WO2020135390A1 - 表达干扰素的溶瘤病毒及其应用 - Google Patents

表达干扰素的溶瘤病毒及其应用 Download PDF

Info

Publication number
WO2020135390A1
WO2020135390A1 PCT/CN2019/127782 CN2019127782W WO2020135390A1 WO 2020135390 A1 WO2020135390 A1 WO 2020135390A1 CN 2019127782 W CN2019127782 W CN 2019127782W WO 2020135390 A1 WO2020135390 A1 WO 2020135390A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
ifn
δ24bp
rad
tumor
Prior art date
Application number
PCT/CN2019/127782
Other languages
English (en)
French (fr)
Inventor
章康健
方先龙
顾锦法
刘新垣
Original Assignee
上海元宋生物技术有限公司
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 上海元宋生物技术有限公司 filed Critical 上海元宋生物技术有限公司
Priority to CN201980007573.8A priority Critical patent/CN112384614B/zh
Priority to CA3124877A priority patent/CA3124877A1/en
Priority to KR1020217023174A priority patent/KR102667433B1/ko
Priority to EP19902481.1A priority patent/EP3907281A4/en
Priority to AU2019412342A priority patent/AU2019412342B2/en
Priority to US17/418,759 priority patent/US20220064670A1/en
Priority to JP2021538122A priority patent/JP7378840B2/ja
Publication of WO2020135390A1 publication Critical patent/WO2020135390A1/zh

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
    • 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
    • A61K35/761—Adenovirus
    • 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
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • 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
    • C07K14/555—Interferons [IFN]
    • 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
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • 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
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta
    • 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
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10321—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
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10332—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
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10344—Chimeric viral vector comprising heterologous viral elements for production of another viral 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10345—Special targeting system for viral 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
    • 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
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16145—Special targeting system for viral 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • 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

  • the invention belongs to the technical field of tumor treatment using oncolytic viruses. Specifically, the invention relates to an oncolytic adenovirus expressing interferon, a preparation method and application thereof.
  • oncolytic viruses can replicate and lyse cells in tumor cells, thereby continuously killing tumor cells. Moreover, oncolytic viruses can also carry anti-cancer genes, etc., while using the virus to lyse cells, and exert the ability of genes to kill tumor cells, thereby improving the therapeutic effect.
  • the present invention first replaces the wild-type promoter of the E1A gene in the viral genome with the survivin promoter by improving the structure of the oncolytic adenovirus genome, and then replaces the 24 bases in the E1A gene responsible for encoding amino acids 122 to 129 of the E1A protein
  • the base pair (364th to 387bp) is deleted, making E1A unable to bind Rb protein, which cannot release E2F and promote the host cell to enter the cell division cycle.
  • the oncolytic adenovirus can only be selected in tumor cells with abnormal Rb copy.
  • the present invention introduces a nucleic acid sequence encoding an interferon (for example, a compound interferon) into the viral genome, especially the nucleic acid sequence shown in SEQ ID NO: 2, 3, or 4.
  • an interferon for example, a compound interferon
  • the present invention can effectively inhibit tumor growth at a location far from the injection site and can also effectively prevent tumor Recurrence, which has inestimable value for clinical application. Based on the above findings, the present invention has been completed.
  • the first aspect of the present invention provides an oncolytic virus comprising a nucleic acid sequence encoding an interferon.
  • the virus is an adenovirus.
  • the virus comprises the E1A gene driven by a survivin promoter, preferably the endogenous promoter of E1A in the viral genome is replaced by a survivin promoter; and/or
  • the E1A gene sequence is modified so that the activity of the E1A protein binding to the Rb protein is reduced or completely deleted.
  • the modification is to delete the base sequence encoding the amino acids 122-129 of the E1A protein in the E1A gene, for example, SEQ ID NO : The sequence from 364 to 387 bp in the sequence of 1 is deleted;
  • the interferon is interferon alpha, interferon beta, or a complex interferon (eg, dry interferon).
  • a complex interferon eg, dry interferon.
  • the nucleic acid sequence encoding the interferon is shown in SEQ ID NO: 2, 3, or 4. Show.
  • the nucleic acid sequence is operably linked to a promoter.
  • the promoter is a CMV promoter.
  • the oncolytic virus is an oncolytic virus with a deposit number of CCTCC NO: V201957, and the deposit information is as follows: depository unit: Chinese Type Culture Collection Center, address: Wuhan, China, deposit date: 2019 8 Deposit name: Recombinant human adenovirus type 5 rAd-IFN-1-SP-E1A ( ⁇ 24bp)-E1B, deposit number CCTCC NO: V201957; or
  • the oncolytic virus is an oncolytic virus with the deposit number CCTCC NO: V201958, and the deposit information is as follows: depository unit: Chinese Type Culture Collection Center, address: Wuhan, China, deposit date: August 27, 2019, deposit name: Recombinant human adenovirus type 5 rAd-IFN-2-SP-E1A ( ⁇ 24bp)-E1B, deposit number CCTCC NO: V201858; or
  • the oncolytic virus is an oncolytic virus with the deposit number CCTCC NO: V201871, and the deposit information is as follows: Depository unit: Chinese Type Culture Collection Center, address: Wuhan, China, deposit date: December 12, 2018, deposit name: Recombinant human adenovirus type 5 rAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1B, accession number CCTCC NO: V201871.
  • the second aspect of the present invention provides the use of the oncolytic virus in the first aspect in the preparation of a medicament for treating proliferative diseases.
  • the proliferative diseases are cancers, such as prostate cancer, breast cancer, and colorectal Cancer, lung cancer, liver cancer, melanoma, lymphoma, gastric cancer, esophageal cancer, ovarian cancer, head and neck squamous cell carcinoma, bladder cancer, glioma, cervical cancer, or kidney cancer.
  • the third aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically effective amount of the oncolytic virus of the first aspect, optionally further comprising a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is formulated for administration by intravenous, nebulized inhalation, infusion, or intratumoral route.
  • a fourth aspect of the present invention provides a method for treating proliferative diseases, which comprises administering the oncolytic virus of the first aspect or the pharmaceutical composition of the third aspect to a subject in need thereof
  • proliferative diseases are cancers, such as prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, melanoma, lymphoma, gastric cancer, esophageal cancer, ovarian cancer, head and neck Squamous cell carcinoma, bladder cancer, glioma, cervical cancer or kidney cancer.
  • a fifth aspect of the present invention provides a method for preventing or inhibiting cancer cell metastasis, which includes the oncolytic virus according to any one of claims 1 to 6 or the any one of claims 8 to 10
  • the pharmaceutical composition is administered to a subject in need, such as a mammal, preferably a human; the cancer such as prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, melanoma, lymphoma, gastric cancer, esophageal cancer, Ovarian cancer, head and neck squamous cell carcinoma, bladder cancer, glioma, cervical cancer or kidney cancer.
  • a sixth aspect of the present invention provides a method for preventing cancer recurrence, which comprises combining the oncolytic virus according to any one of claims 1 to 6 or the drug according to any one of claims 8 to 10 Administration to a subject in need, such as a mammal, preferably a human, the cancers such as prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, melanoma, lymphoma, gastric cancer, esophageal cancer, ovarian cancer, Squamous cell carcinoma of the head and neck, bladder cancer, glioma, cervical cancer or kidney cancer.
  • the cancers such as prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, melanoma, lymphoma, gastric cancer, esophageal cancer, ovarian cancer, Squamous cell carcinoma of the head and neck, bladder cancer, glioma, cervical cancer or kidney cancer.
  • the subject is administered about 10 8 to 10 12 vp (eg 1.5 ⁇ 10 10 vp by intravenous, nebulized inhalation, perfusion or intratumoral route)
  • the number of applications per course of treatment is 1-6 times (for example, 1 time, 2 times, 3 times, 4 times, 5 times or 6 times)
  • the administration may be every 1 day, 2 days , 3 days, 4 days, 5 days, 6 days, 7 days or more; or 1 time, 2 times, 3 times, 4 times, 5 times, 6 times or more within 1 day
  • the number of medication courses administered by the subject is 1-12 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) .
  • the recombinant oncolytic virus provided by the present invention has good safety and excellent tumor-suppressing effect, and the tumor-suppressing effect is significantly better than the existing clinical medicines sorafenib and gemcitabine.
  • the recombinant oncolytic virus provided by the present invention achieves unexpected synergistic effects.
  • the oncolytic virus of the present invention also exhibits the ability to inhibit tumor metastasis and recurrence, and thus has broad clinical application prospects.
  • Figure 1 shows a schematic representation of the structure of various oncolytic adenovirus genomes.
  • Figure 2 shows the in vitro expression map of the target protein carried by each oncolytic adenovirus of the present invention.
  • FIG. 3 shows the effect of each oncolytic adenovirus of the present invention on increasing the expression of MHC I.
  • FIG. 3A is the result in the SW780 cell line
  • FIG. 3B is the result in the MCF-7 cell line.
  • Fig. 4 shows the specific replication ability of each oncolytic adenovirus of the present invention in breast cancer cell line MDA-MB-231(A) and normal breast cell line MCF-10A.
  • Figure 5 shows a comparison of the inhibitory effects of recombinant oncolytic adenoviruses with different structures on tumor cells.
  • Figures 5A and 5B show the inhibition of hepatoma cell line Huh-7 and intestinal cancer cell line SW620 by replicative and non-replicative types, respectively. Comparison of effects;
  • Figures 5C and 5D respectively show whether the oncolytic viruses carrying the interferon sequence inhibit the breast cancer cell line MDA-MB-231 and the lung cancer cell line HCC827;
  • Figure 5E shows the recombinant oncolytic adenovirus to normal The killing effect of liver fibroblast cell line HLF.
  • Figure 6 shows the inhibitory effect of recombinant oncolytic adenovirus rAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1B on tumor growth in a nude mouse xenograft model.
  • Figure 6A shows the results of the SW620 nude mouse xenograft in vivo model
  • Figures 6B and C show the results of the MDA-MB-231 nude mouse xenograft in vivo model
  • Figures 6D and E show the HCC827 nude mouse xenograft in vivo model the result of.
  • Fig. 7 shows the combination of the recombinant oncolytic adenovirus of the present invention with a blank control, rIFN protein alone, empty virus alone, and rIFN protein combined with empty virus in a nude mouse xenograft model of breast cancer cell line HCC1806 Comparison of the protocol, in which Figure 7A shows the dosing regimen, and Figure B shows the volume of the transplanted tumor in each experimental group and control group.
  • Fig. 8 shows the results of the in vivo efficacy of each recombinant oncolytic adenovirus of the present invention
  • Fig. 8A shows the change in tumor volume on the injection side
  • Fig. 8B shows the change in tumor volume on the non-injection side.
  • Fig. 9 shows the effect of recombinant oncolytic adenovirus rAd-IFN-3-SP-E1A (?24bp)-E1B to prevent tumor recurrence.
  • 9A shows the effect of re-implantation of HCC827 cells in mice that subsided after injection of rAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1B in the HCC827 nude mouse xenograft in vivo model, compared with the control (PBS) group Compare.
  • Figure 9B shows the data of tumor changes of different mice in the experimental group;
  • Figure 5C is the statistical comparison of the final tumor volume of the mice in the experimental group and the control group.
  • Fig. 10 shows the inhibitory effect of recombinant oncolytic adenovirus rAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1B on tumor growth in a humanized immune system mouse xenograft model.
  • 10A and B show the inhibitory effect on the tumor on the administration side (right side)
  • FIG. 10C and D show the inhibitory effect on the tumor on the non-administration side (left side).
  • Figure 11 shows the inhibitory effect of recombinant oncolytic adenovirus rAd-IFN-1-SP-E1A ( ⁇ 24bp)-E1B on tumor growth in a nude mouse PDX model.
  • Figure 11A shows the inhibitory effect of the recombinant oncolytic adenovirus on lung squamous cell carcinoma PDX
  • Figure 11B shows the inhibitory effect of the recombinant oncolytic adenovirus on lung adenocarcinoma PDX
  • Figure 11C shows the recombinant oncolytic adenovirus on Inhibitory effect of PDX in triple negative breast cancer.
  • oncolytic virus refers to a virus that can selectively replicate in cancer or cells with excessive proliferation, thereby slowing its growth or dying, while having no or little effect on normal cells.
  • exemplary oncolytic viruses include vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), herpes simplex virus (HSV), reovirus, measles virus, retrovirus, influenza virus, Simbis virus, Vaccinia virus, adenovirus, etc. (see, for example, Kirn et al., Nat. Med. 7:781). (2001); Coffey et al. Science 282: 1332 (1998); Lorence et al. Cancer Res. 54: 6017 (1994); and Peng et al. Blood 98: 2002 (2001)).
  • interferon refers to a family of secreted proteins produced by a variety of eukaryotic cells after exposure to various environmental stimuli, including viral infections or exposure to mitogens. In addition to its antiviral properties, interferon has also been shown to affect a variety of cellular functions. The three main interferons are IFN-a, IFN- ⁇ and IFN- ⁇ . Interferon was originally classified according to its cell source (leukocytes, fibroblasts, or T cells). Leukocyte interferon is currently called IFN- ⁇ , fibroblast interferon is IFN- ⁇ , and T cell interferon is IFN- ⁇ .
  • the term "consensus interferon” refers to a synthetic interferon whose amino acid sequence is a roughly average sequence of all known human interferon alpha subtypes. It has been reported that composite interferon has better (about 5 times) antiviral, antiproliferative and NK cell activation activity than any natural human IFN- ⁇ subtype.
  • An exemplary compound interferon such as ganfujin, see the sequences disclosed in US patents US4695623 and US4897471.
  • the present disclosure can use interferon sequences known in the prior art, and in a preferred embodiment, the present disclosure uses composite interferons.
  • interferon protein such as compound interferon
  • the oncolytic effect of the oncolytic virus in vivo will have no small difference.
  • this difference in effect is not related to the preference of codons in the organism, that is, this difference in effect is not caused by whether the most suitable codons for human expression are used.
