WO2022012028A1 - 构建体、溶瘤病毒及其应用 - Google Patents

构建体、溶瘤病毒及其应用 Download PDF

Info

Publication number
WO2022012028A1
WO2022012028A1 PCT/CN2021/074620 CN2021074620W WO2022012028A1 WO 2022012028 A1 WO2022012028 A1 WO 2022012028A1 CN 2021074620 W CN2021074620 W CN 2021074620W WO 2022012028 A1 WO2022012028 A1 WO 2022012028A1
Authority
WO
WIPO (PCT)
Prior art keywords
construct
virus
cells
hil12
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2021/074620
Other languages
English (en)
French (fr)
Inventor
张旭辉
罗显麟
何亮亮
宁维瑜
陈小锋
李文佳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dongguan Hec Biopharmaceutical R&d Co Ltd
Sunshine Lake Pharma Co Ltd
Original Assignee
Dongguan Hec Biopharmaceutical R&d Co Ltd
Sunshine Lake Pharma Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dongguan Hec Biopharmaceutical R&d Co Ltd, Sunshine Lake Pharma Co Ltd filed Critical Dongguan Hec Biopharmaceutical R&d Co Ltd
Publication of WO2022012028A1 publication Critical patent/WO2022012028A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/208—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/869—Herpesviral vectors
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16621—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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16632—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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16643—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention relates to the fields of biotechnology and gene therapy, in particular, the present invention relates to a construct, an oncolytic virus with improved sensitivity and pharmaceutical use.
  • Oncolytic virus has good anti-tumor effect. It selectively replicates on tumor cells and directly lyses and kills tumor cells. The released tumor-related antigens activate the body's anti-tumor immunity through antigen presentation, and achieve the goal of inhibiting tumor growth or even tumor regression. Effect. Antitumor immune responses elicited by infected tumor cell debris can be amplified by the addition of immunostimulatory factors. In order to further improve the therapeutic effect of oncolytic virus, some immune regulation or immune activation genes can usually be inserted into the virus genome, so that the virus can express the corresponding genes while replicating on tumor cells, and release them around the tumor to improve the tumor immune microenvironment and play a role in Enhance anti-tumor immunity.
  • IL12 is interleukin 12, a cytokine with immunomodulatory activity.
  • oncolytic virus to express IL12 increases the level of IL12 in tumors, which can avoid the systemic side effects caused by traditional intravenous administration and improve safety.
  • oncolytic viruses expressing IL12 in the prior art, such as patent CN202010145491.4, the specific construction strategies of oncolytic viruses and the insertion sites of exogenous genes (such as IL12) are different. Bioactive effects are unknown. To date, there have been no reports on the sensitivity of oncolytic viruses expressing IL12.
  • the new oncolytic virus HSV1-IL12(+) constructed has the advantages of high efficiency and strong specificity, and can also induce a specific anti-tumor immune response and improve the sensitivity to tumor cells.
  • the present application aims to solve one of the above technical problems in the background art at least to a certain extent.
  • the present invention proposes a construct.
  • the vector of the construct is derived from HSV, and the construct carries an immunostimulatory factor gene coding frame, and the immunostimulatory factor gene coding frame is set at the position of the UL23 gene.
  • the inventors unexpectedly and unexpectedly found that when the coding frame of the immune stimulator gene is set at the position of the UL23 gene, the construct can continuously express the immune stimulator at a high level.
  • the above-mentioned construct may further include at least one of the following additional technical features:
  • ICP47 and double copy ICP34.5 of the construct are knocked out
  • Knockout of the replication non-essential gene ICP47 in the HSV genome can improve the expression of MHC-I on the surface of virus-infected tumor cells and the ability to present cellular antigens; knockout of double-copy ICP34.5 can make herpes simplex virus (HSV) in The replication in normal cells is limited, and it selectively replicates in tumor cells, thereby improving the drug safety of HSV virus.
  • HSV herpes simplex virus
  • the coding frame of the immunostimulatory factor gene is set at the position between the 387th and 521st nucleotides of the UL23 gene sequence.
  • the UL23 gene sequence coding described in this application is coded with the first nucleotide of the UL23 gene initiation codon as the first nucleotide sequence.
  • the coding frame of the immunostimulatory factor gene is set between the 387-521 nucleotides of the UL23 gene sequence by knocking out the 388-520 nucleotides of the UL23 gene sequence, and then the immune
  • the stimulator gene coding frame was inserted between nucleotides 387-521 of the UL23 gene sequence.
  • the IL-12 gene coding frame is operably linked to a promoter comprising a sequence selected from the group consisting of CMV, CAG, EF1 ⁇ , Rous sarcoma virus long terminal repeat (RSV LTR), metal sulfide At least one of protein I (MTI), the immunostimulatory agent is selected from at least one of GM-CSF, IL2, IL5, IL7, IL12, IL15, IL24 and IL27.
  • a promoter comprising a sequence selected from the group consisting of CMV, CAG, EF1 ⁇ , Rous sarcoma virus long terminal repeat (RSV LTR), metal sulfide At least one of protein I (MTI), the immunostimulatory agent is selected from at least one of GM-CSF, IL2, IL5, IL7, IL12, IL15, IL24 and IL27.
  • the promoter is CMV.
  • the immunostimulatory factor is IL12.
