32, 49, 51, 54, 133, 221, 225 and 226.
  • the M protein may comprise amino acid mutations at positions 32, 33, 49, 51, 54, 133, 221, 225 and 226.
  • the M protein may comprise amino acid mutations at positions 21, 32, 49, 51, 54, 111, 225 and 226.
  • the M protein may also contain amino acid substitutions at other positions.
  • the oncolytic virus also includes a G protein; compared with the amino acid sequence shown in SEQ ID NO 31, the G protein contains amino acid substitutions at one or more of the following sites: 438th, 453rd, 471st and the 487th.
  • the amino acid substitution of the G protein comprises the mutation of valine at position 53 to isoleucine (V53I); and/or, the mutation of alanine at position 141 to valine (A141V ); and/or, the aspartic acid at position 172 is mutated to tyrosine (D172Y); and/or, the lysine at position 217 is mutated to glutamic acid (K217E); and/or, at Aspartic acid at position 232 is mutated to glycine (D232G); and/or, valine at position 331 is mutated to alanine (V331A); and/or, valine at position 371 is mutated to glutamic acid (V371E); and/or, mutation of glycine at position 436 to aspartic acid (G436D); and/or, mutation of threonine at position 438 to serine (T438S); and/or, Mutation of phenylalanine at position 4
  • the G protein may contain an amino acid mutation at position 53.
  • the G protein may comprise amino acid mutations at positions 53 and 141.
  • the G protein may comprise amino acid mutations at positions 53, 141 and 172.
  • the G protein may comprise amino acid mutations at position 53, position 141, position 172 and position 217.
  • the G protein may comprise amino acid mutations at positions 53, 141, 172, 217 and 232.
  • the G protein may comprise amino acid mutations at positions 53, 141, 172, 217, 232 and 331.
  • the G protein may comprise amino acid mutations at positions 53, 141, 172, 217, 232, 331 and 371.
  • the G protein may comprise amino acid mutations at positions 53, 141, 172, 217, 232, 331, 371 and 436.
  • the G protein may comprise amino acid mutations at positions 53, 141, 172, 217, 232, 331, 371, 436 and 438.
  • the G protein may comprise the 53rd, 141st, 172nd, 217th, 232nd, 331st, 371st, 436th, 438th and 453rd positions amino acid mutations.
  • the G protein may comprise the 53rd, 141st, 172nd, 217th, 232nd, 331st, 371st, 436th, 438th, 453rd and amino acid mutation at position 471.
  • the G protein may comprise the 53rd, 141st, 172nd, 217th, 232nd, 331st, 371st, 436th, 438th, 453rd , amino acid mutations at positions 471 and 487.
  • the G protein may comprise the 141st, 172nd, 217th, 232nd, 331st, 371st, 436th, 438th, 453rd, 471st and amino acid mutation at position 487.
  • the G protein may comprise the 172nd, 217th, 232nd, 331st, 371st, 436th, 438th, 453rd, 471st and 487th amino acid mutations.
  • the G protein may comprise amino acid mutations at positions 217, 232, 331, 371, 436, 438, 453, 471 and 487.
  • the G protein may comprise amino acid mutations at positions 232, 331, 371, 436, 438, 453, 471 and 487.
  • the G protein may comprise amino acid mutations at positions 331, 371, 436, 438, 453, 471 and 487.
  • the G protein may comprise amino acid mutations at positions 371, 436, 438, 453, 471 and 487.
  • the G protein may comprise amino acid mutations at positions 436, 438, 453, 471 and 487.
  • the G protein may comprise amino acid mutations at positions 438, 453, 471 and 487.
  • the G protein may comprise amino acid mutations at positions 453, 471 and 487.
  • the G protein may contain amino acid mutations at positions 471 and 487.
  • the G protein may contain an amino acid mutation at position 487.
  • the G protein may also include amino acid substitutions at other positions.
  • the G protein comprises at least one or more amino acid substitutions in conserved regions.
  • the conserved region may comprise amino acids 437-461 of the G protein.
  • the G protein comprises at least one or more amino acid substitutions in the truncated region of the cytoplasmic domain.
  • the truncated region of the cytoplasmic domain may comprise amino acids 483-511 of the G protein.