  • using a codon suitable for prokaryotic expression for example, the IFN-3 sequence shown in SEQ ID NO: 4
  • the coding sequence of the composite interferon used in this disclosure is as follows:
  • pharmaceutical composition refers to a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient combined together to achieve a specific purpose.
  • the pharmaceutical composition includes a combination that is separated in time and/or space as long as it can work together to achieve the object of the present invention.
  • the ingredients contained in the pharmaceutical composition may be administered to the subject as a whole or separately.
  • the ingredients contained in the pharmaceutical composition may be administered to the subject simultaneously or sequentially.
  • the pharmaceutically acceptable carrier is water, a buffered aqueous solution, an isotonic saline solution such as PBS (phosphate buffer), glucose, mannitol, dextran, lactose, starch, magnesium stearate, cellulose , Magnesium carbonate, 0.3% glycerin, hyaluronic acid, or polyalkylene glycols such as polypropylene glycol, triglycerides, etc.
  • PBS phosphate buffer
  • glucose mannitol
  • dextran dextran
  • lactose starch
  • magnesium stearate cellulose
  • Magnesium carbonate 0.3% glycerin, hyaluronic acid
  • polyalkylene glycols such as polypropylene glycol, triglycerides, etc.
  • the type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intratumoral, perfusion, intradermal, subcutaneous, intramus
  • composition according to the present invention may contain lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, coloring substances, flavoring substances, and/or aromatic substances, etc. as additives.
  • administering means providing a substance, such as a pharmaceutical composition, to the subject in a pharmacologically usable manner.
  • the dose of the pharmaceutical composition provided to the subject refers to a dose sufficient to show its benefit to the administered subject, and may also be referred to herein as a "pharmaceutically effective amount” or “effective amount”.
  • the actual amount administered, as well as the rate and time course of administration will depend on the subject's own condition and severity.
  • the prescription of treatment (for example, the decision on dosage, etc.) is ultimately the responsibility of general practitioners and other doctors and depends on them to make decisions, usually considering the disease to be treated, the individual patient's condition, delivery site, method of administration, and Know other factors.
  • the pharmaceutical composition comprises 108 to 10 12 vp oncolytic viruses, e.g. 1 ⁇ 10 8, 1.5 ⁇ 10 8, 2 ⁇ 10 8, 2.5 ⁇ 10 8, 3 ⁇ 10 8, 3.5 ⁇ 10 8 , 4 ⁇ 10 8 , 4.5 ⁇ 10 8 , 5 ⁇ 10 8 , 5.5 ⁇ 10 8 , 6 ⁇ 10 8 , 6.5 ⁇ 10 8 , 7 ⁇ 10 8 , 7.5 ⁇ 10 8 , 8 ⁇ 10 8 , 8.5 ⁇ 10 8 , 9 ⁇ 10 8 , 9.5 ⁇ 10 8 , 1 ⁇ 10 9 , 1.5 ⁇ 10 9 , 2 ⁇ 10 9 , 2.5 ⁇ 10 9 , 3 ⁇ 10 9 , 3.5 ⁇ 10 9 , 4 ⁇ 10 9 , 4.5 ⁇ 10 9 , 5 ⁇ 10 9 , 5.5 ⁇ 10 9 , 6 ⁇ 10 9 , 6.5 ⁇ 10 9 , 7 ⁇ 10 9 , 7.5 ⁇ 10 9 , 8 ⁇ 10 9 , 8.5 ⁇ 10 9 , 9 ⁇ 10 9 , 9.5 ⁇ 10 9 , 1
  • a single administration of oncolytic virus to the subject a dose of 108 to 10 12 vp, for example, 1 ⁇ 10 8, 1.5 ⁇ 10 8, 2 ⁇ 10 8, 2.5 ⁇ 10 8, 3 ⁇ 10 8 , 3.5 ⁇ 10 8 , 4 ⁇ 10 8 , 4.5 ⁇ 10 8 , 5 ⁇ 10 8 , 5.5 ⁇ 10 8 , 6 ⁇ 10 8 , 6.5 ⁇ 10 8 , 7 ⁇ 10 8 , 7.5 ⁇ 10 8 , 8 ⁇ 10 8 , 8.5 ⁇ 10 8 , 9 ⁇ 10 8 , 9.5 ⁇ 10 8 , 1 ⁇ 10 9 , 1.5 ⁇ 10 9 , 2 ⁇ 10 9 , 2.5 ⁇ 10 9 , 3 ⁇ 10 9 , 3.5 ⁇ 10 9 , 4 ⁇ 10 9 , 4.5 ⁇ 10 9 , 5 ⁇ 10 9 , 5.5 ⁇ 10 9 , 6 ⁇ 10 9 , 6.5 ⁇ 10 9 , 7 ⁇ 10 9 , 7.5 ⁇ 10 9 , 8 ⁇ 10 9 , 8.5 ⁇ 10 9 , 9 ⁇ 10 9 , 9.5 ⁇
  • the number of applications for each course of treatment is 1-6 times, such as 1 time, 2 times, 3 times, 4 times, 5 times or 6 times
  • the interval between two administrations is 1-7 days, such as 1 day, 2 days, 3 Days, 4 days, 5 days, 6 days or 7 days.
  • the unit of virus dosage is vp (viral particles), which represents the number of virus particles contained in the virus solution, and is the virus particle titer.
  • subject means animals, including warm-blooded mammals, such as humans and primates; birds; domesticated domestic or farm animals, such as cats, dogs, sheep, goats, cattle, horses, and pigs ; Laboratory animals, such as mice, rats and guinea pigs; fish; reptiles; zoo animals and wild animals.
  • any components, elements, attributes, or steps disclosed with respect to one embodiment of the methods and products may be applied to any other methods and products disclosed herein.
  • rAd-IFN-1-SP-E1A ( ⁇ 24bp)-E1 uses the coding shown in SEQ IN NO: 2 Sequence; rAd-IFN-2-SP-E1A ( ⁇ 24bp)-E1 uses the coding sequence shown in SEQ ID NO: 3; rAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1 uses SEQ ID NO: 4 The coding sequence shown.
  • the following takes the construction process of rAd-IFN-3-SP-E1A( ⁇ 24bp)-E1 as an example to describe the construction process of recombinant oncolytic adenovirus, as long as rAd-IFN-3-SP-E1A( ⁇ 24bp)-E1
  • the coding sequence of the compound interferon was replaced with the other two sequences, that is, two other oncolytic adenovirus construction methods were obtained.
  • the digestion system is:
  • Double digestion of plasmid pShuttle (Shanghai Jiran Biotechnology Co., Ltd.) and adenovirus vector plasmid pXC2 gift from Academician Liu Xinyuan, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai)
  • the system is as follows:
  • the above reaction system was placed in a water bath at 37°C for 2h, and then subjected to agarose gel electrophoresis to separately recover the large fragment digested with pShuttle plasmid and the small fragment digested with pXC2 plasmid. Then use ligase to ligate the recovered product to construct pShuttle-E1A-E1B plasmid, in which the ligation reaction system is as follows:
  • the above reaction system was placed in a 16°C water bath and reacted for 2 hours.
  • the ligation product is the plasmid pShuttle-E1A-E1B.
  • the pShuttle-E1A-E1B plasmids were digested with NotI and XbaI to recover large fragments (vector fragments). Then use Ligation High Ligase (TOYOBO) to connect the digested nucleic acid fragment and the vector fragment, the connection system is:
  • the ligation product was pShuttle-IFN-3-SP-E1A( ⁇ 24bp)-E1B after 16 hours at 16°C water bath.
  • reaction system (1) Using PmeI enzyme to linearize pShuttle-IFN-3-SP-E1A( ⁇ 24bp)-E1B by single enzyme digestion, the reaction system is as follows:
  • Digest the pBHGE3 plasmid with HindIII (purchased from Jieyi Bio), use SpeI to digest the pAdEasy-1 plasmid (purchase from Shanghai Jiran Biotechnology Co., Ltd.), and then transfer the two digested plasmids to E. coli BJ5183 Homologous recombination was carried out to obtain a recombinant adenovirus skeleton plasmid carrying the E3 region; then, the recombinant adenovirus skeleton plasmid was transferred into E.
  • coli DH5 ⁇ for amplification to obtain an amplified recombinant adenovirus skeleton plasmid; then, the The recombinant adenovirus skeletal plasmid is transformed into E. coli BJ5183 competent state to obtain E. coli BJ5183 carrying the recombinant adenovirus skeletal plasmid, and then the E. coli is prepared into a competent state, thereby obtaining the BJ5183 competent cells used in step 1.
  • the recombinant plasmid pAd-IFN-3-SP-E1A( ⁇ 24bp)-E1B plasmid was transformed into E. coli DH5 ⁇ competent state. After plating, monoclonal colonies were picked out, and the plasmid was extracted after shaking culture and identified by enzyme digestion.
  • the digestion reaction system is as follows:
  • the above reaction system was placed in a 37°C water bath and reacted for 30 minutes, and then identified by electrophoresis.
  • HEK-293 cells were cultured in 6-well plates, and the cell density reached 60%-80% on the second day.
  • step (2) Following the instructions of Effectene Transfection Reagent Transfection Kit, take 1 ⁇ g of linearized recombinant plasmid pAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1B in step (2), and transfect it into step (1) to obtain Of HEK-293 cells plated in 6-well plates. After about 7-10 days, the cells were completely lesioned. At this time, the virus solution was collected and stored at -80°C until use.
  • the principle of virus titer determination is to determine the number of viruses with infectious activity according to the number of cells with positive hexon staining according to immunocytochemistry. According to the instructions on the adenovirus titer kit, the titer of small and purified adenovirus is determined .
  • MDA-MB-231 cells were cultured in 6-well plates, 5 ⁇ 10 5 cells per well, and cultured in the CO 2 incubator until the next day. Take the empty control virus rAd-SP-E1A( ⁇ 24bp)-E1B and recombinant oncolytic adenovirus rAd-IFN-1-SP-E1A( ⁇ 24bp)-E1B, rAd-IFN-2-SP-E1A( ⁇ 24bp)-E1B , RAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1B infects MDA-MB-231 cells cultured in six-well plates at the inoculation ratio of 10 MOI, each virus infects one well and continues in the CO 2 incubator Incubate for 24 hours. Then discard the supernatant and lyse the cells with 100ul of RIPA lysate per well, and collect the lysate as the test sample.
  • BCA protein quantification kit (purchased from Thermo) was used to measure the protein concentration of each sample collected in step (1).
  • the specific operation method please refer to the instruction manual of the kit.
  • RIPA lysate to adjust the concentration of each sample according to the measurement results to make the protein concentration consistent.
  • add an appropriate volume of 5 ⁇ SDS protein loading buffer (purchased from Biyuntian) to each sample so that the final concentration is 1 ⁇ , mix well, and cook in a metal bath at 100°C for 10 min.
  • the sample is directly used for western analysis or stored in the refrigerator at -20°C.
  • SW780 and MCF-7 cells were cultured in a 24-well plate, 1.5 ⁇ 10 5 cells per well, 500ul/well system, and cultured in the CO 2 incubator until the next day.
  • rAd-SP-E1A( ⁇ 24bp)-E1B and recombinant oncolytic adenovirus rAd-IFN-1-SP-E1A( ⁇ 24bp)-E1B, rAd-IFN-2-SP-E1A( ⁇ 24bp)-E1B , RAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1B infects the cancer cells cultured in the 24-well plate at the inoculation ratio of 1 MOI, the inoculation volume is 100ul/well.
  • a blank control group (PBS group) without virus was set for each cell. After treatment, the cells were shaken gently and cultured in a CO 2 incubator for 24 hours.
  • Breast cancer cell line MDA-MB-231 and normal breast cell line MCF-10A were cultured in 6-well plates, and plated at a density of 1.2 ⁇ 10 6 and 8 ⁇ 10 5 cells/well, 2 ml/well system, in CO 2 Incubate in the incubator until the next day.
  • the cells treated for 72h as described above were taken, and after sealing the parafilm around the 6-well plate, they were stored in a refrigerator at -80°C.
  • the freezing and thawing treatment was repeated three times between room temperature and -80°C, and after the third thawing, the cells were blown off with a pipette and mixed well with the culture medium.
  • the total volume of the sample in each well needs to be made up to 2ml with the culture medium, mixed thoroughly, and then divided into 3 parts (1 part is used for titer detection, the remaining 2 parts are reserved), Store in 80°C refrigerator.
  • the detection principle is to determine the number of viruses with infectious activity according to the number of cells with positive hexon staining according to immunocytochemical method.
  • the specific operation method is as follows:
  • mice When the experimental nude mice reached 6 weeks of age, 1 ⁇ 10 6 SW620 tumor cells were injected for tumor formation experiment.
  • the tumor size was found to be around 80-100 mm 3 and the health was good, the mice were randomly divided into 3 groups, namely PBS Group, rAd-IFN-3-SP-E1A( ⁇ 24bp)-E1B group and Ad-IFN-3-SP-E1A( ⁇ 24bp)-E1B group, all were injected intratumorally with a dose of 1.5 ⁇ 10 10 vp/ Only/times, every other day, a total of 4 injections. Observe and measure tumor size every 3 days.
  • mice When the nude mice reached 5 weeks of age, 2 ⁇ 10 6 MDA-MB-231 tumor cells were injected for tumor formation experiment. After measurement, when the tumor size was found to be around 80-100 mm 3 and the body was in good health, the mice were randomly divided into 3 Group, namely PBS group (intratumoral administration, once every other day, a total of 4 times), positive drug Sorafenib group (gavage, a single dose of 30mg/kg, once a day, a total of 14 days) and rAd-IFN -3-SP-E1A( ⁇ 24bp)-E1B group (intratumoral administration, the dose is 1.5 ⁇ 10 10 vp/pc/time, once every 1 day, 4 times in total), 8 mice per group, every other time Observe and measure tumor size at 3 days.
  • PBS group Intratumoral administration, once every other day, a total of 4 times
  • positive drug Sorafenib group gavage, a single dose of 30mg
  • mice When the experimental nude mice reached 5 weeks of age, 2 ⁇ 10 6 HCC827 tumor cells were injected for tumor formation experiment.
