WO2018120496A1 - Procédé de préparation et application d'un virus grippal oncolytique recombinant - Google Patents

Procédé de préparation et application d'un virus grippal oncolytique recombinant Download PDF

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WO2018120496A1
WO2018120496A1 PCT/CN2017/080010 CN2017080010W WO2018120496A1 WO 2018120496 A1 WO2018120496 A1 WO 2018120496A1 CN 2017080010 W CN2017080010 W CN 2017080010W WO 2018120496 A1 WO2018120496 A1 WO 2018120496A1
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rna
cancer
influenza virus
dna molecule
recombinant
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Chinese (zh)
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杨鹏辉
王希良
张绍庚
任天宇
张培瑞
王兆海
孙芳
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中国人民解放军第三〇二医院
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Priority to ZA2019/04164A priority Critical patent/ZA201904164B/en

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    • 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
    • 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
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16121—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
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to a preparation method and application of recombinant oncolytic influenza virus in the field of biotechnology.
  • Tumor is a "killer" that seriously endangers human health.
  • Existing clinical treatments include surgery, radiotherapy, chemotherapy, targeted drugs, etc.
  • Although there is a certain degree of clinical efficacy there are large side effects, limited effects, and normal for the human body. Cells also cause problems such as great lethality. Therefore, the great challenge facing cancer treatment is how to kill tumor cells efficiently and specifically, but has no killing ability to normal cell tissues.
  • gene therapy began to be used in the treatment of a variety of diseases, and achieved some efficacy in some genetic diseases.
  • the first generation of tumor gene therapy vector uses a viral vector to introduce a tumor suppressor gene into a tumor cell of a human body, so that the tumor suppressor gene can inhibit and kill tumor cells.
  • the tumor mechanism is complex and the gene mutation is diverse.
  • the introduction of one or two tumor suppressor genes is not sufficient to inhibit the growth of tumor cells, and some viral vectors cannot be transfected in tumor cells, and the therapeutic effect is not obvious. Therefore, a novel viral vector is designed which can specifically replicate in tumor cells and selectively grow in tumor cells, resulting in specific killing of tumor cells.
  • the surrounding cells are caused to continue to kill and lyse the tumor cells.
  • Such viral vectors can overcome the shortcomings of the first generation of tumor gene therapy vectors.
  • This viral vector is called an oncolytic virus.
  • Tumor cells have their own independent genetic characteristics different from normal cells, such as genetic mutations, abnormal gene expression and other physiological characteristics. However, tumor cells are more likely to become hosts of viruses while gaining special growth privileges. For example, many tumor cells have tolerance to Ras-activated apoptosis, but retroviruses are proliferated in such cells due to inhibition of double-stranded RNA-activated protein kinase R (PKR). Studies have shown that activation of Ras is associated with inhibition of PKR. At the same time, the expression of viral receptors on the surface of malignant tumor cells is high, which makes them more susceptible to becoming a host of viruses.
  • PKI double-stranded RNA-activated protein kinase R
  • oncolytic viruses effectively kill tumor cells mainly by the following mechanisms: First, the virus directly replicates, packages and releases in tumor cells, and the accumulation of virions and the transcriptional translation pathways of tumor cells lead to cancer cell death. Second, viral proteins have cytotoxic effects or affect cellular functions. For example, antigens expressed by cells induce the body to produce cytotoxic T lymphocyte immune responses. Third, after the virus infects the cells, the sensitivity of the cells to the immune response is increased, that is, the immune effect caused by the cytokines on the cells is increased, thereby causing the killing of the tumor cells.
  • influenza virus has the characteristics of easy modification, and utilizes different genetic characteristics of tumor cells and normal cells to target cells in tumor cells for tumor treatment. New research strategy; (2) able to induce strong body production Strong humoral, cellular and mucosal immune responses, and a more comprehensive cross-immunoprotective response; (3) reverse genetics technology tends to mature, and directed transformation and utilization of influenza viruses has become a new approach to anti-tumor research.
  • the human H1N1 influenza virus strain A/PR/8/34 (abbreviated as PR8) is a chicken embryo-adapted virus strain that can efficiently replicate in chicken embryos.
  • PR8 is a single stranded, segmented RNA consisting of 8 independent RNA fragments encoding 10 proteins.