  • the above construct can continuously express IL-12 at a high level, which not only can reproduce and infect in tumor cells with high selectivity, but also has an immunomodulatory effect. , to ensure that tumor cells are completely removed by the body's own immune response.
  • the HSV is HSV1.
  • the inventors found that HSV2 may cause genital infection, and then selected HSV1 to further improve the safety.
  • the IL12 gene coding frame has the nucleotide sequence shown below, 1) the nucleotide sequence shown in SEQ ID NO: 1; 2) compared with 1), it has at least 70%, at least 75% %, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical nucleotide sequences.
  • the construct has the nucleotide sequence shown below, a) the nucleotide sequence shown in SEQ ID NO: 2; b) compared with a) at least 70%, at least 75% %, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical nucleotide sequences.
  • the present invention provides an oncolytic virus.
  • the oncolytic virus carries the aforementioned construct.
  • the modified virus compared with the wild-type virus, the modified virus often has a significantly reduced sensitivity to tumors.
  • researches such as Mckie E A show that it can be reduced by 100 times (Mckie E A, Maclean A R, Lewis A D, et al.Selective in vitro replication of herpes simplex virus type 1(HSV-1)ICP34.5 null mutants in primary human CNS tumors--evaluation of a potentially effective clinical therapy.[J].British Journal of Cancer,1996,74 (5):745-752.).
  • HSV1 carries IL12, which not only maintains strong replication ability in tumor cells and strong killing power to tumor cells, but also enhances the targeting of virus strains to tumor cells.
  • the expression of IL12 protein through exogenous IL12 gene activates the body's anti-tumor immune response.
  • the oncolytic virus according to the embodiments of the present invention expressing IL12 can replicate the virus in the tumor by intratumoral injection and continuously express IL12 locally at a high level, avoiding systemic side effects caused by intravenous administration.
  • the oncolytic virus can continuously express IL12 at a high level.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises the aforementioned construct or the aforementioned oncolytic virus.
  • the pharmaceutical composition according to the embodiments of the present invention can take effect at a low dose, and has a good killing effect on tumor cells.
  • the present invention proposes the use of the aforementioned construct, the aforementioned oncolytic virus or the aforementioned pharmaceutical composition in the preparation of a medicament for treating or preventing tumors.
  • the drug is used to selectively kill tumor cells.
  • the tumor is pancreatic cancer, ovarian cancer, pharyngeal squamous cell carcinoma, lung cancer, liver cancer, melanoma, esophageal cancer, and colon cancer.
  • Fig. 1 is the pMD18-HOM-hIL12 plasmid map according to the embodiment of the present invention
  • Fig. 2 is the PCR gel electrophoresis detection diagram of the recombinant virus constructed according to the embodiment of the present invention
  • FIG. 3 is a sequence diagram of a recombinant virus constructed according to an embodiment of the present invention.
  • FIG. 4 is a graph showing the expression results of hIL12 in Vero cells according to an embodiment of the present invention.
  • Fig. 5 is the inhibition curve of 2k-hIL12, 2K on different tumor cells according to the embodiment of the present invention.
  • Fig. 6 is the inhibition curve of 2k-hIL12, 2K on different tumor cells according to an embodiment of the present invention.
  • Fig. 7 is a graph showing the results of proliferation of 2k-hIL12 and 2K in different tumor cells according to the embodiment of the present invention.
  • first and second are only used for descriptive purposes, and should not be construed as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature delimited with “first”, “second” may expressly or implicitly include at least one of that feature.
  • plurality means at least two, such as two, three, etc., unless otherwise expressly and specifically defined.
  • the "immunostimulatory factor” mentioned in this application refers to a polypeptide that can regulate the function of immune cells.
  • the immune Stimulatory factors are immunostimulatory factors that can be used for tumor therapy.
  • immunostimulatory factors include, but are not limited to, cytokines (eg, IL2, IL7, IL12, IL15, GM-CSF, IFN ⁇ , etc.), chemokines (eg, IL8), growth factors (eg, IL5).
  • IL12 IL-12 or interleukin-12 or interleukin-12
  • hIL12 human interleukin-12
  • the "IL12 gene coding frame" described in this application refers to a nucleic acid sequence capable of expressing functional IL12, that is, the IL12 segment encoded by the nucleic acid sequence is a segment capable of realizing the IL12 function, or in other words, the nucleic acid
  • the IL12 segment encoded by the sequence is an essential functional region of IL12.
  • ICP47 and ICP34.5 are knocked out means that the ICP47 and ICP34.5 genes are silenced. significantly reduced.
  • the "UL23 gene inactivation" mentioned in this application refers to insertion inactivation, that is, after inserting an exogenous gene at the position of the UL23 gene, the original function of the UL23 gene is lost.
  • operably linked in this application refers to linking the exogenous gene to the vector, so that the control elements in the vector, such as promoter sequences, etc., can exert their intended functions to regulate the transcription and translation of the exogenous gene.
  • the following examples utilize the CRISPR/Cas9 system to construct recombinant HSV-1 virus, which specifically includes plasmid construction, recombinant screening, and virus identification.