  • mice When the experimental nude mice reached 5 weeks of age, 2 ⁇ 10 6 HCC1806 tumor cells were injected for tumor formation experiment.
  • This model was commissioned by Shanghai Lidi Biotechnology Co., Ltd. Cut the tumor mass of one case of triple-negative breast cancer, one case of lung squamous cell carcinoma and one case of lung adenocarcinoma PDX transplantation tumor into tumor tissues with a size of about 3mm ⁇ 3mm ⁇ 3mm (about 50-90mg) and inoculate them It was subcutaneously at 5 weeks old nude mice. Observe the mice after inoculation and monitor the growth of the tumor. When the average tumor volume of the tumor-bearing mice reaches about 150 mm 3 , grouping and administration observation are performed. Each PDX model was randomly divided into 2 groups with 3 mice in each group.
  • Vehicle group Virus cryopreservation solution, intratumoral administration, once every 1 day, a total of 5 times
  • rAd-IFN-1-SP-E1A ( ⁇ 24bp)-E1B group intraatumoral administration Medicine, dose 1.5 ⁇ 10 10 vp/piece/time, once every other day, a total of 5 times. Observe and measure tumor size twice a week.
  • the subcultured HEK-293 cells were taken and expanded to 17 T175 (purchased from Corning) culture flasks using DMEM medium (containing 10% FBS), taking care to keep the cells in each flask evenly distributed. Before virus inoculation, randomly take 1 T175 culture flask, discard the original culture medium, add 5ml trypsin digestion and collect the cells, use appropriate cell counter to measure the cell concentration, and then calculate the T175 culture flask in this time The number of cells.
  • the recombinant oncolytic adenoviruses to be measured rAd-IFN-1-SP-E1A ( ⁇ 24bp)-E1B and rAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1B were subjected to VP detection (HPLC method) to obtain the virus Seed concentration.
  • the two oncolytic adenoviruses to be tested were inoculated with 4 T175 culture flasks at a virus/cell ratio of 400:1 and 800:1, respectively, and grouped and labeled, for a total of 16 T175 culture flasks.
  • each group harvested 2 T175 square bottles, and after 54h, each group harvested the remaining 2 T175 square bottles.
  • the structure and construction method of the recombinant oncolytic adenovirus carrying the compound interferon gene are as follows: On the basis of the wild-type adenovirus, the E1A protein encoding gene is responsible for encoding 24 base pairs of amino acids 122-129 of the E1A protein (p.364 To 387bp). After deleting the above segment, the E1A protein cannot bind to the Rb protein, and thus cannot release E2F and cause the host cell to enter the cell division cycle. The modified virus can only selectively replicate in tumor cells with abnormal Rb.
  • the tumor-specific Survivin promoter was used to replace the wild-type promoter of E1A, further enhancing the safety and targeting of adenovirus.
  • the depositing unit is the Chinese Type Culture Collection Center (located in Wuhan, China).
  • the specific deposit information is as follows:
  • MHC I major histocompatibility complex
  • MHC I The expression of MHC I was almost undetectable in the PBS group and the no-load virus rAd-SP-E1A ( ⁇ 24bp)-E1B group; all recombinant oncolytic adenoviruses capable of expressing rIFN were able to significantly increase MHC I expression. This indicates that rIFN expressed by recombinant oncolytic adenovirus has normal activity.
  • rAd-IFN-1-SP-E1A( ⁇ 24bp)-E1B and rAd-IFN-2-SP-E1A( ⁇ 24bp)-E1B can be more effective than rAd-IFN-3-SP-E1A( ⁇ 24bp)-E1B Significantly increased the expression of MHC I.
  • the same result was obtained in the human breast cancer cell line MCF-7 ( Figure 3B).
  • the replication ability of the virus is measured by "specific replication ability” and "replication targeting coefficient".
  • Specific replication ability is the ratio of the number of progeny viruses produced after the oncolytic adenovirus infects the host cell for a certain period of time and the number of originally infected viruses, which is used to quantitatively describe the replication ability of the oncolytic adenovirus in the host cell;
  • replication The "targeting coefficient” is the specific replication ability of oncolytic adenovirus in tumor cells divided by its specific replication ability in normal cells, and is used to quantitatively describe the tumor-targeted replication of oncolytic adenovirus.
  • each recombinant oncolytic adenovirus infects the host cell with 1 MOI, and the progeny virus is collected 72 hours after infection, and then the titer of the progeny virus is measured, and the specific replication power is obtained compared with the original infection titer.
  • the replication targeting coefficient is calculated from the specific replication power of each virus in tumor cells and normal cells.
  • rAd-IFN-1-SP-E1A ( ⁇ 24bp)-E1B although the specific replication in breast cancer cell line MDA-MB-231 is slightly lower than rAd-IFN-2-SP-E1A ( ⁇ 24bp)-E1B and rAd -IFN-3-SP-E1A( ⁇ 24bp)-E1B, but due to the significantly lower specific replication force in breast normal cells MCF-10A, so that rAd-IFN-1-SP-E1A( ⁇ 24bp)-E1B has The highest replication targeting coefficient is 23.36, much higher than the other two recombinant oncolytic adenoviruses. In other words, rAd-IFN-1-SP-E1A ( ⁇ 24bp)-E1B has the best tumor cell targeting and the highest safety.
  • Table 1 The replication targeting coefficient of each recombinant oncolytic adenovirus
  • Virus name Replication targeting coefficient rAd-IFN-1-SP-E1A( ⁇ 24bp)-E1B 23.36 rAd-IFN-2-SP-E1A( ⁇ 24bp)-E1B 8.15 rAd-IFN-3-SP-E1A( ⁇ 24bp)-E1B 9.64
  • Example 4 Recombinant oncolytic adenovirus can specifically kill cancer cells
  • Example 2 the recombinant oncolytic adenovirus rAd-IFN-3-SP-E1A( ⁇ 24bp)-E1B with moderate replication targeting coefficient verified in Example 2 was taken as an example to study the recombinant oncolytic adenovirus of the present invention for cancer The role of cells and ordinary cells.
  • this example further constructed a non-replicating adenovirus that deleted the E1 region, and inserted the IFN-3 expression frame into the non-replicating recombinant adenovirus Ad-IFN-3 carrying the IFN-3 gene. (see picture 1).
  • rAd-IFN-3-SP-E1A( ⁇ 24bp)-E1B and Ad-IFN-3 on liver cancer cell line Huh-7 were studied.
  • rAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1B has a stronger killing ability on liver cancer cells than non-replicating virus Ad-IFN-3.
  • MOI is 1
  • rAd-IFN-3- SP-E1A( ⁇ 24bp)-E1B can kill more than half of Huh-7 cells, which is better than Ad-IFN-3 when MOI is 10.
  • rAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1B has a significantly stronger ability to kill colon cancer cell line SW620 than non-replicating virus Ad-IFN-3.
  • MOI is 0.1
  • rAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1B can kill about half of SW620 cells, which is better than Ad-IFN-3 when MOI is 10.
  • this example further compares the killing effect of oncolytic adenoviruses carrying cancer cells with interferon sequences.
  • rAd-IFN-3-SP-E1A( ⁇ 24bp)-E1B kills breast cancer
  • the capacity of the cell line MDA-MB-231 is significantly stronger than that of the no-load control virus rAd-SP-E1A ( ⁇ 24bp)-E1B.
  • MOI is 0.5
  • rAd-IFN-3-SP-E1A( ⁇ 24bp)-E1B can kill about half of MDA-MB-231 cells, which is basically the same as rAd-SP-E1A( ⁇ 24bp)-E1B when MOI is 5. Consistent.
  • rAd-IFN-3-SP-E1A( ⁇ 24bp)-E1B can kill almost all HCC827 cells, which is stronger than rAd-SP-E1A( ⁇ 24bp)-E1B when MOI is 20.
  • the recombinant oncolytic adenovirus of the present invention can specifically kill tumor cells, but has little effect on normal cells, and has good safety.
  • Example 5 Recombinant oncolytic adenovirus significantly inhibits the growth of transplanted tumors in nude mice
  • the tumor inhibition of SW620 nude mouse xenograft model was tested by rAd-IFN-3-SP-E1A( ⁇ 24bp)-E1B.
  • the results are shown in Figure 6A.
  • the recombinant oncolytic adenovirus rAd-IFN-3-SP -E1A( ⁇ 24bp)-E1B also showed significantly better efficacy than the non-replicating adenovirus Ad-IFN-3 carrying the IFN-3 gene in the SW620 nude mouse xenograft in vivo model.
  • the effect of recombinant oncolytic adenovirus rAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1B in the HCC827 nude mouse xenograft in vivo model was tested.
  • the oncolytic adenovirus showed Excellent drug efficacy, significantly better than the positive control drugs sorafenib and gemcitabine, completely eliminated the transplanted tumor, and the tumor inhibition rate reached 100%.
  • Example 6 The inhibitory effect of recombinant oncolytic adenovirus on tumor showed a synergistic effect
  • a three-negative breast cancer cell line HCC1806 with less immune cell infiltration was selected for transplantation tumor in nude mice for in vivo drug efficacy test.
  • Each nude mouse was subcutaneously injected with 2 ⁇ 10 6 HCC1806 cells for subcutaneous tumor formation.
  • the dosing regimen is shown in Figure 7A.
  • Vehicle control and each recombinant oncolytic adenovirus are administered on the day of grouping.
  • Intratumoral injection is administered at a dose of 1.5 ⁇ 10 10 VP/time/piece (50 ⁇ L/time/piece ), once every other day, a total of five times.
  • Recombinant interferon protein was administered one day after grouping, and intraperitoneal injection was given. The dosage was 20 ⁇ g/time/piece, and it was given every other day for a total of five times.
  • the tumor size was measured every three days.
  • Example 7 Recombinant oncolytic adenovirus can achieve global inhibition of tumor cells in vivo
  • the three-negative breast cancer cell line MDA-MB-231 nude mouse xenograft model was selected, and at the same time, a recombinant oncolytic adenovirus rAd-IFN- ⁇ -SP-E1A ( ⁇ 24bp)-E1B with natural IFN- ⁇ was further constructed As a control of drug efficacy.
  • Each nude mouse developed bilateral subcutaneous tumors on the left and right sides, and injected 2 ⁇ 10 6 MDA-MB-231 cells on each side. When the left tumor volume reached about 90 mm 3 , it was randomly divided into groups and started the drug treatment. Vehicle control and each recombinant oncolytic adenovirus were administered on the day of grouping.
  • the left side of the tumor was administered by intratumoral injection.
  • the dose was 1.5 ⁇ 10 10 VP/time/piece (50 ⁇ L/time/piece), given every other day Give the medicine once and give it a total of five times.
  • the right side is not administered, and the left and right tumors are measured every three days.
  • each recombinant oncolytic adenovirus carrying the interferon gene can almost completely eliminate the transplanted tumor, and the empty oncolytic adenovirus also showed Significant inhibition of the growth of transplanted tumors.
  • Table 2 on the 11th day after administration (ie, Day 8 in FIG.
  • the unloaded oncolytic adenovirus has no tumor suppressing effect at all, and the recombinant oncolytic adenovirus rAd-IFN- ⁇ -SP-E1A ( ⁇ 24bp) carrying natural IFN- ⁇ )-E1B has a certain tumor suppressing effect.
  • the three recombinant oncolytic viruses constructed by the present invention rAd-IFN-1-SP-E1A( ⁇ 24bp)-E1B, rAd-IFN-2-SP -E1A( ⁇ 24bp)-E1B, rAd-IFN-3-SP-E1A( ⁇ 24bp)-E1B produced significantly better tumor suppressing effects, and obtained statistically significant differences (*). There is no obvious difference in drug efficacy between the above three recombinant oncolytic adenoviruses.
  • the recombinant oncolytic virus of the present invention has a global killing effect on tumor cells, and is not limited to the local injection site, and has inestimable value for clinical treatment of cancer, for example, for the treatment of tumor metastasis.
  • Example 8 recombinant oncolytic adenovirus can effectively prevent tumor recurrence
  • the HCC827 nude mouse xenograft tumor model in which the intratumoral injection of rAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1B completely eliminated the transplanted tumor was selected in Example 5.
  • Mice were injected unilaterally with 2 ⁇ 10 6 HCC827 tumor cells for tumor formation experiment.
  • untreated nude mice of the same week age were injected unilaterally with 2 ⁇ 10 6 HCC827 tumor cells for tumor formation experiment. After tumor formation, both groups were treated without any treatment, and the tumor volume was measured twice a week.
  • Example 9 Recombinant oncolytic adenovirus rAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1B significantly inhibits the growth of humanized mouse xenografts
  • this example tested the tumor suppressive ability of the recombinant oncolytic adenovirus of the present invention in a mouse transplanted tumor model of the humanized immune system (
  • rAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1B was selected as the test object).
  • Human HCC827 cells were injected subcutaneously on the back of mice with humanized immune system to form a transplanted tumor, but only the right tumor was administered with recombinant oncolytic adenovirus, and no treatment was performed on the left.
  • Fig. 10 The results are shown in Fig. 10.
  • the transplanted tumor on the right side (administration side) of the test group was injected with recombinant oncolytic adenovirus rAd-IFN-3-SP-E1A ( ⁇ 24bp)-E1B, and the volume began to gradually decrease.
  • the tumor inhibition rate reached 96.4%, which was significantly stronger than the positive control drugs gemcitabine and PD-1 antibody protein ( Figure 10A and B).
  • Example 10 Recombinant oncolytic adenovirus rAd-IFN-1-SP-E1A ( ⁇ 24bp)-E1B significantly inhibits the growth of PDX transplanted tumors in nude mice
  • this example was tested in a xenograft tumor model of clinical patient-derived transplanted tumor (PDX) in nude mice
  • PDX patient-derived transplanted tumor
  • the anti-tumor ability of the recombinant oncolytic adenovirus of the present invention (rAd-IFN-1-SP-E1A ( ⁇ 24bp)-E1B was selected as the test object in this example), a total of lung adenocarcinoma PDX, lung squamous cell carcinoma PDX and three One case of each PDX breast cancer was tested.