  • Fragment 1-3 encodes an RNA-dependent RNA polymerase
  • fragment 1 encodes a polymerase subunit PB2
  • fragment 2 encodes a polymerase subunit PB1
  • fragment 3 encodes a polymerase subunit PA
  • fragment 4 encodes hemagglutinin HA, which is a Surface glycoprotein associated with virus-attached infection
  • Fragment 5 encodes nuclear protein NP, which is the major structural part of viral RNA
  • Fragment 6 encodes neuraminidase NA, which is an envelope glycoprotein
  • Fragment 7 encodes two matrix proteins, M1 And M2, a non-glycosylated structural protein
  • fragment 8 encodes two non-structural proteins, NS1 and NS2.
  • the present invention first provides a recombinant influenza virus (named rFLU-HTRP, recombinant oncolytic influenza virus), which is 1) or 2):
  • the HTRP1 is a protein of the following A1) or A2) or A3):
  • amino acid sequence is the protein of sequence 1;
  • A2 a protein derived from A1) having the same function after substitution and/or deletion and/or addition of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO:
  • A3 a fusion protein obtained by ligating the N-terminus or/and C-terminus of A1) or A2);
  • the HTRP2 is a protein of the following B1) or B2) or B3):
  • B2 a protein derived from B1) having the same function after substitution and/or deletion and/or addition of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO:3;
  • B3 A fusion protein obtained by ligating the N-terminus or/and C-terminus of B1) or B2).
  • the genome of the above recombinant influenza virus is a single stranded, segmented RNA, and the negative strand RNA of the recombinant influenza virus transcribes a set of positive strand RNA complementary to the minus strand RNA, the set of positive strand RNA including PB1 -RNA, PB2-RNA, PA-RNA, NP-RNA, M-RNA, NA-RNA, HTRP1-RNA and HTRP2-RNA;
  • the PB1-RNA is an RNA encoding PB1 in an influenza virus strain
  • the PB2-RNA is an RNA encoding PB2 in the influenza virus strain
  • the PA-RNA is an RNA encoding PA in the influenza virus strain
  • the NP-RNA is an RNA encoding NP in the influenza virus strain
  • the M-RNA is an RNA encoding M1 and M2 in the influenza virus strain
  • the NA-RNA is an RNA encoding NA in the influenza virus strain
  • the HTRP1-RNA is an RNA encoding the HTRP1;
  • the HTRP2-RNA is an RNA encoding the HTRP2.
  • the kit of positive strand RNA may also be composed only of the PB1-RNA, the PB2-RNA, the PA-RNA, the NP-RNA, the M-RNA, the NA- RNA, the HTRP1-RNA and the HTRP2-RNA composition.
  • the sequence of the HTRP1-RNA is a sequence obtained by replacing all T in positions 41-289 of the sequence 2 in the sequence listing with U, and the other nucleotides are unchanged;
  • the sequence of the HTRP2-RNA is a sequence obtained by replacing all of the T positions 47-3817 of the sequence 4 in the sequence listing with U, and the other nucleotides are unchanged.
  • influenza virus strain may be any subtype of influenza A virus (H1-H16, N1-N9) or any lineage of influenza B virus (B/Victoria, B/Yamagata), specifically A/PR/ 8/34.
  • the present invention also provides a method for constructing the recombinant influenza virus, which comprises a recombinant vector containing a DNA molecule encoding the PB1-RNA, and a DNA molecule encoding the PB2-RNA.
  • a recombinant vector comprising a DNA molecule encoding the PA-RNA, a recombinant vector comprising a DNA molecule encoding the NP-RNA, a recombinant vector containing a DNA molecule encoding the M-RNA, and a coding vector comprising the NA
  • a recombinant vector of a DNA molecule of RNA, a recombinant vector containing a DNA molecule encoding the HTRP1-RNA, and a recombinant vector containing a DNA molecule encoding the HTRP2-RNA are introduced into a packaging cell to obtain a recombinant influenza virus.