  • the G protein may at least include amino acid substitutions at positions 438, 453, 471 and 487.
  • the oncolytic virus also includes an N protein; compared with the amino acid sequence shown in SEQ ID NO 55, the N protein contains amino acid substitutions at one or more of the following sites: 14th, 155th, and 353rd .
  • the amino acid substitution of the N protein comprises the mutation of isoleucine at position 14 to valine (I14V); and/or, the mutation of arginine at position 155 to lysine (R155K) and/or, the serine at position 353 is mutated to asparagine (S353N).
  • the N protein comprises the amino acid sequence shown in SEQ ID NO 58.
  • the N protein may contain an amino acid mutation at position 14.
  • the N protein may contain amino acid mutations at positions 14 and 155.
  • the N protein may comprise amino acid mutations at positions 14, 155 and 353.
  • the N protein may contain amino acid mutations at positions 155 and 353.
  • the N protein may contain an amino acid mutation at position 353.
  • the N protein may also contain amino acid substitutions at other positions.
  • the oncolytic virus also includes a P protein; compared with the amino acid sequence shown in SEQ ID NO 61, the P protein includes amino acid substitutions at one or more of the following positions: 50th, 76th, and 99th , 126th, 140th, 151st, 168th, 170th, 189th, 237th.
  • the amino acid substitution of the P protein comprises the mutation of arginine at position 50 to lysine (R50K); and/or, the mutation of valine at position 76 to alanine (V76A) and/or, asparagine at position 99 is mutated to glutamic acid (D99E); and/or, leucine at position 126 is mutated to serine (L126S); and/or, at position 140 Leucine is mutated to serine (L140S); and/or, histidine at position 151 is mutated to tyrosine (H151Y); and/or isoleucine at position 168 is mutated to methionine (I168M); and/or, the lysine at position 170 is mutated to glutamic acid (K170E); and/or, the tyrosine at position 189 is mutated to serine (Y189S); and/or, at The asparagine at position 237 was mutated to aspartic
  • the P protein may contain an amino acid mutation at position 50.
  • the P protein may contain amino acid mutations at positions 50 and 76.
  • the P protein may comprise amino acid mutations at positions 50, 76 and 99.
  • the P protein may comprise amino acid mutations at positions 50, 76, 99 and 126.
  • the P protein may comprise amino acid mutations at positions 50, 76, 99, 126 and 140.
  • the P protein may comprise amino acid mutations at positions 50, 76, 99, 126, 140 and 151.
  • the P protein may comprise amino acid mutations at positions 50, 76, 99, 126, 140, 151 and 168.
  • the P protein may comprise amino acid mutations at positions 50, 76, 99, 126, 140, 151, 168 and 170.
  • the P protein may comprise amino acid mutations at positions 50, 76, 99, 126, 140, 151, 168, 170 and 189.
  • the P protein may comprise the 50th, 76th, 99th, 126th, 140th, 151st, 168th, 170th, 189th and 237th amino acid mutations.
  • the P protein may comprise amino acid mutations at positions 76, 99, 126, 140, 151, 168, 170, 189 and 237.
  • the P protein may comprise amino acid mutations at positions 99, 126, 140, 151, 168, 170, 189 and 237.
  • the P protein may comprise amino acid mutations at positions 126, 140, 151, 168, 170, 189 and 237.
  • the P protein may comprise amino acid mutations at positions 140, 151, 168, 170, 189 and 237.
  • the P protein may comprise amino acid mutations at positions 151, 168, 170, 189 and 237.
  • the P protein may comprise amino acid mutations at positions 168, 170, 189 and 237.
  • the P protein may comprise amino acid mutations at positions 170, 189 and 237.
  • the P protein may contain amino acid mutations at positions 189 and 237.
  • the P protein may contain an amino acid mutation at position 237.
  • the P protein may also include amino acid substitutions at other positions.
  • the oncolytic virus also includes L protein; compared with the amino acid sequence shown in SEQ ID NO 81, the L protein includes amino acid substitutions at one or more of the following positions: 87th and 487th.
  • the L protein comprises the amino acid sequence shown in SEQ ID NO 83.
  • the amino acid substitution of the L protein includes the mutation of serine at position 87 to proline (S87P); and/or the mutation of isoleucine at position 487 to threonine (I487T).