  • Recombinant oncolytic adenovirus is a drug that requires fermentation to be mass-produced.
  • the pilot-scale cell yield is not only a basis for judging whether it can be produced by fermentation, but also an important indicator of drug economy. Therefore, this example tested the cell yield of recombinant oncolytic adenovirus at the pilot scale level.
  • the engineering cell lines used for cell yield testing are HECC-293 cells of ATCC origin commonly used in the industry.
  • the virus to be tested is inoculated into a certain number of engineering cell lines at different inoculation ratios (VP/cell). Cells were lysed for 54 hours to collect progeny virus, and the VP count of the progeny virus was measured and divided by the number of cells inoculated at that time to obtain cell yield.
  • the recombinant oncolytic adenovirus rAd-IFN-1-SP-E1A ( ⁇ 24bp)-E1B reached the highest cell yield after 42 hours of inoculation, but the cell yield decreased 54 hours after inoculation, indicating that the recombinant oncolytic
  • the fermentation speed of adenovirus is faster, which means that in industrial production, fermentation can be completed in a shorter time, which can save costs; in comparison, rAd-IFN-3-SP-E1A( ⁇ 24bp)-E1B
  • the cell yield reached a peak 54 hours after inoculation, and the fermentation rate was lower than rAd-IFN-1-SP-E1A ( ⁇ 24bp)-E1B.
  • the peak yield of rAd-IFN-1-SP-E1A( ⁇ 24bp)-E1B at 42 hours is higher than the peak yield of rAd-IFN-3-SP-E1A( ⁇ 24bp)-E1B at 56 hours. It means that the production efficiency of the virus is also higher. Thus, in terms of cell yield, recombinant oncolytic adenovirus rAd-IFN-1-SP-E1A ( ⁇ 24bp)-E1B is more advantageous.

Landscapes

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

Abstract

提供一种表达干扰素序列的溶瘤病毒，特别提供了一种包含了表达干扰素的融合蛋白的溶瘤腺病毒，该溶瘤病毒能够在体外和体内高效抑制肿瘤。

Description

表达干扰素的溶瘤病毒及其应用
交叉引用
本申请要求发明名称为“表达干扰素的溶瘤病毒及其应用”于2018年12月26日提交到中国专利局的中国专利申请201811603052.2的优先权，其内容通过引用以整体并入本文。
技术领域
本发明属于利用溶瘤病毒进行肿瘤治疗的技术领域，具体的，本发明涉及一种表达干扰素的溶瘤腺病毒、其制备方法以及应用。
背景技术
据统计，全世界每年有超过1200万人诊断出癌症，癌症严重影响着全人类的健康和发展。受医疗和环境条件的影响，中国癌症死亡率高于全球平均水平。传统的肿瘤疗法存在疗效差、死亡率高及预后复发率高等缺点，因此对癌症的治疗提出了重大挑战。例如早在肿瘤发展的极早期，微转移就已经产生从而定位于远离肿瘤原发部位的组织中。因此在诊断发现癌症时，许多癌症患者已出现了微转移的现象。
作为一个新兴的具有广阔前景的疗法，溶瘤病毒能够在肿瘤细胞中复制并裂解细胞，从而持续杀死肿瘤细胞。而且溶瘤病毒中还能够携带抗癌基因等，在利用病毒裂解细胞的同时，发挥基因杀伤肿瘤细胞的能力，从而提高治疗效果。
目前仍然急需获得新的手段增强溶瘤病毒的效力，从而增加临床上获得成功的机会。
发明内容
本发明首先通过改进溶瘤腺病毒基因组的结构，将病毒基因组中的E1A基因的野生型启动子替换为survivin启动子，随后将E1A基因中负责编码E1A蛋白第122至129位氨基酸的24个碱基对(第364至387bp) 删除，使得E1A不能结合Rb蛋白，从而不能释放E2F并促使宿主细胞进入细胞分裂周期，经过上述改造后的溶瘤腺病毒仅能在Rb异常的肿瘤细胞中选择性复制。
进一步的，本发明在病毒基因组中引入编码干扰素(例如复合干扰素)的核酸序列，特别是如SEQ ID NO：2、3或4所示的核酸序列。结果发现这样的溶瘤腺病毒具有极佳的体外和体内抑瘤效果，特别令人惊讶的是，本发明的溶瘤腺病毒能够在远离注射部位的位置高效抑制肿瘤生长并且还能够有效防止肿瘤的复发，这对于临床应用具有不可估量的价值。正是基于上述发现，完成了本发明。
本发明的第一个方面提供了一种溶瘤病毒，其包含编码干扰素的核酸序列。
在一个实施方案中，所述病毒是腺病毒。
在另一个实施方案中，所述病毒包含以survivin启动子驱动的E1A基因，优选病毒基因组中E1A的内源启动子被survivin启动子所替换；和/或
E1A基因序列被修饰从而使E1A蛋白结合Rb蛋白的活性降低或完全缺失，优选的，所述修饰为将E1A基因中编码E1A蛋白第122-129位氨基酸的碱基序列删除，例如将SEQ ID NO：1的序列中第364至387bp的序列删除；
Figure PCTCN2019127782-appb-000001
Figure PCTCN2019127782-appb-000002
在另一个实施方案中，干扰素为α干扰素、β干扰素或复合干扰素(例如干复津)，优选的，编码所述干扰素的核酸序列如SEQ ID NO：2、3或4所示。
在另一个实施方案中，所述核酸序列与启动子可操作连接，优选的，所述启动子为CMV启动子。
在另一个实施方案中，所述溶瘤病毒为保藏编号为CCTCC NO：V201957的溶瘤病毒，保藏信息如下：保藏单位：中国典型培养物保藏中心，地址：中国武汉，保藏日期：2019年8月27日，保藏名称：重组人5型腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B，保藏编号为CCTCC NO：V201957；或者
所述溶瘤病毒为保藏编号为CCTCC NO：V201958的溶瘤病毒，保藏信息如下：保藏单位：中国典型培养物保藏中心，地址：中国武汉，保藏日期：2019年8月27日，保藏名称：重组人5型腺病毒rAd-IFN-2-SP-E1A(Δ24bp)-E1B，保藏编号为CCTCC NO：V201858；或者
所述溶瘤病毒为保藏编号为CCTCC NO：V201871的溶瘤病毒，保藏信息如下：保藏单位：中国典型培养物保藏中心，地址：中国武汉，保藏日期：2018年12月12日，保藏名称：重组人5型腺病毒rAd-IFN-3-SP-E1A(Δ24bp)-E1B，保藏编号为CCTCC NO：V201871。
本发明的第二个方面提供了第一个方面中的溶瘤病毒在制备治疗增生性疾病的药物中的用途，优选的，所述增生性疾病是癌症，例如前列腺癌、乳腺癌、结直肠癌、肺癌、肝癌、黑色素瘤、淋巴癌、胃癌、食管癌、 卵巢癌、头颈部鳞癌、膀胱癌、神经胶质瘤、宫颈癌或者肾癌。
本发明的第三个方面提供了一种药物组合物，其包含药物有效量的第一个方面中的溶瘤病毒，任选的还包含药学上可接受的载体。
在一个实施方案中，所述药物组合物被配制为通过静脉内、雾化吸入、灌注或者瘤内途径施用。
在另一个实施方案中，其包含约10 8至10 12vp(例如1.5×10 10vp)的所述溶瘤病毒。
本发明的第四个方面提供了一种治疗增生性疾病的方法，其包括将第一个方面中的溶瘤病毒或第三个方面中的药物组合物施予有需要的对象，所述对象例如哺乳动物，优选为人；更优选的，所述增生性疾病是癌症，例如前列腺癌、乳腺癌、结直肠癌、肺癌、肝癌、黑色素瘤、淋巴癌、胃癌、食管癌、卵巢癌、头颈部鳞癌、膀胱癌、神经胶质瘤、宫颈癌或者肾癌。
本发明的第五个方面提供了一种防止或抑制癌细胞转移的方法，其包括将权利要求1至6中任一项所述的溶瘤病毒或权利要求8至10中任一项所述的药物组合物施予有需要的对象，所述对象例如哺乳动物，优选为人；所述癌例如前列腺癌、乳腺癌、结直肠癌、肺癌、肝癌、黑色素瘤、淋巴癌、胃癌、食管癌、卵巢癌、头颈部鳞癌、膀胱癌、神经胶质瘤、宫颈癌或者肾癌。
本发明的第六个方面提供了一种防止癌症复发的方法，其包括将权利要求1至6中任一项所述的溶瘤病毒或权利要求8至10中任一项所述的药物组合物施予有需要的对象，所述对象例如哺乳动物，优选为人，所述癌症例如前列腺癌、乳腺癌、结直肠癌、肺癌、肝癌、黑色素瘤、淋巴癌、胃癌、食管癌、卵巢癌、头颈部鳞癌、膀胱癌、神经胶质瘤、宫颈癌或者肾癌。
其中，在上述第四至第六方面的一些具体实施方案中，其中通过静脉内、雾化吸入、灌注或者瘤内途径向所述对象施用约10 8至10 12vp(例如1.5×10 10vp)的所述溶瘤病毒，每个疗程施用次数为1-6次(例如1次、2次、3次、4次、5次或6次)，所述施用可以是每1天、2天、3天、4天、5天、6天、7天或更多天进行；或者是1天内施用1次、2次、3次、4次、5次、6次或更多次，向所述对象施用的用药疗程数为1-12个(例如1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、 12个)。
本发明所提供的重组溶瘤病毒，具有良好的安全性和优异的抑瘤效果，抑瘤效果显著优于现有临床用药索拉菲尼和吉西他滨。相比于单独施用干扰素蛋白和溶瘤病毒空载体，本发明提供的重组溶瘤病毒获得了预料不到的协同效果。此外，本发明的溶瘤病毒还展现出了抑制肿瘤转移和复发的能力，因而具有广阔的临床应用前景。
附图说明
图1显示了各种溶瘤腺病毒基因组的结构示意图。
图2显示了本发明各溶瘤腺病毒所携带目标蛋白的体外表达图。
图3显示了本发明各溶瘤腺病毒对MHC I表达的提高效果，其中图3A为SW780细胞系中的结果，图3B为MCF-7细胞系中的结果。
图4显示了本发明各溶瘤腺病毒在乳腺癌细胞系MDA-MB-231(A)和正常乳腺细胞系MCF-10A中的比复制力。
图5显示了具有不同结构的重组溶瘤腺病毒对肿瘤细胞的抑制效果比较，其中图5A和5B分别显示了复制型和非复制型对肝癌细胞系Huh-7和肠癌细胞系SW620的抑制效果比较；图5C和5D分别显示了是否携带干扰素序列的溶瘤病毒对乳腺癌细胞系MDA-MB-231和肺癌细胞系HCC827的抑制效果比较；图5E显示了重组溶瘤腺病毒对正常肝成纤维细胞系HLF的杀伤作用。
图6显示了重组溶瘤腺病毒rAd-IFN-3-SP-E1A(Δ24bp)-E1B在裸鼠移植瘤模型中对肿瘤生长的抑制作用。其中，图6A显示了SW620裸鼠移植瘤体内模型的结果，图6B和C显示了MDA-MB-231裸鼠移植瘤体内模型的结果；图6D和E显示了HCC827裸鼠移植瘤体内模型中的结果。
图7显示了在乳腺癌细胞系HCC1806裸鼠移植瘤模型中，本发明重组溶瘤腺病毒与空白对照、单用rIFN蛋白、单用空载病毒、以及rIFN蛋白联用和空载病毒联用的方案的比较，其中图7A显示了给药方案，图B显示了各实验组和对照组中移植瘤的体积。
图8显示了本发明各重组溶瘤腺病毒在体内的药效结果；图8A显示了注射侧的肿瘤体积变化；图8B显示了非注射侧的肿瘤体积变化。
图9显示了重组溶瘤腺病毒rAd-IFN-3-SP-E1A(Δ24bp)-E1B防止肿 瘤复发的效果。图9A显示了在HCC827裸鼠移植瘤体内模型中经过注射rAd-IFN-3-SP-E1A(Δ24bp)-E1B后消退的小鼠体内重新移植HCC827细胞后的效果，与对照(PBS)组的比较。图9B显示了实验组不同小鼠个体的肿瘤变化数据；图5C为实验组与对照组中小鼠最终肿瘤体积的统计学比较。
图10显示了重组溶瘤腺病毒rAd-IFN-3-SP-E1A(Δ24bp)-E1B在人源化免疫系统小鼠移植瘤模型中对肿瘤生长的抑制作用。其中图10A和B显示了对给药侧(右侧)肿瘤的抑制效果，图10C和D显示了对非给药侧(左侧)肿瘤的抑制效果。