  • the present invention also provides any of the following products:
  • P2a and P2b a complete carrier for treating tumors, consisting of P2a and P2b:
  • P2a a vector containing the gene encoding the HTRP1;
  • P2b a vector containing the gene encoding the HTRP2
  • P3a and P3b a complete expression cassette for treating tumors, consisting of P3a and P3b:
  • P3a an expression cassette containing the gene encoding the HTRP1;
  • P3b an expression cassette containing the gene encoding the HTRP2;
  • P4a and P4b a set of genes for treating tumors, consisting of P4a and P4b:
  • P5a and P5b a complete set of proteins for treating tumors, consisting of P5a and P5b:
  • the present invention also provides a biological material related to the recombinant influenza virus, which is any one of the following E1) to E6):
  • E2a The complete carrier consists of the following E2a), E2b), E2c), E2d), E2e), E2f), E2g) and E2h):
  • E2a a recombinant vector comprising a DNA molecule encoding said PB1-RNA
  • E2b a recombinant vector comprising a DNA molecule encoding said PB2-RNA
  • E2c a recombinant vector comprising a DNA molecule encoding the PA-RNA
  • E2d a recombinant vector comprising a DNA molecule encoding the NP-RNA
  • E2e a recombinant vector comprising a DNA molecule encoding the M-RNA
  • E2f a recombinant vector comprising a DNA molecule encoding the NA-RNA
  • E2g a recombinant vector comprising a DNA molecule encoding the HTRP1-RNA
  • E2h a recombinant vector comprising a DNA molecule encoding the HTRP2-RNA
  • E4 an animal cell line containing the recombinant influenza virus
  • the set of DNA molecules comprises a DNA molecule encoding the PB1-RNA, a DNA molecule encoding the PB2-RNA, a DNA molecule encoding the PA-RNA, a DNA molecule encoding the NP-RNA, encoding the M a DNA molecule of RNA, a DNA molecule encoding the NA-RNA, a DNA molecule encoding the HTRP1-RNA, and a DNA molecule encoding the HTRP2-RNA.
  • the DNA molecule encoding the HTRP1-RNA may be a DNA molecule whose nucleotide sequence is shown in positions 41 to 289 of SEQ ID NO: 2 in the Sequence Listing; the DNA encoding the HTRP2-RNA The molecule may be a DNA molecule whose nucleotide sequence is shown at positions 47-3817 of SEQ ID NO:4 in the Sequence Listing.
  • the animal cell line, the animal tissue, and the animal organ may each include a propagation material, or may not include a propagation material.
  • E3) the microorganism, E4) the animal cell line, E5) the animal tissue, and E6) the animal organ may be the host of the influenza virus.
  • the recombinant vector of E2a) is pHW-PB1; E2b) the recombinant vector is pHW-PB2; E2c) the recombinant vector is pHW-PA; E2d) the recombinant vector is pHW - NP; E2e) the recombinant vector is pHW-M; E2f) the recombinant vector is pHW-NA; E2g) The recombinant vector is pHW-HTRP1. E2h) The recombinant vector is pHW-HTRP2. pHW-HTRP1 expresses HTRP1 shown in SEQ ID NO: 1, and pHW-HTRP2 expresses HTRP2 shown in SEQ ID NO: 3.
  • the present invention also provides a therapeutic tumor drug, wherein the active ingredient of the drug is the recombinant influenza virus.
  • the active ingredient of the above drug may also be a composition obtained by combining the recombinant influenza virus and other therapeutic tumor drugs as an active ingredient.
  • the present invention also provides any of the following applications:
  • the present invention also provides a method of treating and/or preventing a tumor, which comprises administering to a recipient animal any one of the following M1)-M3):
  • the recipient animal can be a mammal.
  • the mammal can be a human (Homo sapiens).
  • the tumor of the present invention may be a solid tumor.
  • the tumor may specifically be liver cancer, lung cancer, colon cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, kidney cancer, pancreatic cancer, prostate cancer, lymphoma, glioma or melanoma.
  • the liver cancer may specifically be hepatocellular carcinoma.
  • Figure 1 shows the morphology and particle size distribution of rFLU-HTRP electron microscopy.
  • the A picture shows the morphology of rFLU-HTRP virus particles under electron microscope
  • the B picture shows the size distribution of virus particles.
  • Figure 2 shows the effect of recombinant influenza virus rFLU-HTRP on liver cancer cells by MTS assay.
  • Figure 3 is a graph showing the effects of 48 hours after infection with recombinant influenza virus rFLU-HTRP on liver cancer cell lines (SMMC-7721, HepG2, MHCC-97L, HUH7.5) and normal liver cells (L02) by staining with crystal violet.
  • Figure 4 is a graph showing the anti-tumor effect of recombinant influenza virus rFLU-HTRP in human hepatoma cells HepG2 tumor nude mice.