  • the biological activity of the recombinant HSV-1 virus specifically includes the expression of hIL12, the ability of the virus to infect and kill tumor cells, etc.
  • the KOS virus strain used in this example has 99% nuclear weight compared with the wild type KOS virus strain. nucleotide sequence similarity.
  • Examples 2 to 4 carried out PCR and ELISA validation of HSV-1-hIL12 from a single viral plaque after multiple rounds of purification, and evaluated the cytotoxicity of HSV1-hIL12 in different tumor cells.
  • the primers were B45577UL22F/B48431UL24R, and the PCR product size was 4051bp.
  • the experimental group HSV1-hIL12 refers to 2K-hIL12-lacZ11-3-1, and the negative control is HSV-1/2K.
  • Vero African green monkey kidney cells
  • HSV1-hIL12 HSV-1/2K infection MOI (ratio of virus to cell number) was 0.01, respectively after infection with Vero cells , 24, 48, 72 hours to collect the cultured virus culture supernatant.
  • 2k-hIL12 that is, the aforementioned HSV1-hIL12, which refers to 2K-hIL12-lacZ11-3-1), HSV-1/2K (abbreviated as 2K), and the cells are ASPC-1 ( Pancreatic cancer cells); SKOV3 (human ovarian cancer cells); Fadu (human pharyngeal squamous cell carcinoma cells); Hep3B2.1-7 (liver cancer cells); Skmel-28 (melanoma cells) HepG2 (human liver cancer cells); HCT-116 (colon cancer cells); SW620 (colorectal cancer cells); T.Tn (human esophageal squamous cell carcinoma cells); NCI-H460 (human large cell lung cancer).
  • ASPC-1 Pancreatic cancer cells
  • SKOV3 human ovarian cancer cells
  • Fadu human pharyngeal squamous cell carcinoma cells
  • Hep3B2.1-7 liver cancer cells
  • Skmel-28 melanoma cells
  • HepG2 human
  • 2k-hIL12 virus at lower doses was effective against Hep3B2.1-7 liver cancer cells, Skmel-28 melanoma cells, ASPC-1 human pancreatic cancer cells, Fadu nasopharyngeal cancer cells, Skov3 human ovarian cancer cells, HepG2 human liver cancer cells, HCT- 116 colon cancer cells, SW620 colorectal cancer cells, T.Tn human esophageal squamous cell carcinoma cells, and NCI-H460 human large cell lung cancer cells all have good killing effects.
  • the lethality of -1 tumor cells and Fadu nasopharyngeal carcinoma cells was significantly stronger than that of 2K virus. It can be concluded that the recombinant oncolytic virus 2k-hIL12 constructed by the present invention has an increased sensitivity selectively on certain tumor cells, as shown in Figure 5 and Figure 6 .
  • 2k-hIL12 that is, the aforementioned HSV1-hIL12
  • HSV-1/2K abbreviated as 2K
  • the cells are Hep3B2.1-7 liver cancer cells, Skmel-28 melanoma cells, ASPC- 1 Tumor cells, Fadu nasopharyngeal carcinoma cells, Skov3 human ovarian cancer cells, NCI-H460 large cell lung cancer cells, HepG2 liver cancer cells, HCT-116 human colon cancer cells, and SW620 human colon cancer cells.
  • Virus titer virus dilution factor*(1000/300)*number of plaques.
  • the titer of 2k virus was higher than that of 2k-hIL12.
  • Skmel-28 cells were completely infected and the titer increased.
  • two viruses can be obtained. The titers were not much different, and the proliferative and replication abilities were comparable; and the two viruses were completely infected within 48 hours, and the titers were both at the 7th power of 10, indicating that their infectivity was strong.
  • the virus titer of 2K was not much different from that of hIL12 virus.
  • the 2k-hIL12 virus titer was slightly lower than that of 2k at 48h, but ASPC-1 at 48h
  • the cells were completely infected, and the floating of some cells had a great influence on the subsequent titer determination, and the titer decreased greatly.
  • the viral titer of 2K was slightly higher than that of hIL12 at 24 h, the viral titer of hIL12 was 1.00E+08 at 48 h, the viral titer of 2K was lower than that of hIL12, and the two viruses It was completely infected at 48 hours, indicating that its infection ability was strong.
  • the viral titer of 2K was 7.65E+07
  • the viral titer of hIL12 was 8.65E+07
  • the viral titer of 2K at 48h was 1.43E+08
  • the viral titer of hIL12 was 2.11E +08
  • the 2k-hIL12 virus titer was slightly higher than the 2k virus titer, and the two viruses had the same ability to replicate and proliferate on NCI-H460 cells, and the two viruses were completely infected within 48 hours, indicating that their infectivity was very high. powerful.
  • the mean virus titer of 2K was 9.88E+07
  • the mean virus titer of hIL12 was 1.86E+07
  • the virus titer of 2K was slightly higher than that of hIL12
  • the virus titer of 2K The mean titer was 6.62E+07
  • the mean viral titer of hIL12 was 9.03E+07. From the data, the 2k-hIL12 virus titer was lower than the 2k virus titer at 24h, but 2k-hIL12 was greater than 2k at 48h. The virus titer is not much different from the maximum value of 2k.
  • HCT-116 cells For HCT-116 cells, at 48h, HCT-116 cells were completely infected, the viral titer of 2K was 4.70E+07, and the viral titer of hIL12 was 7.61E+07. From the data, the viral titer of 2k was lower than 2k
  • the virus titer of -hIL12 shows that the replication and proliferation of 2k-hIL12 virus on HCT-116 cells is slightly higher than that of 2k virus; and the two viruses have been completely infected at 48h, and the titers are both in the 7th power of 10, indicating infection. Strong ability.
  • SW620 cells For SW620 cells, at 48h, SW620 cells were completely infected, the mean viral titer of 2K was 1.92E+07, and the mean viral titer of hIL12 was 1.45E+07.
  • the viral replication and proliferation of 2k-hIL12 on SW620 cells were slightly lower than 2k virus, the ability to replicate and proliferate is equivalent; the two viruses have been completely infected within 48 hours, and the titers are both in the 7th power of 10, indicating that the infection ability is strong.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