图11显示了重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B在裸鼠PDX模型中对肿瘤生长的抑制作用。其中图11A显示了该重组溶瘤腺病毒对肺鳞癌PDX的抑制效果，图11B显示了该重组溶瘤腺病毒对肺腺癌PDX的抑制效果，图11C显示了该重组溶瘤腺病毒对三阴乳腺癌PDX的抑制效果。
具体实施方式
本申请所用术语具有与现有技术中该术语相同的含义。为了清楚地表明所用术语的含义，以下给出一些术语在本申请中的具体含义。当本文定义与该术语的常规含义有冲突时，以本文定义为准。
定义
除非另外定义，在此使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常所理解的相同的含义。所有在此提及的出版物引入本文作为参考，以用于描述和公开在所述出版物中所报道的细胞系、载体和方法，其可用于本发明。在此没有任何事物可被认为承认了，本发明没有位于依据在前发明的此类公开内容之前的权利。
术语“溶瘤病毒”是指能够在癌症或者具有过度增殖的细胞中选择性复制，从而减缓其生长或者使其死亡的病毒，同时对正常细胞没有影响或影响很小。示例性的溶瘤病毒包括水泡性口炎病毒(VSV)，新城疫病毒(NDV)，单纯疱疹病毒(HSV)，呼肠孤病毒，麻疹病毒，逆转录病毒，流感病毒，辛比斯病毒，痘苗病毒和腺病毒等(参见，例如，Kirn等，Nat.Med.7：781)。(2001)；Coffey等，Science 282：1332(1998)；Lorence等，Cancer Res.54：6017(1994)；和Peng等，Blood 98：2002(2001))。”
术语“干扰素”指由多种真核细胞在暴露于各种不同的环境刺激(包括病毒感染或者接触促细胞分裂原)后产生的分泌型蛋白质家族。除了具有抗病毒特性以外，干扰素还显示出影响多种细胞功能。三种主要的干扰素是IFN-a、IFN-β和IFN-γ。干扰素最初是根据其细胞来源(白细胞、纤维原细胞或T细胞)来分类的。白细胞干扰素目前称为IFN-α，纤维原细胞干扰素为IFN-β，T细胞干扰素为IFN-γ。
术语“复合干扰素(consensus interferon)”指一种合成干扰素,其氨基酸序列是所有已知人α干扰素亚型的大致平均的序列。已经报道了，复合干扰素具有比任何天然人IFN-α亚型更好(约5倍)的抗病毒、抗增殖和激活NK细胞的活性。一种示例性的复合干扰素例如干复津，参见美国专利US4695623和US4897471中所公开的序列。
本公开可以使用现有技术中已知的干扰素序列，在一个优选的实施方案中，本公开使用了复合干扰素。
申请人经过深入研究后发现，选择合适的核酸编码序列会影响干扰素蛋白在体内的治疗效果。针对同一个干扰素蛋白，例如复合干扰素，当溶瘤病毒载体中携带不同的干扰素核酸编码序列时，溶瘤病毒体内溶瘤效果会具有不小的差别。并且申请人发现这种效果的差异与生物体密码子的偏好性无关，即这种效果的差异并非由是否采用最适合于人体表达的密码子而导致的。在一个实施方案中，使用了适合原核表达的密码子(例如SEQ ID NO:4所示的IFN-3序列)，同样获得了优异的技术效果，获得了预料不到的技术效果。
本公开使用的复合干扰素的编码序列如下：
IFN-1:
Figure PCTCN2019127782-appb-000003
Figure PCTCN2019127782-appb-000004
IFN-2:
Figure PCTCN2019127782-appb-000005
IFN-3:
Figure PCTCN2019127782-appb-000006
Figure PCTCN2019127782-appb-000007
本文使用的术语“药物组合物”表示组合在一起以实现某种特定目的的至少一种药物以及任选地可药用载体或辅料的组合。在某些实施方案中，所述药物组合物包括在时间和/或空间上分开的组合，只要其能够共同作用以实现本发明的目的。例如，所述药物组合物中所含的成分可以以整体施用于对象，或者分开施用于对象。当所述药物组合物中所含的成分分开地施用于对象时，所述成分可以同时或依次施用于对象。优选地，所述药学上可接受的载体是水、缓冲水溶液、等渗盐溶液如PBS(磷酸盐缓冲液)、葡萄糖、甘露醇、右旋葡萄糖、乳糖、淀粉、硬脂酸镁、纤维素、碳酸镁、0.3％甘油、透明质酸、或聚亚烷基二醇如聚丙二醇、甘油三酯等。所用可药用载体的类型尤其依赖于根据本发明的组合物是否配制为用于经口、鼻、瘤内、灌注、皮内、皮下、肌内或静脉施用。根据本发明的组合物可包含润滑剂、防腐剂、稳定剂、湿润剂、乳化剂、影响渗透压的盐、缓冲剂、着色物质、矫味物质和/或芳香物质等作为添加剂。
“施与”或者“施用”意指以在药理学上可用的方式向对象提供物质，例如药物组合物。
向对象提供的药物组合物的剂量，是指足以显示其对于所施用对象产生益处的剂量，在本文中也可以被称为“药物有效量”或“有效量”。施用的实际量，以及施用的速率和时间过程会取决于所治疗者的自身情况和严重程度。治疗的处方(例如对剂量的决定等)最终是全科医生及其它医生的责任并依赖其做决定，通常考虑所治疗的疾病、患者个体的情况、递送部位、施用方法以及对于医生来说已知的其它因素。
在本发明的一个实施方案中，药物组合物包含10 8至10 12vp的溶瘤病毒，例如1×10 8、1.5×10 8、2×10 8、2.5×10 8、3×10 8、3.5×10 8、4×10 8、4.5×10 8、5×10 8、5.5×10 8、6×10 8、6.5×10 8、7×10 8、7.5×10 8、8×10 8、8.5×10 8、9×10 8、9.5×10 8、1×10 9、1.5×10 9、2×10 9、2.5×10 9、3×10 9、3.5×10 9、4×10 9、4.5×10 9、5×10 9、5.5×10 9、6×10 9、6.5×10 9、7×10 9、7.5×10 9、8×10 9、8.5×10 9、9×10 9、9.5×10 9、1×10 10、1.5×10 10、2×10 10、2.5×10 10、3×10 10、3.5×10 10、4×10 10、4.5×10 10、5×10 10、5.5×10 10、6×10 10、6.5×10 10、7×10 10、7.5×10 10、8×10 10、8.5×10 10、9×10 10、9.5×10 10、1×10 11、1.5×10 11、2×10 11、2.5×10 11、3×10 11、3.5×10 11、4×10 11、4.5×10 11、5×10 11、5.5×10 11、6×10 11、6.5×10 11、7×10 11、7.5×10 11、8×10 11、8.5×10 11、9×10 11、9.5×10 11或1×10 12vp；以及上述两点 间任意的剂量。
在另一个实施方案中，单次向所述对象施用溶瘤病毒的剂量为10 8至10 12vp，例如1×10 8、1.5×10 8、2×10 8、2.5×10 8、3×10 8、3.5×10 8、4×10 8、4.5×10 8、5×10 8、5.5×10 8、6×10 8、6.5×10 8、7×10 8、7.5×10 8、8×10 8、8.5×10 8、9×10 8、9.5×10 8、1×10 9、1.5×10 9、2×10 9、2.5×10 9、3×10 9、3.5×10 9、4×10 9、4.5×10 9、5×10 9、5.5×10 9、6×10 9、6.5×10 9、7×10 9、7.5×10 9、8×10 9、8.5×10 9、9×10 9、9.5×10 9、1×10 10、1.5×10 10、2×10 10、2.5×10 10、3×10 10、3.5×10 10、4×10 10、4.5×10 10、5×10 10、5.5×10 10、6×10 10、6.5×10 10、7×10 10、7.5×10 10、8×10 10、8.5×10 10、9×10 10、9.5×10 10、1×10 11、1.5×10 11、2×10 11、2.5×10 11、3×10 11、3.5×10 11、4×10 11、4.5×10 11、5×10 11、5.5×10 11、6×10 11、6.5×10 11、7×10 11、7.5×10 11、8×10 11、8.5×10 11、9×10 11、9.5×10 11或1×10 12vp；以及上述两点间任意的剂量。每个疗程的施用次数为1-6次，例如1次、2次、3次、4次、5次或6次，两次施用的间隔为1-7天，例如1天、2天、3天、4天、5天、6天或7天。
本发明中，病毒剂量的单位为vp(viral particle)，表示病毒溶液中所含有的病毒颗粒数，是病毒的颗粒滴度。
本文所使用的术语“对象”意指动物，包括温血哺乳动物，例如人和灵长类动物；鸟类；驯养的家养或农场动物，例如猫、狗、绵羊、山羊、牛、马和猪；实验室动物，例如小鼠、大鼠和豚鼠；鱼；爬行动物；动物园动物和野生动物等。
应注意，如果本公开提到一个特定的数值，至少会包括该数值，除非文章清楚的表明了其另有所指。当数值表示近似值，应理解为特定的数值形成了另一个实施方案。就如所使用的，“约X”(其中X是一个数值)是指±10％(包含)所列出值。如果存在，所有范围都是包括和可以组合的。
本文使用的术语例如“包含”、“含”、“含有”和“包括”不意在限制。此外，除非另有说明，“或”、“或者”意指“和/或”。
除非另有说明，任何关于本方法和产品一种实施方案公开的组分、元素、属性或者步骤可以应用于任何其他在此公开的方法和产品。
本公开的每项专利、专利申请、引用的出版物或者本文件中的描述以参考的方式整体并入文本中。
本发明在下面的实施例中进一步的定义。应了解这些实施例仅仅以举 例的方式来说明，并不旨在限制本发明的范围。从上面的讨论以及这些例子中，本领域技术人员可以确定本发明的本质特征，而且在不脱离其本质和范围的情况下，能够对本发明做出各方面的改变和修改来使之适应各种各样的用法和条件。
材料与方法
一、重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B、rAd-IFN-2-SP-E1A(Δ24bp)-E1B和rAd-IFN-3-SP-E1A(Δ24bp)-E1B的构建
上述三个病毒的构建过程类似，其区别仅在于构建中使用的复合干扰素的编码序列不同：rAd-IFN-1-SP-E1A(Δ24bp)-E1使用了SEQ IN NO：2所示的编码序列；rAd-IFN-2-SP-E1A(Δ24bp)-E1使用了SEQ ID NO：3所示的编码序列；rAd-IFN-3-SP-E1A(Δ24bp)-E1使用了SEQ ID NO：4所示的编码序列。下面以rAd-IFN-3-SP-E1A(Δ24bp)-E1的构建过程为例，详述重组溶瘤腺病毒的构建过程，只要将rAd-IFN-3-SP-E1A(Δ24bp)-E1中的复合干扰素的编码序列替换为其他两种序列，即得到另外两种溶瘤腺病毒的构建方法。
1、重组质粒pShuttle-IFN-3-SP-E1A(Δ24bp)-E1B的构建
委托基因合成公司合成5’端带有NotI酶切位点和3’端带有XbaI酶切位点的核苷酸序列(如SEQ ID NO：5所示)，其中复合干扰素使用了如SEQ ID NO:4所示的编码序列：
Figure PCTCN2019127782-appb-000008
Figure PCTCN2019127782-appb-000009
使用NotI、XbaI双酶切，回收酶切后核酸片段，酶切体系为：
Figure PCTCN2019127782-appb-000010
37℃水浴2小时。
构建pShuttle-E1A-E1B质粒
用Xho Ⅰ和Mfe Ⅰ双酶切质粒pShuttle(上海吉然生物科技有限公司)和腺病毒载体质粒pXC2(中国科学院上海生命科学研究院生物化学与细胞生物学研究所刘新垣院士惠赠)，酶切体系如下：
Xho Ⅰ和Mfe Ⅰ双酶切质粒pShuttle体系：
Figure PCTCN2019127782-appb-000011
Xho Ⅰ和Mfe Ⅰ酶切质粒pXC2体系：
Figure PCTCN2019127782-appb-000012
将上述反应体系置于37℃水浴2h，然后经琼脂糖凝胶电泳，分别回收pShuttle质粒酶切后的大片段和pXC2质粒酶切后的小片段。然后使用连接酶将上述回收产物连接，构建成pShuttle-E1A-E1B质粒，其中连接反应体系如下：
Figure PCTCN2019127782-appb-000013
将上述反应体系置于16℃水浴锅中反应2小时。连接产物即为质粒pShuttle-E1A-E1B。
使用NotI、XbaI双酶切pShuttle-E1A-E1B质粒，回收大片段(载体片段)。然后使用Ligation High连接酶(TOYOBO)将酶切后的核酸片段和载体片段连接起来，连接体系为：
Figure PCTCN2019127782-appb-000014
16℃水浴2小时，连接产物即为pShuttle-IFN-3-SP-E1A(Δ24bp)-E1B。
2、重组质粒pAd-IFN-3-SP-E1A(Δ24bp)-E1B的构建
(1)利用PmeI酶对pShuttle-IFN-3-SP-E1A(Δ24bp)-E1B进行单酶切线性化，反应体系如下：
Figure PCTCN2019127782-appb-000015
将上述反应体系置于37℃水浴锅中反应1小时，随后继续进行下一步去磷酸化实验。