  • Figure 5 is a pathological observation of each tissue of nude mice.
  • Figure 6 shows the replication of nude mouse recombinant influenza virus rFLU-HTRP in different tissues.
  • the pHW2000 (Hoffmann E, Mrauss S, Perez D, et al. Eight-Sladium system for rapid generation of influenza virus vaccines. Vaccine, 2002, 20: 3165-70, in the following examples) is publicly available from the applicant.
  • Biological material the The biological material is used only for the repeated experiments of the present invention and cannot be used for other purposes.
  • the African green monkey SV40 transformed kidney cells (COS-1) in the following examples are ATCC (American Type Culture Collection) cell bank products, and dog kidney cells (MDCK) are ATCC (American Type Culture Collection) cell bank products, SPF.
  • the chicken embryo is a product of the Beijing Experimental Animal Research Center.
  • Recombinant vectors pHW-PB1, pHW-PB2, pHW-PA, pHW-NP, pHW-M and pHW-NA in the following examples (Hoffmann E, Mrauss S, Perez D, et al.Eight-Plasmid system for rapid generation Of influenza virus vaccines. Vaccine, 2002, 20: 3165-70) is publicly available from the Applicant, and these biological materials are used only for the repeated experiments of the present invention and cannot be used for other purposes.
  • the recombinant vectors pHW-PB1, pHW-PB2, pHW-PA, pHW-NP, pHW-M and pHW-NA were respectively contained with influenza virus strain A/PR/8/34 (Hoffmann E, Mrauss S, Perez D, etal).
  • HepG2, SMMC-7721, MHCC-97L, HuH-7.5 and L02 in the following examples are products of the Third Hospital of the People's Liberation Army.
  • the present invention provides a recombinant influenza virus (recombinant oncolytic influenza virus) which expresses a protein of HTRP1 and HTRP2, respectively;
  • HTRP1 is a protein whose amino acid sequence is sequence 1
  • HTRP2 is a protein whose amino acid sequence is sequence 3.
  • HTRP1 is encoded by the HTRP1 gene indicated by nucleotides 41 to 289 of SEQ ID NO: 2 in the Sequence Listing
  • HTRP2 is encoded by the HTRP2 gene indicated by nucleotides 47 to 3817 of SEQ ID NO: 4 in the Sequence Listing.
  • the DNA molecule shown in SEQ ID NO: 2 was digested with AarI to obtain a DNA fragment containing the HTRP1 gene, and the pHW2000 was digested with AarI to obtain a pHW2000 skeleton vector 1, and the DNA fragment containing the HTRP1 gene was ligated with the pHW2000 skeleton vector 1.
  • the vector was recombined, and the recombinant vector was named as HTRP1 shown in pHW-HTRP1, pHW-HTRP1 expression sequence 1.
  • the DNA molecule shown in SEQ ID NO:4 was digested with BsmBI to obtain a DNA fragment containing the HTRP2 gene, and the pHW2000 was digested with BsmBI to obtain a pHW2000 skeleton vector 2, and the DNA fragment containing the HTRP2 gene was ligated to the pHW2000 skeleton vector 2 to obtain
  • the vector was recombined, and the recombinant vector was named HTRP2 shown in pHW-HTRP2, pHW-HTRP2 expression sequence 3.
  • pHW-HTRP1 and pHW-HTRP2 of step 1 were mixed with the recombinant carriers pHW-PB1, pHW-PB2, pHW-PA, pHW-NP, pHW-M and pHW-HA in an amount of 0.2 ⁇ g each, and then added to 10 ⁇ L.
  • the transfection reagent (Effectene, Qiagen) was used for 10 min at room temperature and co-transfected into African green monkey SV40 transformed kidney cells (COS-1) and dog kidney cells (MDCK) at 37 ° C, 5% CO 2 After culture for 48-60 hours, the cell suspension was obtained, and the cell suspension was inoculated into 9-11 day old SPF chicken embryos, cultured at 37 ° C for 72 hours, and the chicken embryo allantoic fluid was harvested and subjected to blood coagulation (HA) test according to OIE standard. The test result of HA blood coagulation titer is 1:256-512.
  • the successfully obtained recombinant influenza virus was named rFLU-HTRP, and further expanded, cultured, concentrated, and purified to obtain rFLU-HTRP, which was frozen at -70 °C.