提供一种构建体、溶瘤病毒及其应用。该构建体将I型单纯疱疹病毒的ICP34.5基因、ICP47基因敲除，并在UL23基因上插入免疫刺激因子表达序列，构建得到的溶瘤病毒，可选择性在肿瘤细胞中感染复制增殖，并且对肿瘤细胞的敏感性提高。

Description

构建体、溶瘤病毒及其应用 技术领域
本发明涉及生物技术和基因治疗领域，具体地，本发明涉及一种构建体、敏感性提高的溶瘤病毒以及制药用途。
背景技术
溶瘤病毒具有良好的抗肿瘤效果，其选择性在肿瘤细胞上复制直接裂解杀伤肿瘤细胞，释放的肿瘤相关抗原通过抗原呈递作用，激活了机体抗肿瘤免疫，达到抑制肿瘤生长甚至肿瘤完全消退的效果。由受感染的肿瘤细胞碎片引起的抗肿瘤免疫响应可通过加入免疫刺激因子而被放大。为了进一步提升溶瘤病毒治疗效果，通常可以将一些免疫调节或免疫激活基因插入病毒基因组，使病毒在肿瘤细胞上复制的同时表达相应基因，并释放在肿瘤周围，改善肿瘤免疫微环境，起到增强抗肿瘤免疫的作用。
IL12即白介素12，一种具有免疫调节活性的细胞因子。
利用溶瘤病毒表达IL12，使肿瘤中IL12的水平提高，可以避免传统通过静脉给药带来的全身性毒副作用，提高安全性。虽然现有技术中不乏溶瘤病毒表达IL12的报道，如专利CN202010145491.4，但是溶瘤病毒的具体构建策略及外源基因(如IL12)的插入位点不同，对所构建的溶瘤病毒的生物活性影响是未可知的。至今还没有关于表达IL12的溶瘤病毒的敏感性的报道。
将IL12运用在抗肿瘤领域，构建的新型溶瘤病毒HSV1-IL12(+)，具有高效、特异性强等优点，还可引起特异的抗肿瘤免疫反应，提升对肿瘤细胞的敏感性。
发明内容
本申请旨在至少在一定程度上解决上述背景技术中的技术问题之一。
为此，在本发明的第一方面，本发明提出了一种构建体。根据本发明的实施例，所述构建体的载体来源于HSV，所述构建体携带免疫刺激因子基因编码框，所述免疫刺激因子基因编码框设置于UL23基因位置。发明人在实验中意外而惊喜发现，当免疫刺激因子基因编码框设置于UL23基因位置时，构建体可持续高表达免疫刺激因子。
根据本发明的实施例，上述构建体还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述构建体的ICP47和双拷贝ICP34.5被敲除，
敲除HSV基因组中的复制非必需基因ICP47，可提高被病毒感染的肿瘤细胞表面MHC-I的表达和细胞抗原呈递的能力；敲除双拷贝ICP34.5，可使单纯疱疹病毒(HSV)在正常细胞内复制受限，选择性地在肿瘤细胞中复制，进而提高HSV病毒的用药安全性。
根据本发明的实施例，所述免疫刺激因子基因编码框设置于UL23基因序列的第387-521位核苷酸之间位置。需要说明的是，本申请所述的UL23基因序列编码是以UL23基因起始密码子的第一位核苷酸为第1位进行顺序编码的，UL23基因的序列可参考 https://www.ncbi.nlm.nih.gov/nuccore/NC_001806.2？report＝genbank&from＝46609 &to＝47803&strand＝true。根据本发明的实施例，免疫刺激因子基因编码框设置于UL23基因序列的第387-521位核苷酸之间是通过将UL23基因序列的第388-520位核苷酸敲除，之后将免疫刺激因子基因编码框插入UL23基因序列的第387-521位核苷酸之间获得的。
根据本发明的实施例，IL-12基因编码框与启动子可操作地连接，所述启动子包括选自CMV、CAG、EF1α、劳斯肉瘤氏病毒长末端重复序列(RSV LTR)、金属硫蛋白I(MTI)的至少之一,所述免疫刺激剂选自GM-CSF、IL 2、IL5、IL7、IL 12、IL 15、IL 24和IL 27的至少之一。
根据本发明的实施例，所述启动子为CMV。
根据本发明的实施例，所述免疫刺激因子为IL12。
经试验验证，当IL-12基因编码框设置于HSV载体的UL23基因位置时，上述构建体可持续高表达IL-12，不仅高选择地在肿瘤细胞内繁殖感染，而且还可具有免疫调节作用，确保肿瘤细胞被机体自身的免疫反应彻底清除。
根据本发明的实施例，所述HSV为HSV1。发明人发现，HSV2可能引起生殖器感染，进而选择HSV1，安全性进一步提高。
根据本发明的实施例，IL12基因编码框具有如下所示的核苷酸序列，1)SEQ ID NO:1所示的核苷酸序列；2)与1)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同一性的核苷酸序列。
Figure PCTCN2021074620-appb-000001
根据本发明的实施例，所述构建体具有如下所示的核苷酸序列，a)SEQ ID NO:2所示的核苷酸序列；b)与a)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同一性的核苷酸序列。
Figure PCTCN2021074620-appb-000002
Figure PCTCN2021074620-appb-000003
Figure PCTCN2021074620-appb-000004
在本发明的第二方面，本发明提出了一种溶瘤病毒。
根据本发明的实施例，所述溶瘤病毒携带前面所述的构建体。
现有技术中，相较于野生型病毒，改造后的病毒往往对肿瘤敏感性会明显降低，例如Mckie E A等的研究显示可降低100倍(Mckie E A,Maclean A R,Lewis A D,et al.Selective in vitro replication of herpes simplex virus type 1(HSV-1)ICP34.5 null mutants in primary human CNS tumours--evaluation of a potentially effective clinical therapy.[J].British Journal of Cancer,1996,74(5):745-752.)。
根据本发明实施例的溶瘤病毒，HSV1携带IL12，不仅保持了在肿瘤细胞中较强的复制能力和对肿瘤细胞较强的杀伤力，增强了病毒株对肿瘤细胞的靶向性，还可通过外源IL12基因表达IL12蛋白，激活机体抗肿瘤免疫反应。表达IL12的根据本发明实施例的溶瘤病毒，可通过瘤内注射的方式使病毒在肿瘤复制并在局部持续高表达IL12，避免通过静脉给药带来的全身性毒副作用。