(2)利用FastAp对步骤(1)中经过线性化的pShuttle-IFN-3-SP-E1A(Δ24bp)-E1B质粒进行去磷酸化处理，反应体系如下：
Figure PCTCN2019127782-appb-000016
将上述反应体系置于37℃水浴锅中1小时，随后进行下一步转化实验。
(3)在BJ5183感受态细胞中重组腺病毒骨架质粒与线性化的pShuttle-IFN-3-SP-E1A(Δ24bp)-E1B，从而获得重组质粒pAd-IFN-3-SP-E1A(Δ24bp)-E1B，具体步骤如下：
①取上步实验中的线性化的pShuttle-IFN-3-SP-E1A(Δ24bp)-E1B，转化BJ5183感受态细胞，涂板，挑菌，并摇菌过夜；
②使用质粒少量提取试剂盒对培养的菌液进行质粒提取。
其中，BJ5183感受态细胞的制备方法可参见中国专利申请CN201810651914.2，此处简要描述如下：
用HindIII酶切pBHGE3质粒(购自捷易生物)，使用SpeI酶切pAdEasy-1质粒(购自上海吉然生物科技有限公司)，然后将上述两种酶切后的质粒共转至大肠杆菌BJ5183中进行同源重组，获得携带E3区的重组腺病毒骨架质粒；接着，将该重组腺病毒骨架质粒转入大肠杆菌DH5α中扩增，获得扩增后的重组腺病毒骨架质粒；接着，将所述重组腺病毒骨架质粒转入大肠杆菌BJ5183感受态中，得到携带重组腺病毒骨架质粒的大肠杆菌BJ5183，再将该大肠杆菌制备成感受态，从而得到步骤①中使用的BJ5183感受态细胞。
(4)酶切鉴定。将重组质粒pAd-IFN-3-SP-E1A(Δ24bp)-E1B质粒转入大肠杆菌DH5α感受态中，涂板后挑出单克隆菌落，摇菌培养后提取质粒进行酶切鉴定。酶切反应体系如下：
Figure PCTCN2019127782-appb-000017
将上述反应体系置于37℃水浴锅中反应30min，然后电泳鉴定。
3、重组病毒rAd-IFN-3-SP-E1A(Δ24bp)-E1B的包装
(1)细胞铺板
在6孔板中培养HEK-293细胞，第2天细胞密度达到60％-80％。
(2)利用PacI酶切重组质粒pAd-IFN-3-SP-E1A(Δ24bp)-E1B进行线性化，反应体系如下：
Figure PCTCN2019127782-appb-000018
(3)质粒转染和病毒包装
按照Effectene Transfection Reagent转染试剂盒的说明书进行，取1μg步骤(2)中经过线性化的重组质粒pAd-IFN-3-SP-E1A(Δ24bp)-E1B，将其转染入步骤(1)得到的铺在6孔板中的HEK-293细胞。约7-10天后细胞完全病变，此时收集病毒液，于-80℃中保存备用。
4、重组病毒的滴度测定
病毒滴度测定的原理是依据免疫细胞化学法统计hexon染色阳性的细胞数来判定具有感染活性的病毒数量，根据腺病毒滴度试剂盒上的说明书对小扩和纯化后的腺病毒测定滴度。
二、重组溶瘤腺病毒的功能检测
1、目标蛋白表达水平的western检测
(1)细胞感染与样品收获
在6孔板中培养MDA-MB-231细胞，每孔5×10 5个细胞，于CO 2培养箱中培养至第二天。取空载对照病毒rAd-SP-E1A(Δ24bp)-E1B及重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B、rAd-IFN-2-SP-E1A(Δ24bp)-E1B、rAd-IFN-3-SP-E1A(Δ24bp)-E1B分别以10MOI的接种比例感染培养在六孔板中的MDA-MB-231细胞，每种病毒感染一孔，于CO 2培养箱中继续培养24小时。然后弃上清，每孔使用100ul RIPA裂解液裂解细胞，收集裂解液作为测试样品。
(2)蛋白样品预处理
使用BCA蛋白定量试剂盒(购自Thermo)测量(1)步收集的各样品的蛋白浓度，具体操作方法参见试剂盒的使用说明书。然后根据测量结果使用RIPA裂解液调整各样品浓度，使蛋白浓度一致。然后向各样品中加入适量体积的5×SDS蛋白上样缓冲液(购自碧云天)，使终浓度为1×， 充分混匀，于100℃金属浴煮10min。样品直接用于western分析或置于-20℃冰箱保存。
(3)Western分析
取预处理完毕的各蛋白样品，以每孔20ug总蛋白的上样量进行SDS-PAGE凝胶电泳，然后通过Biorad电转槽将凝胶中的样品转移至PVDF膜上。以5％脱脂牛奶封闭30min，然后加入稀释至使用浓度的rIFN抗体(委托华安生物制备)、β-tubulin抗体(购自康为世纪)，4℃摇床孵育过夜。弃一抗，PBST缓冲液洗涤3次，加入稀释至使用浓度的相应二抗(购自碧云天)，室温摇床孵育2h。弃二抗，PBST缓冲液洗涤3次，使用ECL发光显色试剂盒(购自翌圣)显色，具体操作参见试剂盒使用说明书。
2、干扰素调节MHC I表达检测。
(1)细胞铺板与病毒接种
在24孔板中培养SW780、MCF-7细胞，每孔1.5×10 5个细胞，500ul/孔体系，于CO 2培养箱中培养至第二天。取空载对照病毒rAd-SP-E1A(Δ24bp)-E1B及重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B、rAd-IFN-2-SP-E1A(Δ24bp)-E1B、rAd-IFN-3-SP-E1A(Δ24bp)-E1B分别以1MOI的接种比例感染培养在上述24孔板中的癌细胞，接种体积为100ul/孔。另外每种细胞均设置了不加病毒的空白对照组(PBS组)，处理完细胞后轻轻摇匀，于CO 2培养箱中继续培养24h。
(2)样品处理及流式细胞术分析
取上述处理了24h的细胞，弃上清，用1*PBS洗1遍后，以200ul/孔的消化液accutase加入样品孔中。待所有细胞收缩变圆，轻摇飘起后，加入800ul的1*PBS，反复吹散细胞5次，将细胞混合液转移至1.5ml EP管中。以1500r/min离心5min，弃上清，加1ml 1*PBS再洗涤离心一次。然后以60ul/样的抗体工作液(1ul抗体原液：240ul 1*PBS)对加入上述洗涤离心后的细胞中，混匀后室温避光染色30min，同时以未染色的细胞做阴性对照。染色结束后，用1*PBS洗涤离心一次，用2％PFA室温固定10min后进行流式细胞术分析(按仪器操作说明书操作)，上样体积为50ul/样。
3、比复制力测定
(1)细胞铺板与病毒接种
在6孔板中培养乳腺癌细胞系MDA-MB-231、正常乳腺细胞系MCF-10A，分别以1.2×10 6、8×10 5细胞/每孔的密度铺板，2ml/孔体系，于CO 2培养箱中培养至第二天。取重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B、rAd-IFN-2-SP-E1A(Δ24bp)-E1B、rAd-IFN-3-SP-E1A(Δ24bp)-E1B分别以1MOI的接种比例感染培养在上述6孔板中的细胞，每种病毒感染3个复孔，接种体积为100ul/孔，处理完细胞后轻轻摇匀，于CO 2培养箱中继续培养72h。
(2)样品收获、预处理及分装
取上述处理了72h的细胞，在6孔板四周封上封口膜后，存入-80℃冰箱。在室温与-80℃之间反复冻融3次处理，并在第3次融化后，用移液枪将细胞吹落，与培养基充分混匀。考虑到培养过程中的液体蒸发，最终需要使用培养基将每孔样品的总体积补至2ml，充分混匀，然后分装为3份(1份用于滴度检测，剩余2份备用)，存入80℃冰箱。
(3)样品滴度测定及复制倍数计算
使用Cell biolabs公司的QuickTiter TM Adenovirus Titer Immunoassay Kit对上述分装的样品选取1份进行滴度检测。检测原理是依据免疫细胞化学法统计hexon染色阳性的细胞数来判定具有感染活性的病毒数量，具体操作方法如下：
1)以2.5*10 5个细胞/孔的密度将293A细胞接种于24孔板中(1ml/孔)，待细胞贴壁(约24h)后每孔加入100ul梯度稀释后的待测定病毒。以不加病毒的孔作为空白对照，每个梯度做两个复孔，37℃，5％CO 2条件下继续培养细胞；
2)培养2天后，取出24孔板，在生物安全柜内吸弃上清，以500ul/孔的量加入-20℃预冷的甲醇，于-20℃冰箱中放置20min；
3)用1*PBS轻柔洗涤细胞3遍，用1％BSA封闭1h；
4)吸弃1％BSA，以250ul/孔的量加入预先稀释好的一抗anti-Hexon于室温下孵育1h；
5)用1*PBS轻柔洗涤细胞3遍，以250ul/孔的量加入预先稀释好的二抗(HRP)于室温下孵育1h；
6)用1*PBS轻柔洗涤细胞3遍，加入1*DAB工作液，室温染色15min；
7)用1*PBS轻柔洗涤细胞2遍，最后再以200ul/孔的量加入1*PBS避免细胞干掉；
8)用显微镜进行拍照，使用10倍物镜及10倍的目镜，每个孔分别选取9个点(上、下、左、右、左上、左下、右上、右下以及正中位置)拍摄照片；
9)计数对应稀释梯度下每个视野有多少病毒单位，以每个视野50个左右病毒单位的梯度为最佳计数梯度，若假设实际计数得到的各视野平均阳性点数为Y，则病毒滴度(ifu/ml)为：135.2×Y×稀释倍数。
每个样品的滴度值乘以样品总体积2ml，即为每个样品孔的总产量。最后计算出每个样品复制倍数，复制倍数＝总产量/起始接种量。
4、结晶紫法检测溶瘤病毒的安全性
将细胞铺于24孔板，一天后用适当MOI的待测重组腺病毒感染细胞，37℃继续培养4天后弃培养基，每孔加入500μL结晶紫染色液(2％结晶紫溶于20％甲醇溶液)，染色30min，在净水中将多余的染液洗净，晾干后拍照。
5、体内药效试验
SW620细胞裸鼠移植瘤模型
实验裸鼠达到6周龄，注射1×10 6个SW620肿瘤细胞进行成瘤实验，经测量后发现肿瘤大小在80-100mm 3左右且体质健康良好时，将小鼠随机分成3组，即PBS组、rAd-IFN-3-SP-E1A(Δ24bp)-E1B组和Ad-IFN-3-SP-E1A(Δ24bp)-E1B组，均采用瘤内注射的方式，剂量为1.5×10 10vp/只/次，每隔1天注射一次，共4次。每隔3天观察并测量肿瘤大小。
MDA-MB-231细胞裸鼠移植瘤模型
实验裸鼠达到5周龄，注射2×10 6个MDA-MB-231肿瘤细胞进行成瘤实验，经测量后发现肿瘤大小在80-100mm 3左右且体质健康良好时，将小鼠随机分成3组，即PBS组(瘤内给药，每隔1天1次，共4次)、阳性药Sorafenib组(灌胃，单次剂量30mg/kg，每天1次，共14天)和rAd-IFN-3-SP-E1A(Δ24bp)-E1B组(瘤内给药，剂量为为1.5×10 10vp/只/次，每隔1天1次，共4次)，每组8只，每隔3天观察并测量肿瘤大小。
HCC827细胞裸鼠移植瘤模型
实验裸鼠达到5周龄，注射2×10 6个HCC827肿瘤细胞进行成瘤实验，经测量后发现肿瘤大小在80-100mm 3左右且体质健康良好时，将小鼠随机分成4组，即PBS组(n＝8，瘤内给药，每隔1天1次，共4次)、阳性药Sorafenib组(n＝5，灌胃，剂量30mg/kg，每天1次，共14天)、阳性药Gemcitabine组(n＝5，尾静脉注射，剂量120mg/kg，每4天1次，共4次)rAd-IFN-3-SP-E1A(Δ24bp)-E1B组(n＝5，瘤内给药，剂量1.5×10 10vp/只/次，每隔1天1次，共4次)，每隔3天观察并测量肿瘤大小。
HCC1806细胞裸鼠移植瘤模型
实验裸鼠达到5周龄，注射2×10 6个HCC1806肿瘤细胞进行成瘤实验，经测量后发现肿瘤大小在80-100mm 3左右且体质健康良好时，将小鼠随机分成5组，即病毒保存液(Vehicle)组(n＝5，瘤内给药，每隔1天1次，共5次)、重组干扰素rIFN(20ug/只/次)组(n＝5，腹腔注射，每隔1天1次，共5次)，空载溶瘤病毒rAd-SP-E1A(Δ24bp)-E1B组(n＝5，瘤内给药，剂量1.5×10 10vp/只/次，每隔1天1次，共5次)，空载与重组干扰素联合用药rIFN+rAd-SP-E1A(Δ24bp)-E1B组(n＝5；空载病毒瘤内给药，剂量1.5×10 10vp/只/次，每隔1天1次，共5次；rIFN腹腔注射，每隔1天1次，共5次)，重组溶瘤病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B组(n＝5，瘤内给药，剂量1.5×10 10vp/只/次，每隔1天1次，共5次)，溶瘤病毒第一次给药为分组当天，而重组干扰素第一次给药为分组后第二天，溶瘤病毒给药后每3天观察一次并测量肿瘤大小。
HCC827细胞人源化免疫系统小鼠移植瘤模型
本模型委托澎立生物医药技术(上海)有限公司完成。由澎立生物前期制备的CD34 +人源化小鼠将被用于此实验，实验开始动物为无特定病原体(SPF)级别，周龄约22-23周，经鉴定免疫系统人源化构建成功(人CD45+细胞比例大于15％)后，每只小鼠左右两侧各注射2×10 6个HCC827肿瘤细胞进行成瘤实验，经测量后发现右侧肿瘤大小在80-100mm 3左右且体质健康良好时，将小鼠随机分成4组，即Vehicle组(病毒冻存液，n＝6，瘤内给药，每隔1天1次，共5次)、阳性药Gemcitabine组(
Figure PCTCN2019127782-appb-000019
吉西它滨，n＝6，尾静脉注射，剂量120mg/kg，每4天一次，共4次)、阳性对照PD-1抗体组(受赠于礼进生物医药科技(上海)有限公司，n＝6，腹腔注射，剂量10mg/kg，每周2次，共6次)和rAd-IFN-3-SP-E1A(Δ24bp)-E1B组(n＝6，瘤内给药，剂量1.5×10 10vp/只/次，每隔1天1次，共5次)， 两侧成瘤，仅右侧给药。每周观察并测量肿瘤大小2次。