  • the P protein may contain an amino acid mutation at position 87.
  • the P protein may contain amino acid mutations at positions 87 and 487.
  • the P protein may contain an amino acid mutation at position 487.
  • the P protein may also include amino acid substitutions at other positions.
  • the oncolytic virus may also comprise a nucleic acid molecule and an exogenous protein of interest.
  • the nucleic acid molecule comprises a nucleic acid sequence encoding the M protein with amino acid substitutions, and/or a nucleic acid sequence encoding the G protein with amino acid substitutions, and/or a nucleic acid sequence encoding the N protein with amino acid substitutions, and/or Or the nucleic acid sequence encoding the P protein with amino acid substitution, and/or the nucleic acid sequence encoding the L protein with amino acid substitution.
  • the nucleic acid molecule comprises a nucleic acid sequence encoding the exogenous protein of interest.
  • the nucleic acid sequence encoding the exogenous target protein is located in the nucleic acid sequence encoding the M protein with amino acid substitutions, and/or the nucleic acid sequence encoding the G protein with amino acid substitutions, and/or encoding the amino acid substitutions Between the nucleic acid sequence of the N protein, and/or the nucleic acid sequence encoding the P protein with amino acid substitutions, and/or the nucleic acid sequence encoding the L protein with amino acid substitutions.
  • the oncolytic virus described in this application can be obtained through a virus packaging process and a virus rescue process.
  • the specific process can include infecting and inoculating BSR-T7 cells with poxvirus vTF7-3 expressing T7 RNA polymerase, using expression plasmids and backbone plasmids for cloned VSV N, VSV P, and VSV L genes to perform lipofectamine transfection to obtain the target oncolytic virus .
  • the present application also provides an oncolytic virus expression vector, a virus production cell and a pharmaceutical composition.
  • the oncolytic virus expression vector may comprise nucleic acid sequences encoding the M protein and G protein of the oncolytic virus; the oncolytic virus expression vector may also comprise the N protein, P protein and L protein encoding the oncolytic virus nucleic acid sequence.
  • the virus production cells can produce the above-mentioned oncolytic virus; the virus production cells may include BSR-T7 cells, Vero cells, 293 cells, MRC-5 cells, WI38 cells.
  • the pharmaceutical composition includes the above-mentioned oncolytic virus, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may include one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or Suitable preparation of preservatives.
  • the acceptable ingredients of the pharmaceutical compositions are preferably nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present application include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents compatible with pharmaceutical administration, generally safe, nontoxic .
  • the pharmaceutical composition includes the above-mentioned oncolytic virus, and optionally other pharmaceutically acceptable drugs.
  • the above pharmaceutical composition can be used for combined treatment of diseases, including but not limited to the treatment of tumors.
  • the pharmaceutical composition may comprise parenteral, subcutaneous, intracavity, intraarterial, intravenous, intrathecal and/or intranasal administration or direct injection into tissue.
  • the pharmaceutical composition can be administered to a patient or subject by infusion or injection.
  • the administration of the pharmaceutical composition can be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the pharmaceutical composition can be administered without interruption. Such uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the influx of the therapeutic agent into the patient, as described in WO2015/036583.
  • the present application also provides a preparation method of the above-mentioned oncolytic virus, which may include a preparation method of an oncolytic virus expression vector, a virus production cell and/or a pharmaceutical composition.
  • Any method suitable for the production of oncolytic viruses can be used to produce the oncolytic viruses of the present application.
  • a poxvirus expressing T7 RNA polymerase can be added to the cells for transfection
  • plasmids expressing the N protein, L protein and P protein of the oncolytic virus and a backbone plasmid can be added for transfection, and the virus of the present application can be obtained through the virus rescue process.
  • Oncolytic virus can be obtained through the virus rescue process.
  • the present application also provides the use of the above-mentioned oncolytic virus, oncolytic virus expression vector, virus production cell and/or pharmaceutical composition in the preparation of medicaments for preventing and/or treating diseases and/or conditions.
  • the oncolytic virus provided by the application performs site-directed mutations on the amino acids on the M protein, G protein, N protein, P protein, and L protein of the oncolytic virus, thereby further improving the effect of the oncolytic virus on abnormally proliferative (tumor) LLC cells. , 4T1 cells, MC38 cells and Hela cells.