根据本发明的实施例，所述溶瘤病毒可持续高表达IL12。
在本发明的第三方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括前面所述的构建体或前面所述的溶瘤病毒。根据本发明的实施例的药物组合物在低剂量就能起效，对于肿瘤细胞杀伤效果佳。
在本发明的第四方面，本发明提出了前面所述的构建体、前面所述的溶瘤病毒或前面所述的药物组合物在制备用于治疗或预防肿瘤的药物中的用途。
根据本发明的实施例，所述药物用于选择性杀伤肿瘤细胞。
根据本发明的实施例，所述肿瘤为胰腺癌、卵巢癌、咽鳞癌、肺癌、肝癌、黑色素瘤、食道癌、结肠癌。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是根据本发明实施例的pMD18-HOM-hIL12质粒图谱；
图2是根据本发明实施例的构建的重组病毒的PCR凝胶电泳检测图；
图3是根据本发明实施例的构建的重组病毒的测序结构图；
图4是根据本发明实施例的hIL12在Vero细胞中的表达结果图；
图5是根据本发明实施例的2k-hIL12、2K对不同肿瘤细胞的抑制曲线；
图6是根据本发明实施例的2k-hIL12、2K对不同肿瘤细胞的抑制曲线；
图7是本发明实施例的2k-hIL12、2K在不同肿瘤细胞中的增殖结果图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
需要说明的是，本申请所述的“免疫刺激因子”是指能调节免疫细胞功能的多肽，在本发明中，当使用本发明的溶瘤病毒来治疗肿瘤时，特别优选地，所述免疫刺激因子为能够用于肿瘤治疗的免疫刺激因子。“免疫刺激因子”的实例包括但不限于，细胞因子(如IL2、IL7、IL12、IL15、GM-CSF、IFNγ等)、趋化因子(如IL8)、生长因子(如IL5)。
本申请所述的“IL12”又称IL-12或白介素12或白细胞介素12，是一种具有免疫调节作用的细胞因子。各种癌症类型影响的早期研究揭示了其广泛的抗肿瘤潜力。其中“hIL12”是人白介素12。
本申请所述的“IL12基因编码框”是指能够表达功能性IL12的核酸序列，也 就说，该核酸序列所编码的IL12区段是能够实现该IL12功能的区段，或者说，该核酸序列所编码的IL12区段是IL12必要功能区。
本申请所述的“ICP47和ICP34.5被敲除”是指ICP47和ICP34.5基因被沉默，这两种基因的表达量相比于未被敲除ICP47和ICP34.5的HSV-1，显著降低。
本申请所述的“UL23基因失活”是指插入失活，也就是说在UL23基因位置插入外源基因后，UL23基因的原有功能丧失。
本申请所述的“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如启动子序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。
以下实施例利用CRISPR/Cas9系统构建重组HSV-1病毒，具体包括质粒构建，重组筛选，及病毒鉴定等。重组HSV-1病毒的生物学活性，具体包括hIL12的表达情况，病毒在肿瘤细胞上感染杀伤能力等。
实施例1构建重组单纯疱疹病毒
将编码hIL12基因序列插入至含UL23基因左右两侧同源臂序列之间，获得pMD18T-HOM-hIL12质粒，如图1，通过Crisper-Cas9系统在HSV-1/ICP47-/ICP34.5-双敲除KOS病毒株的UL23基因上的LacZ位点替换为治疗性基因hIL12(人类IL12)，构建重组病毒，并用X-gal蓝白斑筛选方法筛选构建成功的病毒。
需要说明的是，由于在病毒的保存和使用过程中，病毒在一些碱基位点会发生变异，本实施例中用到的KOS病毒株相较于野生型KOS病毒株，具有99％的核苷酸序列相似性。
以下实施例2～4对多轮纯化后单一病毒斑的HSV-1-hIL12进行PCR和ELISA验证，并评价HSV1-hIL12在不同肿瘤细胞的细胞毒性。
实施例2 PCR验证
(1)实验材料：
多轮筛选后的病毒单克隆2K-hIL12-lacZ11-3-1。
(2)PCR验证：
引物为B45577UL22F/B48431UL24R,PCR产物大小4051bp。
引物序列：
B45577UL22F GAGGCGGTTGGATTGTGCAAGAGAC
B48431UL24R ACGAGAACTGCGGTCGTTGTCCTAA
PCR体系：
成分 体积(uL)
5xPS GXL Buffer 10
Template(Virus gDNA) 1
Forward primer(B45577UL22F) 1
Reverse primer(B48431UL24R) 1
primeSTAR GXL 1
dNTP Mix 4
ddH 2O 32
Total 50
PCR程序：
98℃，3min；(98℃，10s；60℃，15s；68℃，3min)x 35个循环；72℃，5min；12℃，forever。
(3)测序结果与分析：PCR产物进行凝胶电泳检测，如图2所示，产物回收后进行测序，如图3所示，测序验证UL23HOM1以左222bp到UL23HOM2以右500bp序列完整无突变。
实施例3蛋白ELISA鉴定
(1)实验材料：
实验组HSV1-hIL12指代2K-hIL12-lacZ11-3-1，阴性对照为HSV-1/2K。
(2)ELISA验证：以Vero(非洲绿猴肾细胞)为宿主细胞，条件如下：HSV1-hIL12、HSV-1/2K感染MOI(病毒与细胞数量的比值)为0.01，分别在感染Vero细胞后，24、48、72小时收取培养病毒培养上清。
(3)hIL12表达结果：
hIL12在Vero细胞中的表达结果如表1所示。
表1：
Figure PCTCN2021074620-appb-000005
hIL12在Vero细胞中的表达结果如图4所示。
实施例4 2k-hIL12病毒细胞毒性实验
(1)实验材料：2k-hIL12(即前面所述的HSV1-hIL12，其指代2K-hIL12-lacZ11-3-1)，HSV-1/2K(简写为2K)，细胞为ASPC-1(胰腺癌细胞)；SKOV3(人卵巢癌细胞)；Fadu(人咽鳞癌细胞)；Hep3B2.1-7(肝癌细胞)；Skmel-28(黑色素瘤细胞)HepG2(人肝癌细胞)；HCT-116(结肠癌细胞)；SW620(结直肠癌细胞)；T.Tn(人食管鳞癌细胞)；NCI-H460(人大细胞肺癌)。