  • pHW-HTRP1 was replaced with pHW2000, and the other steps were unchanged, and the recombinant influenza virus was rescued. As a result, no recombinant influenza virus was obtained.
  • pHW-HTRP2 was replaced with pHW2000, and the other steps were unchanged, and the recombinant influenza virus was rescued. As a result, no recombinant influenza virus was obtained.
  • pHW-HTRP1 was replaced with pHW2000 and pHW-HTRP2 was replaced with pHW2000.
  • the other steps were unchanged, and the recombinant influenza virus was rescued. As a result, no recombinant influenza virus was obtained.
  • the recombinant influenza virus strain rFLU-HTRP obtained in step 2 was negatively stained and observed by transmission electron microscopy. The results showed that rFLU-HTRP conformed to the typical morphological characteristics of influenza virus, and had envelope and surface with spike structure. The virus particle size was 80-120 nm. The result is shown in Figure 1.
  • the rFLU-HTRP of step 2 was inoculated into 9-11 day old SPF chicken embryos, and the second generation chicken embryo allantoic fluid was used to extract viral RNA. After RT-PCR, the correct PB2, PB1, PA, NP and NA were amplified. And M gene fragments as well as the HTRP1 gene and the HTRP2 gene.
  • the rFLU-HTRP of step 2 was expanded by chicken embryo, concentrated by ultrafiltration, purified by sucrose gradient centrifugation, and then subjected to SDS-PAGE electrophoresis. After gel staining and decolorization, the corresponding size of NP, HA1, HA2, NEP protein can be detected. , indicating that the main components of the antigen were not lost.
  • Example 2 Specific role of recombinant influenza virus rFLU-HTRP in hepatoma cells
  • the rFLU-HTRP of Example 2, Step 2 was resuspended in PBS to give an rFLU-HTRP suspension.
  • the MTS kit Promega was used to detect the cytotoxic specificity of recombinant influenza virus rFLU-HTRP on hepatoma cells at different multiplicity of infection.
  • the liver cancer cells used were SMMC-7721, HepG2, MHCC-97L and HuH-7.5, using human normal hepatocytes. L02 was used as a control. The experiment was repeated three times, and the specific steps of each repeated experiment were as follows:
  • the multiplicity of infection MOI of the living cell TCID50 was replaced by 0.1, 1, 3 and 5, respectively, and each cell was seeded with rFLU-HTRP suspension, and each cell was cultured.
  • the cytotoxic specificity of rFLU-HTRP on hepatoma cells was detected by MTS reagent at 48 hours, 72 hours, and 96 hours after inoculation of each of the above cells, and MTS reagent was added to the sample cells for 1 hour at 37 ° C for 1 hour. Perform 490 nm absorbance detection to calculate cell viability of cells in each well Cell viabillty, %), the results are shown in Figure 2.
  • the liver cancer cells SMMC-7721, HepG2, MHCC-97L and HuH-7.5 were significantly killed at various time points after infection, when the number of infected cells TCID50 was plural.
  • MOI was 0.1 and 1
  • the hepatocarcinoma cells HepG2, MHCC-97L and HuH-7.5 were significantly killed at different time points after infection, and the different infection numbers at different time points had no obvious killing effect on normal liver cells L02.
  • RT-PCR was used to detect the expression of the target gene: after detecting the above 490 nm absorbance, the total RNA of each cell was extracted, and the cDNA was reverse-transcribed into cDNA, and then the primers of HTRP1 were used upstream primer5'-TATTCGTCTCAGGGAGCAAAAGCAGGGTG-3' and downstream5'-ATATCGTCTCGTATTAGTAGAAACAAGGGT-3.
  • liver cancer cell lines SMMC-7721, HepG2, MHCC-97L, HUH7.5
  • normal hepatocytes L02
  • the liver cancer cell line HUH7.5 cells were more than 90%, indicating that rFLU-HTRP has a strong cell killing effect on HUH7.5; when the infection dose is greater than 0.1 MOI
  • the pathological changes of the four hepatocarcinoma cell lines were over 90%, indicating that rFLU-HTRP has obvious killing effect on liver cancer cells; and the infection doses of the virus have no obvious killing effect on normal liver cells L02. And this result is consistent with the MTS results.
  • the results showed that rFLU-HTRP had selective killing effect on liver cancer cells, but had no significant effect on normal cells.