  • the oncolytic virus prepared above has poor infectivity to normal cells and normal MEF cells, indicating that the oncolytic virus prepared by the present application can be better used for infecting cells such as tumors and cancers without damaging normal cells. cells have broad application prospects.
  • the oncolytic virus provided by the application performs site-directed mutations on the amino acids on the M protein, G protein, N protein, P protein, and L protein of the oncolytic virus, thereby further improving the effect of the oncolytic virus on abnormally proliferative (tumor) LLC cells. , 4T1 cells, MC38 cells and Hela cells.
  • abnormally proliferative (tumor) LLC cells 4T1 cells, MC38 cells and Hela cells.
  • the in vitro killing ability of the oncolytic virus on LLC cells, 4T1 cells, MC38 cells and Hela cells was further improved.
  • the oncolytic virus prepared above has almost no effect on normal MEF cells, indicating that the oncolytic virus prepared in the present application can be used to damage and kill abnormal cells such as tumors and cancers without damaging normal cells.
  • the oncolytic virus provided by the present application is not easily cleared in abnormally proliferative (tumor) LLC cells, 4T1 cells, MC38 cells and Hela cells. Relatively speaking, wild-type oncolytic virus is easier to clear in LLC cells, MC38 cells and Hela cells. However, the oncolytic virus provided by this application performs site-directed mutations on the amino acids on the M protein, G protein, N protein, P protein, and L protein of the oncolytic virus, so that the oncolytic virus can be used in LLC cells, 4T1 cells, MC38 cells, and Hela cells.
  • Preparation Examples 1-29 respectively provide an oncolytic virus, the difference mainly lies in: the amino acid site-directed mutation site on the M protein of the wild-type oncolytic virus.
  • the construction method of the oncolytic virus corresponding to each preparation example is as follows:
  • the mutation sites shown in Table 1 were introduced by PCR technology. Perform PCR on the pRV-core plasmid and primers carrying each mutation site; then perform 1% agarose gel electrophoresis on the PCR product; then use the gel recovery kit for gel extraction to obtain different mutation sites with M protein Point the plasmid to obtain the constructed plasmid pRV-core Mut.
  • the constructed plasmid pRV-core Mut was transfected into BSR-T7 cells (purchased from ATCC, American Type Culture Collection, also known as the American model) by cell transfection technology. strain collection center).
  • pP, pN, and pL According to the mass ratio of pRV-core Mut, pP, pN, and pL as 10:5:4:1, the four plasmids were mixed, and the total amount of plasmids was 5 ⁇ g; the plasmids were diluted with 200 ⁇ l opti-MEM medium (Thermo Fisher Scientific), And add 7.5 ⁇ l transfection reagent Plus Reagent (Life Technologies) to obtain transfection plasmid master mix; wherein, pP (plasmid carrying baculovirus phosphoprotein gene), pN (plasmid carrying baculovirus nucleoprotein gene), pL (Plasmid carrying the baculovirus polymerase protein gene); pN, pP, and pL three corresponding parent vectors are all pCAGGS (purchased from ATCC);
  • the cell supernatant obtained by culturing BSR-T7 cells was transferred to Vero cells (Thermo Fisher Scientific), and the Vero cells were cultured at 37°C for 3 days, and the green fluorescence in the cells was observed under a fluorescence microscope to determine the degree of virus rescue. Situation; Further, the rescued mutant baculovirus library was passaged through Vero cells, and monoclonal virus strains were picked in the established phage plaque screening system.
  • the primer sequences are:
  • the product was recovered by 1% agarose gel electrophoresis and sent to a sequencing company for sequencing.
  • the sequencing results are shown in Table 1.
  • Preparation Examples 30-52 respectively provide an oncolytic virus, the main difference of which is: the sites of amino acid site-directed mutations on the M protein and G protein of the wild-type oncolytic virus.
  • the mutation site of the M protein is the same as the corresponding mutation site in Preparation Example 24, and the mutation site of the G protein is shown in Table 2.
  • the construction method of the oncolytic virus provided in the above preparation example is the same as the construction method of preparation example 11, the difference lies in:
  • step (1) PCR technology is utilized to introduce mutation sites as shown in Table 2;
  • Step (3) is G protein gene sequencing.