(2)细胞杀伤验证：以合适的细胞密度接种于96孔培养板，培养过夜后，分别加入7个梯度浓度(MOI＝10、5、1、0.5、0.1、0.05、0.01)的两种病毒，再分别培养24，48或72小时，依照CCK8试剂盒说明书进行细胞活力的检测。
(3)细胞杀伤结果：
2k-hIL12、2K对不同肿瘤细胞的毒性如表2所示。
2k-hIL12病毒在较低剂量对Hep3B2.1-7肝癌细胞、Skmel-28黑色素瘤细胞、ASPC-1人胰腺癌细胞、Fadu鼻咽癌细胞、Skov3人卵巢癌、HepG2人肝癌细胞、HCT-116结肠癌细胞、SW620结直肠癌细胞、T.Tn人食管鳞癌细胞、NCI-H460人大细胞肺癌细胞均具有良好的杀伤作用，48h时2k-hIL12病毒对Hep3B2.1-7肝癌细胞、ASPC-1肿瘤细胞、Fadu鼻咽癌细胞的杀伤力明显强于2K病毒。可以得出本发明所构建的重组溶瘤病毒2k-hIL12选择性地在某些肿瘤细胞上的敏感性有所提高，如图5和图6所示。
表2：
Figure PCTCN2021074620-appb-000006
Figure PCTCN2021074620-appb-000007
实施例4 2k-hIL12病毒在肿瘤细胞系上的复制能力
(1)实验材料：2k-hIL12(即前面所述的HSV1-hIL12)、HSV-1/2K(简写为2K)，细胞为Hep3B2.1-7肝癌细胞、Skmel-28黑色素瘤细胞、ASPC-1肿瘤细胞、Fadu鼻咽癌细胞、Skov3人卵巢癌细胞、NCI-H460大细胞肺癌细胞、HepG2肝癌细胞、HCT-116人结肠癌细胞和SW620人结肠癌细胞。
(2)细胞给药：取对数生长期上述细胞以合适的细胞密度接种于6孔培养板，培养过夜后，对6孔板细胞进行计数，根据每孔细胞数将病毒母液用含1％灭活FBS的高糖DMEM或RPMI-1640培养基稀释，配制成MOI＝0.1的病毒溶液。对应每孔依次加入300μl的病毒溶液，置于37℃，5％CO2条件下孵育，每15min摇动培养板使得病毒更好的吸附细胞，1.25h后添加1ml的培养基。2h后弃掉培养基，再补充2ml的培养基，放置CO 2培养箱中分别孵育24h、48h或72h。
(3)病毒滴度测定：培养结束后，收取病毒液。冻融三次(-80℃、37℃)后，梯度稀释收取的病毒液，取300ul感染Vero细胞(6孔板)，每15min摇动培养板使得病毒更好的吸附细胞，1.25h后添加2ml的培养基。2h后弃掉培养基，添加300ul的DMEM完全培养基、3ml 2％甲基纤维素固定病毒。培养3-4天后，吸弃覆盖培养基，加入10％HCHO溶液，1mL/孔，固定20min。之后吸掉甲醛溶液，加入1％结晶紫染色液，500μL/孔，染色30min。最后倒掉染色液，自来水缓缓冲洗干净，吸水纸倒扣擦干，进行空斑计数，并计算病毒滴度。病毒滴度＝病毒稀释倍数*(1000/300)*空斑数。
(4)2k-hIL12病毒在肿瘤细胞的复制能力评价
2k-hIL12和2k病毒在不同肿瘤系细胞上的增殖结果如表3和图7所示。
表3：
Figure PCTCN2021074620-appb-000008
对于Hep3B2.1-7细胞，24h时，2K的病毒滴度与hILl2病毒滴度相差不大，48h时，从数据上看2k病毒滴度高于hILl2的病毒滴度，但48h时部分细胞已飘起来对后续滴度测定影响很大，滴度下降较大，参考24h时的数据，可以得出2k-hIL12的病毒滴度稍高于2k病毒滴度，复制增殖能力相当；且两种病毒48h、MOI＝0.1时就已完全感染，说明其感染能力很强。
对于Skmel-28细胞，24h时，2k病毒滴度高于2k-hIL12的病毒滴度，48h时Skmel-28细胞已完全感染，滴度增大，参考48h时的数据，可以得出两种病 毒滴度相差不大，增值复制能力相当；且两种病毒48h时就已完全感染，滴度都在10的7次方，说明其感染能力较强。
对于ASPC-1细胞，24h时，2K的病毒滴度与hILl2病毒滴度相差不大，48h时从数据上看2k-hIL12病毒滴度稍低于2k的病毒滴度，但48h时ASPC-1细胞已完全感染，部分细胞飘起来对后续滴度测定影响很大，滴度下降较大，两种病毒48h、MOI＝0.1时就已完全感染，说明其感染增殖能力很强。
对于Fadu细胞，24h时，2K的病毒滴度稍高于hILl2病毒滴度，48h时，hIL12的病毒滴度为1.00E+08，2K的病毒滴度低于hILl2病毒滴度，且两种病毒48h时就已完全感染，说明其感染能力较强。
对于Skov3细胞，24h时，2K的病毒滴度为7.65E+07，hIL12的病毒滴度为8.65E+07，48h时2K的病毒滴度为1.43E+08，hIL12的病毒滴度为2.11E+08，可以得出2k-hIL12病毒滴度稍高于2k病毒滴度，且病毒滴度达到10的8次方，说明其感染能力较强。
对于NCI-H460细胞，2k-hIL12病毒滴度稍高于2k病毒滴度，两种病毒在NCI-H460细胞上复制增殖能力相当，且两种病毒48h时就已完全感染，说明其感染能力很强。
对于HepG2细胞，24h时，2K的病毒滴度均值为9.88E+07，hIL12的病毒滴度均值为1.86E+07，2K的病毒滴度稍高于hILl2病毒滴度；48h时，2K的病毒滴度均值为6.62E+07，hIL12的病毒滴度均值为9.03E+07，从数据上看24h时2k-hIL12病毒滴度低于2k的病毒滴度，但48h时2k-hIL12又大于2k病毒滴度且与2k最大值相差不大，可以得出2k病毒在HepG2细胞上能够更快进行复制，但复制增殖能力2k-hIL12与2k相当；两种病毒48h、MOI＝0.1时就已完全感染，说明其感染能力较很强。
对于HCT-116细胞，48h时，HCT-116细胞已完全感染，2K的病毒滴度为4.70E+07，hIL12的病毒滴度为7.61E+07，从数据上看2k病毒滴度低于2k-hIL12的病毒滴度，得出在HCT-116细胞上2k-hIL12病毒复制增殖稍高于2k病毒；且两种病毒48h时就已完全感染，滴度都在10的7次方，说明感染能力较强。
对于SW620细胞，48h时，SW620细胞已完全感染，2K的病毒滴度均值为1.92E+07，hIL12的病毒滴度均值为1.45E+07，在SW620细胞上2k-hIL12病毒复制增殖稍低于2k病毒，复制增殖能力相当；两种病毒48h时就已完全感染，滴度都在10的7次方，说明感染能力较强。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (10)