PDX裸鼠移植瘤模型
本模型委托上海立迪生物技术股份有限公司完成。将1例三阴乳腺癌、1例肺鳞癌和1例肺腺癌PDX体内移植瘤的瘤块切成大小约为3mm×3mm×3mm(约50～90mg)的肿瘤组织，并将其接种于5周龄裸鼠皮下。观察接种后小鼠并监测肿瘤的生长，在荷瘤小鼠平均肿瘤体积达到约150mm 3时，即进行分组和给药观察。每例PDX模型均随机分成2组，每组3只小鼠。一组为Vehicle组(病毒冻存液，瘤内给药，每隔1天1次，共5次)，一组为rAd-IFN-1-SP-E1A(Δ24bp)-E1B组(瘤内给药，剂量1.5×10 10vp/只/次，每隔1天1次，共5次)。每周观察并测量肿瘤大小2次。
三、中试规模的细胞单产测试
取传代培养的HEK-293细胞，使用DMEM培养基(含10％FBS)扩大培养至17个T175(购自康宁)培养瓶，注意保持各瓶中细胞分配均匀。病毒接种前随机取1个T175培养瓶，弃原培养基，加入5ml胰酶消化后收集细胞，适当稀释后使用细胞计数仪测量细胞浓度，并由此计算出此时1个T175培养瓶中的细胞数。随后，取待测量的重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B、rAd-IFN-3-SP-E1A(Δ24bp)-E1B进行VP检测(HPLC法)，得到病毒种子浓度。两种待测溶瘤腺病毒分别以400：1、800：1的病毒/细胞比各接种4个T175培养瓶，做好分组标记，共计16个T175培养瓶。CO2培养箱中培养42h后每组收获2个T175方瓶，培养54h后每组收获剩下的2个T175方瓶。收获时，先收集瓶中原培养液作为上清样品，然后每瓶加入5ml病毒保存液，刮取所有细胞至15ml离心管中作为细胞样品。室温到-80℃反复冻融各样品3次，离心取上清2ml用于VP检测。根据上清样品和细胞样品中的VP检测结果求和得到每组总产量，然后除以接种时的细胞数，得到每组的单细胞产量。
实施例1携带复合干扰素基因的重组溶瘤腺病毒的构建
携带复合干扰素基因的重组溶瘤腺病毒结构和构建方法如下：在野生型腺病毒的基础上，将E1A蛋白编码基因中负责编码E1A蛋白122-129位氨基酸的24个碱基对(第364至387bp)删除。删除上述区段后，E1A 蛋白不能结合Rb蛋白，从而不能释放E2F并促使宿主细胞进入细胞分裂周期，经过该改造的病毒仅能在Rb异常的肿瘤细胞中选择性复制。使用肿瘤特异性的Survivin启动子替换了E1A的野生型启动子，进一步增强腺病毒的安全性和靶向性。将复合干扰素(干复津)蛋白的表达框序列(分别如SEQ ID NO:2、3和4所示)插入上述腺病毒载体，从而最终得到了获得携带复合干扰素基因的重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B(含有SEQ ID NO:2所示的表达框)、rAd-IFN-2-SP-E1A(Δ24bp)-E1B(含有SEQ ID NO:3所示的表达框)和rAd-IFN-3-SP-E1A(Δ24bp)-E1B(含有SEQ ID NO:4所示的表达框)。
随后将以上三个病毒进行保藏，保藏单位是中国典型培养物保藏中心(地点中国武汉)，具体保藏信息如下：
1)保藏日期：2018年12月12日，保藏名称：重组人5型腺病毒rAd-IFN-3-SP-E1A(Δ24bp)-E1B，保藏编号为CCTCC NO：V201871；
2)保藏日期：2019年08月26日，保藏名称：重组人5型腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B，保藏编号为CCTCC NO：V201957；
3)保藏日期：2019年08月26日，保藏名称：重组人5型腺病毒rAd-IFN-2-SP-E1A(Δ24bp)-E1B，保藏编号为CCTCC NO：V201958。
此外，作为对照，还构建了不带有rIFN表达框的空载病毒rAd-SP-E1A(Δ24bp)-E1B。上述病毒构建体的结构如图1所示。
实施例2重组溶瘤腺病毒的目标蛋白表达水平和目标蛋白功能测试
为了比较实施例1中构建的携带不同复合干扰素表达框的重组溶瘤腺病毒外源治疗基因表达能力，即表达复合干扰素rIFN蛋白的能力，分别取rAd-IFN-1-SP-E1A(Δ24bp)-E1B、rAd-IFN-2-SP-E1A(Δ24bp)-E1B和rAd-IFN-3-SP-E1A(Δ24bp)-E1B，以10MOT感染人乳腺癌细胞系MDA-MB-231，感染24小时后裂解细胞收集蛋白样品，进行Western Blotting分析。
结果如图2所示，所有携带rIFN编码序列的重组溶瘤病毒均能够表达出目标蛋白rIFN，而空载病毒则并没有可检测的蛋白条带，这表明实施例1构建的重组溶瘤病毒能够在细胞内正常的发挥功能。此外，从图2中还可以看出重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B表达外源治疗基因rIFN的水平最高，其次为rAd-IFN-2-SP-E1A(Δ24bp)-E1B， 而rAd-IFN-3-SP-E1A(Δ24bp)-E1B最低。这表明了目标蛋白编码序列的不同会影响目标蛋白的表达量。
接下来检测了上述表达的rIFN蛋白是否具有正常的功能。由于干扰素能够上调I型主要组织相容性复合物(MHC I，又称HLA-ABC)的表达。因此接下来以MHC I的表达量变化为标准，比较了各重组溶瘤腺病毒所表达的外源重组干扰素rIFN的功能。
取重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B、rAd-IFN-2-SP-E1A(Δ24bp)-E1B和rAd-IFN-3-SP-E1A(Δ24bp)-E1B，分别以1MOT感染人膀胱癌细胞系SW780，感染24小时后收集细胞，经染色后利用流式细胞仪分析。结果如图3A所示，PBS组和空载病毒rAd-SP-E1A(Δ24bp)-E1B组几乎检测不到MHC I的表达；而所有能够表达rIFN的重组溶瘤腺病毒均能够明显上调MHC I的表达。这表明重组溶瘤腺病毒表达得到的rIFN具有正常的活性。其中，rAd-IFN-1-SP-E1A(Δ24bp)-E1B与rAd-IFN-2-SP-E1A(Δ24bp)-E1B相比于rAd-IFN-3-SP-E1A(Δ24bp)-E1B能够更显著的上调MHC I的表达。在人乳腺癌细胞系MCF-7中得到了同样的结果(图3B)。
实施例3重组溶瘤腺病毒的肿瘤靶向性测试
如实施例1所述，三个重组溶瘤腺病毒均经过了复制靶向性改造，从而仅在肿瘤细胞中复制。而是否能够有效的靶向肿瘤细胞进行复制，则与溶瘤病毒的实际临床治疗效果密切相关。本实施例则测试了各重组溶瘤腺病毒在乳腺癌细胞系MDA-MB-231以及正常乳腺细胞MCF-10A中的复制能力。
本实施例中，病毒的复制能力以“比复制力”和“复制靶向系数”进行测量。“比复制力”为溶瘤腺病毒感染宿主细胞一定时间后，产生的子代病毒数与原始感染的病毒数之比，用于定量描述溶瘤腺病毒在宿主细胞中的复制能力；“复制靶向系数”为溶瘤腺病毒在肿瘤细胞中的比复制力除以其在正常细胞中的比复制力，用于定量描述溶瘤腺病毒的肿瘤靶向性复制。
具体地，各重组溶瘤腺病毒均以1 MOI感染宿主细胞，感染72小时后收集子代病毒，然后测量子代病毒的滴度，与原始感染滴度相比得到比复制力。再根据复制靶向系数的定义，由各病毒在肿瘤细胞和正常细胞中 的比复制力计算复制靶向系数。
结果如表1和图4所示，这三个重组溶瘤腺病毒均能够更多的在癌细胞系MDA-MB-231中进行复制，比复制力均较高，其中rAd-IFN-2-SP-E1A(Δ24bp)-E1B和rAd-IFN-3-SP-E1A(Δ24bp)-E1B略高于rAd-IFN-1-SP-E1A(Δ24bp)-E1B；而在正常细胞系MCF-10A中的比复制力均较低，其中rAd-IFN-1-SP-E1A(Δ24bp)-E1B的比复制力最低。以上结果表明这三个重组溶瘤腺病毒在正常细胞中的复制能力较弱。而rAd-IFN-1-SP-E1A(Δ24bp)-E1B虽然在乳腺癌细胞系MDA-MB-231中的比复制力稍微低于rAd-IFN-2-SP-E1A(Δ24bp)-E1B和rAd-IFN-3-SP-E1A(Δ24bp)-E1B，但由于在乳腺正常细胞MCF-10A中具有显著更低的比复制力，从而使得rAd-IFN-1-SP-E1A(Δ24bp)-E1B具有最高的复制靶向系数23.36，远高于另外两个重组溶瘤腺病毒。也就是说rAd-IFN-1-SP-E1A(Δ24bp)-E1B的肿瘤细胞靶向性最好，具有最高的安全性。
表1各重组溶瘤腺病毒的复制靶向系数
病毒名称 复制靶向系数
rAd-IFN-1-SP-E1A(Δ24bp)-E1B 23.36
rAd-IFN-2-SP-E1A(Δ24bp)-E1B 8.15
rAd-IFN-3-SP-E1A(Δ24bp)-E1B 9.64
实施例4重组溶瘤腺病毒能够特异性的杀伤癌细胞
本实施例以实施例2中验证的具有中等复制靶向系数的重组溶瘤腺病毒rAd-IFN-3-SP-E1A(Δ24bp)-E1B为例，研究了本发明重组溶瘤腺病毒对于癌细胞和普通细胞的作用。作为对照，本实施例进一步构建了删除E1区的非复制型腺病毒，同时在该载体上插入IFN-3表达框所得到的携带IFN-3基因的非复制型重组腺病毒Ad-IFN-3(见图1)。
首先研究了rAd-IFN-3-SP-E1A(Δ24bp)-E1B和Ad-IFN-3分别对肝癌细胞系Huh-7的杀伤作用。如图5A所示，rAd-IFN-3-SP-E1A(Δ24bp)-E1B 对肝癌细胞的杀伤能力显著强于非复制型病毒Ad-IFN-3，当MOI为1时rAd-IFN-3-SP-E1A(Δ24bp)-E1B即可杀伤一半以上的Huh-7细胞，优于MOI为10时的Ad-IFN-3。
接下来，在结肠癌细胞系SW620中，比较了复制型和非复制型溶瘤腺病毒的杀伤作用。如图5B所示，rAd-IFN-3-SP-E1A(Δ24bp)-E1B杀伤结肠癌细胞系SW620的能力显著强于非复制型病毒Ad-IFN-3。当MOI为0.1时rAd-IFN-3-SP-E1A(Δ24bp)-E1B即可杀伤一半左右的SW620细胞，优于MOI为10时的Ad-IFN-3。
接下来，本实施例进一步比较了溶瘤腺病毒是否携带干扰素序列时，对癌细胞的杀伤作用，如图5C所示，rAd-IFN-3-SP-E1A(Δ24bp)-E1B杀伤乳腺癌细胞系MDA-MB-231的能力显著强于空载对照病毒rAd-SP-E1A(Δ24bp)-E1B。当MOI为0.5时，rAd-IFN-3-SP-E1A(Δ24bp)-E1B即可杀伤一半左右的MDA-MB-231细胞，与MOI为5时的rAd-SP-E1A(Δ24bp)-E1B基本一致。
在肺癌细胞系HCC827中，比较了rAd-IFN-3-SP-E1A(Δ24bp)-E1B和空载对照病毒rAd-SP-E1A(Δ24bp)-E1B的杀伤作用。如图5D所示,结果表明rAd-IFN-3-SP-E1A(Δ24bp)-E1B杀伤肺癌细胞系HCC827的能力显著强于空载对照病毒rAd-SP-E1A(Δ24bp)-E1B。当MOI为5时rAd-IFN-3-SP-E1A(Δ24bp)-E1B即可几乎杀伤全部的HCC827细胞，强于MOI为20时的rAd-SP-E1A(Δ24bp)-E1B的效果。
相反，对于正常细胞人肝成纤维细胞系HLF，如图5E所示，重组溶瘤腺病毒rAd-IFN-3-SP-E1A(Δ24bp)-E1B与相应空载对照病毒均不杀伤HLF细胞。
以上结果表明了本发明的重组溶瘤腺病毒能够特异性的杀伤肿瘤细胞，而对正常细胞几乎没有影响，具有良好的安全性。
实施例5重组溶瘤腺病毒显著抑制裸鼠移植瘤的生长
本实施例测试了rAd-IFN-3-SP-E1A(Δ24bp)-E1B对SW620裸鼠移植瘤模型的肿瘤抑制情况，结果如图6A所示，重组溶瘤腺病毒rAd-IFN-3-SP-E1A(Δ24bp)-E1B在SW620裸鼠移植瘤体内模型中也显示出显著好于携带IFN-3基因的非复制型腺病毒Ad-IFN-3的药效。
进一步测试了重组溶瘤腺病毒rAd-IFN-3-SP-E1A(Δ24bp)-E1B在 MDA-MB-231裸鼠移植瘤体内模型的效果，如图6B和C所示，该重组溶瘤腺病毒显示出极好的药效，几乎全部消灭了移植瘤，抑瘤率高达96.4％。
随后测试了重组溶瘤腺病毒rAd-IFN-3-SP-E1A(Δ24bp)-E1B在HCC827裸鼠移植瘤体内模型中的效果，如图6D和E所示，该溶瘤腺病毒表现出了极好的药效，显著好于阳性对照药索拉菲尼和吉西他滨，完全消灭了移植瘤，抑瘤率达到了100％。
实施例6重组溶瘤腺病毒对肿瘤的抑制作用表现出协同效应
本实施例验证了本发明的重组溶瘤腺病毒(rAd-IFN-1-SP-E1A(Δ24bp)-E1B)，能够获得比单独使用rIFN蛋白、空载溶瘤病毒、以及rIFN蛋白与空载溶瘤病毒联用更好的效果，表明本发明结构的重组溶瘤病毒能够获得预料不到的协同效果。
具体地，选择免疫细胞浸润较少的三阴乳腺癌细胞系HCC1806裸鼠移植瘤进行体内药效测试。每只裸鼠皮下注射2×10 6个HCC1806细胞进行皮下成瘤，待肿瘤体积长到约90mm 3时随机分组，开始给药处理。给药方案如图7A所示，Vehicle对照及各重组溶瘤腺病毒于分组当天开始给药，瘤内注射给药，给药剂量为1.5×10 10VP/次/只(50μL/次/只)，每隔一天给药一次，共给药五次。重组干扰素蛋白于分组后一天开始给药，腹腔注射给药，给药剂量为20μg/次/只，每隔一天给药一次，共给药五次。每隔三天测量一次肿瘤大小。