  • the primer sequences are:
  • the product was recovered by 1% agarose gel electrophoresis and sent to a sequencing company for sequencing.
  • the sequencing results are shown in Table 2.
  • Table 2 The mutation table of oncolytic virus G protein in preparation examples 30-52
  • Preparation Examples 53-57 respectively provide an oncolytic virus, the difference mainly lies in the sites of amino acid site-directed mutations on the M protein, G protein, and N protein of the wild-type oncolytic virus.
  • the mutation sites of M protein and G protein are the same as the corresponding mutation sites in Preparation Example 41, and the mutation sites of N protein are shown in Table 3.
  • the construction method of the oncolytic virus provided in the above preparation example is the same as the construction method of preparation example 41, the difference lies in:
  • step (1) PCR technology is utilized to introduce mutation sites as shown in Table 3;
  • Step (3) is N protein gene sequencing.
  • the primer sequences are:
  • the product was recovered by 1% agarose gel electrophoresis and sent to a sequencing company for sequencing.
  • the sequencing results are shown in Table 3.
  • Preparation Examples 58-76 respectively provide an oncolytic virus, which differs mainly in the sites of amino acid site-directed mutations on the M protein, G protein, N protein, and P protein of the wild-type oncolytic virus. Among them, the mutation sites of M protein, G protein, and N protein are the same as those in Preparation Example 55, and the mutation sites of P protein are shown in Table 4.
  • the construction method of the oncolytic virus provided in the above preparation example is the same as the construction method of preparation example 55, the difference lies in:
  • step (1) PCR technology is utilized to introduce mutation sites as shown in Table 4;
  • Step (3) is P protein gene sequencing.
  • the primer sequences are:
  • the product was recovered by 1% agarose gel electrophoresis and sent to a sequencing company for sequencing.
  • the sequencing results are shown in Table 4.
  • Preparation Examples 77-79 respectively provide an oncolytic virus, which differs mainly in the sites of amino acid site-directed mutations on the M protein, G protein, N protein, P protein, and L protein of the wild-type oncolytic virus.
  • the mutation sites of M protein, G protein, N protein, and P protein are the same as those in Preparation Example 67, and the mutation sites of L protein are shown in Table 5.
  • the construction method of the oncolytic virus provided in the above preparation example is the same as the construction method of preparation example 67, the difference lies in:
  • step (1) PCR technology is used to introduce mutation sites as shown in Table 5;
  • Step (3) is L protein gene sequencing.
  • the primer sequences are:
  • PR TTAATCTCTCCAAGAGTTTTTCCT.
  • the product was recovered by 1% agarose gel electrophoresis and sent to a sequencing company for sequencing.
  • the sequencing results are shown in Table 5.
  • This preparation example provides the packaging process of the oncolytic virus prepared by any one of the above preparation examples 1-79, which specifically includes the following steps:
  • BSR-T7 cells purchased from ATCC
  • poxvirus vTF7-3 expressing T7 RNA polymerase BioVector NTCC Plasmid Vector Strain Cell Gene Collection Center
  • the specific steps are as follows: spread BSR-T7 cells on a 6-well plate, and control the number of cells per well to 3 ⁇ 105 ; add poxvirus vTF7-3 expressing T7 RNA polymerase 14-16 hours after plating, and conduct poxvirus vTF7- 3. Infection of BSR-T7 cells; 6 hours after infection, the BSR-T7 cells were washed once with DPBS buffer (Thermo Fisher Scientific) for transfection.
  • DPBS buffer Thermo Fisher Scientific
  • the oncolytic virus prepared in Preparation Examples 1-79 and the wild-type oncolytic virus were used to test the infectivity of different cells.
  • the detection method is the TCID50 detection method, that is, 200 pfu each of Preparation Examples 1-79 and wild-type oncolytic viruses were added to the culture medium of different cells, and half of the tissue culture infectious dose (Tissue culture infectious dose) produced by each oncolytic virus was detected. TCID50).
  • the detected cells include: LLC cells (murine lung cancer cell line), 4T1 cells (murine breast cancer cell line), MC38 cells (murine colon cancer cell line), Hela cells (human cervical cancer cell line), MEF cells (human fibroblast cell line).