  1. 一种构建体，其特征在于，所述构建体的载体来源于HSV,所述构建体携带免疫刺激因子基因编码框，并将免疫刺激因子基因编码框设置于UL23基因位置。
  2. 根据权利要求1所述的构建体，其特征在于，所述构建体的ICP47和双拷贝的ICP34.5基因被敲除。
  3. 根据权利要求1所述的构建体，其特征在于，免疫刺激因子基因编码框设置于UL23基因序列的第387-521位核苷酸之间位置。
  4. 根据权利要求1所述的构建体，其特征在于，所述免疫刺激因子基因编码框与启动子可操作地连接，所述启动子包括选自CMV、CAG、EF1α、RSV LTR、MTI的至少之一,所述免疫刺激因子选自GM-CSF、IL2、IL5、IL7、IL12、IL15、IL24和IL27的至少之一；
    任选地，所述免疫刺激因子为IL12。
  5. 根据权利要求4所述的构建体，其特征在于，IL12基因编码框具有如下所示的核苷酸序列，
    1)SEQ ID NO:1所示的核苷酸序列；
    2)与1)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同源性的核苷酸序列。
  6. 根据权利要求4所述的构建体，其特征在于，所述构建体具有如下所示的核苷酸序列，
    1)SEQ ID NO:2所示的核苷酸序列；
    2)与1)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同源性的核苷酸序列。
  7. 一种溶瘤病毒，其特征在于，携带权利要求1-6任一项所述的构建体。
  8. 根据权利要求7所述的敏感性提高的溶瘤病毒，其特征在于，所述病毒表达IL12。
  9. 一种药物组合物，其特征在于，包括权利要求1-6任一项所述的构建体或权利要求7-8任一项所述的溶瘤病毒。
  10. 权利要求1-6任一项所述的构建体、权利要求7-8任一项所述的溶瘤病毒或权利要求9所述的药物组合物在制备用于治疗或预防肿瘤的药物中的用途，任选地，所述药物用于选择性杀伤肿瘤细胞，所述肿瘤为胰腺癌、卵巢癌、咽鳞癌、肝癌、黑色素瘤、结肠癌。
PCT/CN2021/074620 2020-07-15 2021-02-01 构建体、溶瘤病毒及其应用 Ceased WO2022012028A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010680925.0A CN113943752A (zh) 2020-07-15 2020-07-15 构建体、敏感性提高的溶瘤病毒及其应用
CN202010680925.0 2020-07-15