  • Example 3 Anti-tumor effect of recombinant influenza virus rFLU-HTRP in nude mice bearing liver cancer transplantation
  • tumor volume (longest diameter x shortest diameter 2 )/2.
  • rFLU-HTRP suspension is a liquid having a rFLU-HTRP concentration of 2 ⁇ 10 9 PFU/50 ⁇ l obtained by adding rFLU-HTRP to PBS
  • rFLU-HTRP was injected at 2 ⁇ 10 9 PFU for 5 consecutive days.
  • Each nude mouse was intravenously injected with rFLU-HTRP suspension for intravenous treatment.
  • Each nude mouse rFLU-HTRP The injection volume was 2 ⁇ 10 9 PFU, and the injection was continued for 5 days.
  • Each nude mouse of the PBS control group was intravenously injected with 50 ⁇ l of PBS for 5 days as a control.
  • Tumor volume change Tumor volume was measured from day 14 and tumor volume changes were observed within 8 weeks after treatment. The results are shown in Figure 4 and Table 1.
  • the nude mice were sacrificed, and the tumors, liver, lung, kidney, heart and brain of each group were observed for pathological damage.
  • the PBS group had significant tumor atypia and multi-nuclear splitting; the tumor insufficiency was not obvious in the intravenous injection group and the intratumoral injection group, and the mitosis phenomenon was not obvious, indicating that the tumor malignancy degree in the PBS group was higher than that in the two rFLU-HTRP treatment groups. Higher, indicating that intravenous injection and intratumoral injection of rFLU-HTRP have significant inhibitory effects on tumors.
  • the tumors, liver, lung, spleen, kidney, heart and brain tissues of each group were ground, RNA was extracted, and real-time quantitative PCR was used to detect the replication of rFLU-HTRP in different tissues.
  • the primer used was: upstream primer. 5'-AAGACCAATCCTGTCACCTCTGA-3'/downstream primer5'-CAAAGCGTCTACGCTGCAGTCC-3'.
  • the internal reference is mouse-GAPDH, and the primer of the internal reference is upstream primer 5'-GGGAAATTCAACGGCACAGT-3'/downstream primer 5'-AGATGGTGATGGGCTTCCC-3'. No replication of the virus strain was detected in each tissue of the PBS control animals. Both the intravenous group and the intratumor injection group (Fig.
  • the recombinant influenza virus of the invention can inhibit the growth of tumors in animals and can inhibit the development of tumors: the recombinant influenza virus of the invention has improved survival rate after treatment of tumor-bearing animals, and tumor growth is effectively inhibited, rFLU-HTRP intratumoral injection group
  • the recombinant influenza virus of the invention has the advantages of killing tumor specificity, high effectiveness and good safety: the recombinant influenza virus selectively kills tumor cells without significant influence on the host normal cells.
  • the recombinant influenza virus of the present invention can be used for the treatment of tumors.

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Abstract

La présente invention concerne un procédé de préparation et l'application d'un virus grippal oncolytique recombinant exprimant HTRP1 et HTRP2, HTRP1 étant : (A1) une protéine représentée par la SEQ ID N° 1 ; (A2) une protéine dérivée de (A1) par substitution, délétion et/ou addition d'un ou de plusieurs résidus d'acides aminés dans la SEQ ID N° 1 ; ou (A3) une protéine de fusion obtenue en rattachant l'extrémité N-terminale et/ou l'extrémité C-terminale de (A1) ou de (A2) à un marqueur ; et HTRP2 étant : (B1) une protéine représentée par la SEQ ID N° 3 ; (B2) une protéine dérivée de (B1) par substitution, délétion et/ou addition d'un ou de plusieurs résidus d'acides aminés dans la SEQ ID N° 3 ; ou (B3) une protéine de fusion obtenue en rattachant l'extrémité N-terminale et/ou l'extrémité C-terminale de (B1) ou de (B2) à un marqueur. Le virus grippal oncolytique recombinant peut être utilisé pour cibler la destruction des cellules d'hépatome sans influencer les cellules hôtes normales, et peut ainsi être utilisé dans une thérapie de ciblage des hépatomes.
PCT/CN2017/080010 2016-12-30 2017-04-11 Procédé de préparation et application d'un virus grippal oncolytique recombinant WO2018120496A1 (fr)

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