  • the specific detection method is:
  • step (3) In a 1.5ml EP tube, make serial 10-fold dilutions of the supernatant harvested in step (2), from 10 -1 to 10 -11 , a total of 11 titers; inoculate the diluted supernatant into 96 wells In the culture plate, inoculate a row of 8 wells for each dilution, and inoculate 100 ⁇ l in each well;
  • TCID50 is calculated according to the Karber method.
  • the test results are shown in Figure 1- Figure 5.
  • the abscissa 0 represents the wild-type oncolytic virus
  • the abscissa 1-79 represents the oncolytic virus prepared in Preparation Examples 1-79
  • the ordinate Log 10 TCID50 represents the TCID50 value calculated by the Karber method
  • the Log 10 TCID50 The larger the value, the better the ability of the oncolytic virus to infect the cell; the smaller the value of Log 10 TCID50, the worse the ability of the oncolytic virus to infect the cell.
  • Fig. 1 is the detection result of the infectivity of the oncolytic virus prepared in the present application and the wild-type oncolytic virus to LLC cells.
  • Fig. 2 is the detection result of the infectivity of the oncolytic virus prepared in the present application and the wild-type oncolytic virus to 4T1 cells.
  • Fig. 3 is the detection result of the infectivity of the oncolytic virus prepared in the present application and the wild-type oncolytic virus to MC38 cells.
  • Fig. 4 is the detection result of the infectivity of the oncolytic virus prepared in the present application and the wild-type oncolytic virus to Hela cells.
  • Fig. 5 is the detection result of the infectivity of the oncolytic virus prepared in the present application and the wild-type oncolytic virus to MEF cells.
  • the oncolytic viruses prepared in Preparation Examples 1-79 of the present application all have good infectivity to LLC cells, 4T1 cells, MC38 cells and Hela cells, especially the oncolytic viruses provided in Preparation Examples 77-79.
  • the ability of the virus to infect LLC cells, 4T1 cells, MC38 cells and Hela cells reached the infectivity of wild-type oncolytic virus.
  • the oncolytic viruses prepared above all have poor ability to infect MEF cells, especially the oncolytic viruses provided in Preparation Examples 77-79, indicating that the oncolytic viruses prepared in this application can be better used for tumors, cancers, etc. Infection of cells without damaging normal cells has broad application prospects.
  • the oncolytic virus prepared in Preparation Examples 1-79 and the wild-type oncolytic virus were used to perform in vitro killing tests on different cells.
  • the detection method is the MTT detection method, that is, 200 pfu each of Preparation Examples 1-79 and wild-type oncolytic virus are added to the culture medium of different cells, and the cell viability is detected by the MTT detection method after 24 hours.
  • the detected cells include: LLC cells, 4T1 cells, MC38 cells, Hela cells, MEF cells.
  • the specific detection method is:
  • step (3) The cell supernatant in the 96-well culture plate of step (2) was taken out, and fresh medium and MTT solution were added to the 96-well culture plate in an amount of 20 ⁇ L/well, and the 96-well culture plate was placed at 37 Cultivate for 4 hours under ambient conditions of °C and 5% CO 2 ;
  • the abscissa 0 represents the wild-type oncolytic virus
  • the abscissa 1-79 respectively represent the oncolytic viruses prepared in Preparation Examples 1-79
  • the ordinate OD 570 represents the OD value of the cells, and the larger the OD 570 value, the oncolytic virus The worse the ability of the virus to kill the cell; the smaller the OD 570 value, the better the ability of the oncolytic virus to kill the cell.
  • Fig. 6 is the detection result of the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on LLC cells.
  • Fig. 7 is the test result of the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on 4T1 cells.
  • Fig. 8 is the test result of the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on MC38 cells.
  • Fig. 9 is the detection result of the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on Hela cells.
  • Fig. 10 is the detection result of the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on MEF cells.
  • the oncolytic viruses prepared in Preparation Examples 1-79 of the present application all have good in vitro killing ability against LLC cells, 4T1 cells, MC38 cells and Hela cells, especially the oncolytic viruses provided in Preparation Examples 77-79.
  • the in vitro killing ability of oncoviruses on LLC cells, 4T1 cells, MC38 cells and Hela cells exceeded that of wild-type oncolytic virus.