Publications (1)

Publication Number Publication Date
WO2022012028A1 true WO2022012028A1 (zh) 2022-01-20

Family

ID=79325847

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/074620 Ceased WO2022012028A1 (zh) 2020-07-15 2021-02-01 构建体、溶瘤病毒及其应用

Country Status (2)

Country Link
CN (1) CN113943752A (zh)
WO (1) WO2022012028A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116555191B (zh) * 2023-05-26 2024-09-17 东北农业大学 编码白细胞介素24和粒细胞-巨噬细胞集落刺激因子的新城疫病毒在肿瘤治疗中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101724608A (zh) * 2008-10-10 2010-06-09 中国疾病预防控制中心病毒病预防控制所 用于重组aav2/8病毒载体生产的重组i型单纯疱疹病毒及其用途
CN101768574A (zh) * 2008-12-29 2010-07-07 北京五加和分子医学研究所有限公司 用于重组aav5/5病毒载体包装的重组单纯疱疹病毒及其用途
CN104877969A (zh) * 2014-12-14 2015-09-02 刘滨磊 重组溶瘤性ii型单纯疱疹病毒及其药物组合物
WO2019236931A1 (en) * 2018-06-08 2019-12-12 The Board Of Trustees Of University Of Illinois Recombinant herpes simplex virus for cancer immunotherapy

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0001475D0 (en) * 2000-01-21 2000-03-15 Neurovex Ltd Virus strains
CN107354136A (zh) * 2017-06-15 2017-11-17 杭州睿可特生物科技有限公司 重组单纯疱疹病毒及其制备方法和应用
CN108004216B (zh) * 2017-12-01 2019-03-05 北京市神经外科研究所 Tspo在治疗脑胶质瘤中的应用及重组单纯疱疹病毒及其制备方法和应用
CN110982794B (zh) * 2020-03-05 2020-06-16 北京唯源立康生物科技有限公司 一种修饰的单纯疱疹病毒
CN111635913B (zh) * 2020-06-16 2022-03-04 广东东阳光药业有限公司 构建体及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101724608A (zh) * 2008-10-10 2010-06-09 中国疾病预防控制中心病毒病预防控制所 用于重组aav2/8病毒载体生产的重组i型单纯疱疹病毒及其用途
CN101768574A (zh) * 2008-12-29 2010-07-07 北京五加和分子医学研究所有限公司 用于重组aav5/5病毒载体包装的重组单纯疱疹病毒及其用途
CN104877969A (zh) * 2014-12-14 2015-09-02 刘滨磊 重组溶瘤性ii型单纯疱疹病毒及其药物组合物
WO2019236931A1 (en) * 2018-06-08 2019-12-12 The Board Of Trustees Of University Of Illinois Recombinant herpes simplex virus for cancer immunotherapy

Also Published As

Publication number Publication date
CN113943752A (zh) 2022-01-18

Similar Documents

Publication Publication Date Title
TWI776858B (zh) 一種重組單純皰疹病毒及其用途
CN103614416B (zh) 一种携带人穿膜肽p53与GM-CSF基因的重组溶瘤腺病毒及其用途
Kirn et al. The emerging fields of suicide gene therapy and virotherapy
WO2005121343A1 (en) Construction of oncolytic adenovirus recombinant specifically expressing immune modulatory factor gm-csf in tumor cells and uses thereof
CN102260712B (zh) 溶肿瘤能力增强的B型人腺病毒Ad11突变体的构建和应用
Faghihkhorasani et al. The role of oncolytic virotherapy and viral oncogenes in the cancer stem cells: a review of virus in cancer stem cells
WO2020239040A1 (zh) 重组溶瘤病毒以及制备方法、应用和药物
JP2014523236A5 (zh)
CA2449013C (en) Viral mutants that selectively replicate in targeted human cancer cells
WO2021253733A1 (en) Construct and use thereof
CN105177045A (zh) 表达人白介素15的重组溶瘤腺病毒及其构建方法
WO2022037020A1 (zh) 构建体、溶瘤病毒及其应用
AU2002346084A1 (en) Viral mutants that selectively replicate in targeted human cancer cells
CN109868263A (zh) 重组单纯疱疹病毒及其制备方法和应用、重组载体的构建方法及其应用
WO2022012028A1 (zh) 构建体、溶瘤病毒及其应用
Gao et al. A systematic comparison of the anti-tumoural activity and toxicity of the three Adv-TKs
WO2022001080A1 (zh) 一种重组单纯疱疹病毒及其构建方法
US11951141B2 (en) Replication-enhanced oncolytic adenoviruses
Zhu et al. Targeting of a conditionally replicative adenovirus agent to human squamous cell carcinomas of the head and neck
US20220218845A1 (en) Virus and tumor therapeutic drug for specifically killing tumor cells
EP3594328A1 (en) Recombinant herpes simplex virus and use thereof
WO2024192681A1 (zh) 一种溶瘤病毒及其应用
CN115747174A (zh) 重组溶瘤病毒rVSVM51R-C在乙肝性肝癌中的应用
CN115806944A (zh) 重组溶瘤病毒rVSVM51R-S在乙肝性肝癌中的应用
CA2627638A1 (en) Conditionally replicating viruses and methods for cancer virotherapy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21842343

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 23.06.2023)

122 Ep: pct application non-entry in european phase

Ref document number: 21842343

Country of ref document: EP

Kind code of ref document: A1