  • the oncolytic virus prepared above has almost no killing effect on MEF cells, indicating that the oncolytic virus prepared in the present application can be used to damage and kill abnormal cells such as tumors and cancers without damaging normal cells.
  • the wild-type oncolytic virus has a good ability to kill LLC cells, MC38 cells and 4T1 cells in vitro, it will also damage and kill MEF cells to a large extent while damaging and killing the above-mentioned cells, which limits Clinical application of wild-type oncolytic virus. Therefore, the transformation of the wild-type oncolytic virus in the present application not only ensures the safety of the oncolytic virus to normal cells, but also ensures the killing ability of the oncolytic virus to tumor and cancer cells, and has broad clinical application prospects.
  • the oncolytic virus prepared in Preparation Examples 1-79 and the wild-type oncolytic virus induced the expression of IFN- ⁇ in different cells.
  • the detection index is the expression of gene IFN- ⁇ in different cells.
  • the gene IFN- ⁇ is a gene of soluble glycoprotein produced by cells with extensive anti-virus, anti-tumor and immunomodulatory effects.
  • the ability of cells to clear oncolytic viruses can be judged by the expression of gene IFN- ⁇ : when the gene IFN- When the expression of ⁇ is high, it indicates that the oncolytic virus is easily cleared in the cell; when the expression of gene IFN- ⁇ is low, it indicates that the oncolytic virus is not easily cleared in the cell.
  • the detected cells include: LLC cells, 4T1 cells, MC38 cells, Hela cells, MEF cells.
  • the specific detection method is:
  • step (3) Disrupt each group of cells obtained in step (2), and use TRIzol (Invitrogen) to extract total RNA from each cell, and use PrimeScript RT Reagent Kit with DNA Eraser (Takara) reverse transcription kit to reverse transcribe into cDNA , and stained with LightCycler 480SYBR Green I Master (Roche) dye, and the Ct value of each gene was detected on a LightCycler 480 quantitative PCR instrument. The relative expression of the target gene IFN- ⁇ was calculated by ⁇ Ct method.
  • the abscissa 0 represents the wild-type oncolytic virus
  • the abscissa 1-79 respectively represent the oncolytic viruses prepared in Preparation Examples 1-79
  • the ordinate IFN- ⁇ level represents the expression of the IFN- ⁇ gene
  • the IFN- ⁇ level The larger the value, the weaker the reproduction ability of the oncolytic virus in the cell, and the easier it is to clear; the smaller the value of the IFN- ⁇ level, the stronger the reproduction ability of the oncolytic virus in the cell, and the easier it is to clear.
  • Figure 11 shows the expression of IFN- ⁇ induced by the oncolytic virus prepared in the present application and the wild-type oncolytic virus in LLC cells.
  • Figure 12 shows the expression of IFN- ⁇ induced by the oncolytic virus prepared in the present application and the wild-type oncolytic virus in 4T1 cells.
  • Figure 13 shows the expression of IFN- ⁇ induced by the oncolytic virus prepared in the present application and the wild-type oncolytic virus in MC38 cells.
  • Figure 14 shows the expression of IFN- ⁇ induced by the oncolytic virus prepared in the present application and the wild-type oncolytic virus in Hela cells.
  • Figure 15 shows the expression of IFN- ⁇ induced by the oncolytic virus prepared in the present application and the wild-type oncolytic virus in MEF cells.
  • the oncolytic viruses provided in Preparation Example 1 and Preparation Example 21 of the present application and the wild-type oncolytic virus have weak reproductive ability in LLC cells, 4T1 cells, MC38 cells and Hela cells, and are easily eliminated.
  • the oncolytic viruses provided in Preparation Examples 2-20 and 22-79 are less easy to clear in LLC cells, 4T1 cells, MC38 cells and Hela cells.
  • the oncolytic viruses provided in Preparation Examples 77-79 are more difficult to clear in LLC cells, 4T1 cells, MC38 cells and Hela cells, further ensuring that the oncolytic viruses can be more effective in LLC cells, 4T1 cells, MC38 cells and Hela cells. Good ability to infect and kill.
  • the oncolytic virus provided by the present application is easier to clear in MEF cells, which further ensures the safety of MEF cells, thereby improving the safety of oncolytic viruses.

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