结果如图7B所示，与阴性对照Vehicle相比，单独施用重组干扰素蛋白无明显抑瘤效果，与之相比，单用空载溶瘤腺病毒能够产生一定的抑瘤效果，而如果进一步将重组干扰素蛋白与空载溶瘤腺病毒联合使用，能够获得比单独使用其中任何一种更强的抑瘤效果。不过，本发明的重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B获得了最佳的抑瘤效果，与二者联用的方案相比具有统计学上的极显著差异(**)。以上结果表明，本发明所构建的携带rIFN治疗基因的重组溶瘤腺病毒产生了协同效果。
实施例7重组溶瘤腺病毒在体内能够实现对肿瘤细胞的全局抑制作用
本实施例验证了重组溶瘤腺病毒在体内对肿瘤细胞的抑制并不局限 于施药注射部位，而是能够在非给药部位同样产生令人惊讶的肿瘤抑制效果。
具体地，选择三阴乳腺癌细胞系MDA-MB-231裸鼠移植瘤模型，同时进一步构建了携带天然IFN-β的重组溶瘤腺病毒rAd-IFN-β-SP-E1A(Δ24bp)-E1B作为药效的对照。每只裸鼠左右双侧皮下成瘤，各侧注射2×10 6个MDA-MB-231细胞，待左侧肿瘤体积长到约90mm 3时随机分组，开始给药处理。Vehicle对照及各重组溶瘤腺病毒均于分组当天开始给药，左侧瘤内注射给药，给药剂量为1.5×10 10VP/次/只(50μL/次/只)，每隔一天给药一次，共给药五次。右侧不给药，左右侧肿瘤每隔三天测量一次。
对于给药侧(左侧)，如图8A所示，至实验结束时，各携带干扰素基因的重组溶瘤腺病毒几乎都可以完全消除移植瘤，空载溶瘤腺病毒同样也表现出对移植瘤生长的显著抑制作用。进一步如表2所示，在给药后的第11天(即图8A和表2中的Day 19)，阴性对照Vehicle组、空载溶瘤腺病毒rAd-SP-E1A(Δ24bp)-E1B组以及重组溶瘤腺病毒rAd-IFN-3-SP-E1A(Δ24bp)-E1B组均无移植瘤被消除，重组溶瘤腺病毒rAd-IFN-2-SP-E1A(Δ24bp)-E1B组和携带天然IFN-β的重组溶瘤腺病毒rAd-IFN-β-SP-E1A(Δ24bp)-E1B组均有40％的移植瘤被消除，只有重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B组所有的移植瘤均被消除。在实验结束时，仅有重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B组和rAd-IFN-2-SP-E1A(Δ24bp)-E1B组所有的移植瘤均被消除。由上可知，在这几种重组溶瘤腺病毒中，rAd-IFN-1-SP-E1A(Δ24bp)-E1B起效最快并且能够达到最佳的肿瘤抑制效果(与rAd-IFN-2-SP-E1A(Δ24bp)-E1B并列，均完全消除肿瘤细胞)。
表2各处理组在19天和28天时的肿瘤消退个体数量
组别 Tumor free on Day 19 Tumor free on Day 28
Vehicle 0/5 0/5
rAd-SP-E1A(Δ24bp)-E1B 0/5 0/5
rAd-IFN-1-SP-E1A(Δ24bp)-E1B 5/5 5/5
rAd-IFN-2-SP-E1A(Δ24bp)-E1B 2/5 5/5
rAd-IFN-3-SP-E1A(Δ24bp)-E1B 0/5 4/5
rAd-IFN-β-SP-E1A(Δ24bp)-E1B 2/5 4/5
对于非给药侧(右侧)，如图8B所示，空载溶瘤腺病毒完全没有抑瘤效果，携带天然IFN-β的重组溶瘤腺病毒rAd-IFN-β-SP-E1A(Δ24bp)-E1B有一定的抑瘤效果。相比rAd-IFN-β-SP-E1A(Δ24bp)-E1B，本发明所构建的三个重组溶瘤病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B、rAd-IFN-2-SP-E1A(Δ24bp)-E1B、rAd-IFN-3-SP-E1A(Δ24bp)-E1B产生了明显更好的抑瘤效果，获得了统计学上的显著差异(*)。上述三种重组溶瘤腺病毒之间无明显的药效差异。
由此可见，本发明的重组溶瘤病毒对肿瘤细胞的杀伤具有全局性，并不仅限于局部注射位置，对于癌症的临床治疗具有不可估量的价值，例如用于肿瘤转移的治疗。
实施例8重组溶瘤腺病毒能够有效防止肿瘤复发
为了了解本发明溶瘤腺病毒的治疗效果持续时间，选取实施例5中经瘤内注射rAd-IFN-3-SP-E1A(△24bp)-E1B使移植瘤全部消退的HCC827裸鼠移植瘤模型小鼠再次单侧注射2×10 6个HCC827肿瘤细胞进行成瘤实验。对照组为相同周龄的未经处理的裸鼠单侧注射2×10 6个HCC827肿瘤细胞进行成瘤实验，成瘤后两组均不作任何处理，每周测量肿瘤体积两次。如图9所示，结果显示，与对照组相比，实验组中移植瘤生长被显著抑制，最大仅能长至平均体积不到100mm 3，然后开始消退直至再次完全消除。以上结果表明rAd-IFN-3-SP-E1A(△24bp)-E1B治疗后的小鼠中存在明显的免疫记忆，可以快速清除后期出现的同样的肿瘤细胞，具有很好的防止肿瘤复发的作用。
实施例9重组溶瘤腺病毒rAd-IFN-3-SP-E1A(Δ24bp)-E1B显著抑制人源化小鼠移植瘤的生长
为了进一步测试重组溶瘤腺病毒在更接近人体免疫环境下的抑瘤效果，本实施例在人源化免疫系统的小鼠移植瘤模型中测试了本发明重组溶瘤腺病毒的抑瘤能力(本实施例选择了rAd-IFN-3-SP-E1A(Δ24bp)-E1B作为测试对象)。在人源化免疫系统的小鼠背部双侧皮下注射人HCC827细胞，形成移植瘤，但只对右侧肿瘤进行重组溶瘤腺病毒给药，左侧不做任何处理。
结果如图10所示，测试组右侧(给药侧)移植瘤在瘤内注射重组溶瘤腺病毒rAd-IFN-3-SP-E1A(Δ24bp)-E1B后体积开始逐步缩小，实验结束时抑瘤率达到了96.4％，显著强于阳性对照药吉西他滨和PD-1抗体蛋白(图10A和B)。与实施例7的结果类似，测试组左侧(非给药侧)移植瘤在右侧瘤内给药后体积也开始逐步缩小，实验结束时抑瘤率也达到了92.5％的水平，同样显著强于阳性对照药吉西他滨和PD-1抗体蛋白(图10C和D)，表明重组溶瘤腺病毒rAd-IFN-3-SP-E1A(Δ24bp)-E1B即使在更接近人体的复杂状态下，仍然表现出极佳的肿瘤杀伤效果且不仅限于局部注射位置，展现了不可估量的临床应用价值。
实施例10重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B显著抑制PDX裸鼠移植瘤的生长
为了进一步测试重组溶瘤腺病毒在更接近临床真实情况(肿瘤存在很强的异质性)下的抑瘤效果，本实施例在临床病人来源的移植瘤(PDX)裸鼠移植瘤模型中测试了本发明重组溶瘤腺病毒的抑瘤能力(本实施例选择了rAd-IFN-1-SP-E1A(Δ24bp)-E1B作为测试对象)，共选取肺腺癌PDX、肺鳞癌PDX和三阴乳腺癌PDX各1例进行测试。
结果如图11所示，重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B在肺腺癌PDX(图11A)、肺鳞癌PDX(图11B)和三阴乳腺癌PDX(图11C)模型中均显示了优异的肿瘤杀伤效果，实验结束时的抑瘤率分别为98.7％、97.0％和97.5％。表明重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B即使用于治疗具有高度异质性的临床来源的肿瘤，仍然表现出优异的肿瘤杀伤效果。
实施例11重组溶瘤腺病毒的中试规模单产检测
重组溶瘤腺病毒作为一种药物，需要通过发酵才能够大规模生产。中 试规模的细胞单产不仅是判断是否能通过发酵生产的依据，也是药物经济性的重要指标。因此本实施例在中试规模水平测试了重组溶瘤腺病毒的细胞单产。
细胞单产测试使用的工程细胞株为业内常用的ATCC来源的HEK-293细胞，待测试病毒以不同的接种比(VP/cell)接种到一定数量的工程细胞株中，然后在接种后的42、54小时裂解细胞收集子代病毒，测量子代病毒的VP数，除以当时接种的细胞数即得到细胞单产。
结果如表3所示，测试的重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B和rAd-IFN-3-SP-E1A(Δ24bp)-E1B均能够实现工业规模的细胞单产。其中，重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B在接种42小时后的细胞单产即达到最高值，接种54小时后的细胞单产反而下降，这表明该重组溶瘤腺病毒的发酵速度更快，这就意味着工业化生产中，可以以更短的时间可完成发酵，能够节省成本；相比较而言，rAd-IFN-3-SP-E1A(Δ24bp)-E1B在接种54小时后的细胞单产才达到峰值，发酵速度低于rAd-IFN-1-SP-E1A(Δ24bp)-E1B。而且rAd-IFN-1-SP-E1A(Δ24bp)-E1B在第42小时时达到的单产峰值高于rAd-IFN-3-SP-E1A(Δ24bp)-E1B在第56小时达到的单产峰值，这意味着病毒的生产效率也更高。由此可见，就细胞单产而言，重组溶瘤腺病毒rAd-IFN-1-SP-E1A(Δ24bp)-E1B更有优势。
表3重组溶瘤腺病毒的细胞单产量
Figure PCTCN2019127782-appb-000020

Claims (14)

  1. 一种溶瘤病毒，其包含编码干扰素的核酸序列。
  2. 根据权利要求1所述的溶瘤病毒，其中所述病毒是腺病毒。
  3. 根据权利要求1或2所述的溶瘤病毒，其中所述病毒包含以survivin启动子驱动的E1A基因，优选病毒基因组中E1A的内源启动子被survivin启动子所替换；和/或
    E1A基因序列被修饰从而使E1A蛋白结合Rb蛋白的活性降低或完全缺失，优选的，所述修饰为将E1A基因中编码E1A蛋白第122-129位氨基酸的碱基序列删除，例如将SEQ ID NO：1的序列中第364至387bp的序列删除。
  4. 根据权利要求1至3中任一项所述的溶瘤病毒，其中干扰素为α干扰素、β干扰素、γ干扰素或复合干扰素(例如干复津)，优选的，编码所述干扰素的核酸序列如SEQ ID NO：2、3或4所示。
  5. 根据权利要求1至4中任一项所述的溶瘤病毒，其中所述核酸序列与启动子可操作连接，优选的，所述启动子为CMV启动子。
  6. 根据权利要求1至5中任一项所述的溶瘤病毒，其中所述溶瘤病毒保藏于中国典型培养物保藏中心，保藏编号分别为CCTCC NO：V201871、CCTCC NO：V201957或CCTCC NO：V201958。
  7. 权利要求1至6中任一项所述的溶瘤病毒在制备治疗增生性疾病的药物中的用途，优选的，所述增生性疾病是癌症，例如前列腺癌、乳腺癌、结直肠癌、肺癌、肝癌、黑色素瘤、淋巴癌、胃癌、食管癌、卵巢癌、头颈部鳞癌、膀胱癌、神经胶质瘤、宫颈癌或者肾癌。
  8. 一种药物组合物，其包含药物有效量的权利要求1至6中任一项所述的溶瘤病毒，任选的还包含药学上可接受的载体。
  9. 根据权利要求8所述的药物组合物，其中所述药物组合物被配制为通过静脉内、雾化吸入、灌注或者瘤内途径施用。
  10. 根据权利要求8或9所述的药物组合物，其包含约10 8至10 12vp(例如1.5×10 10vp)的所述溶瘤病毒。
  11. 一种治疗增生性疾病的方法，其包括将权利要求1至6中任一项 所述的溶瘤病毒或权利要求8至10中任一项所述的药物组合物施予有需要的对象，所述对象例如哺乳动物，优选为人；更优选的，所述增生性疾病是癌症，例如前列腺癌、乳腺癌、结直肠癌、肺癌、肝癌、黑色素瘤、淋巴癌、胃癌、食管癌、卵巢癌、头颈部鳞癌、膀胱癌、神经胶质瘤、宫颈癌或者肾癌。
  12. 一种防止或抑制癌细胞转移的方法，其包括将权利要求1至6中任一项所述的溶瘤病毒或权利要求8至10中任一项所述的药物组合物施予有需要的对象，所述对象例如哺乳动物，优选为人；所述癌例如前列腺癌、乳腺癌、结直肠癌、肺癌、肝癌、黑色素瘤、淋巴癌、胃癌、食管癌、卵巢癌、头颈部鳞癌、膀胱癌、神经胶质瘤、宫颈癌或者肾癌。
  13. 一种防止癌症复发的方法，其包括将权利要求1至6中任一项所述的溶瘤病毒或权利要求8至10中任一项所述的药物组合物施予有需要的对象，所述对象例如哺乳动物，优选为人，所述癌症例如前列腺癌、乳腺癌、结直肠癌、肺癌、肝癌、黑色素瘤、淋巴癌、胃癌、食管癌、卵巢癌、头颈部鳞癌、膀胱癌、神经胶质瘤、宫颈癌或者肾癌。
  14. 根据权利要求11至13任一项所述的方法，其中通过静脉内、雾化吸入、灌注或者瘤内途径向所述对象施用约10 8至10 12vp(例如1.5×10 10vp)的所述溶瘤病毒，每个疗程施用次数为1-6次(例如1次、2次、3次、4次、5次或6次)，所述施用可以是每1天、2天、3天、4天、5天、6天、7天或更多天进行；或者是1天内施用1次、2次、3次、4次、5次、6次或更多次，向所述对象施用的用药疗程数为1-12个(例如1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个)。
PCT/CN2019/127782 2018-12-26 2019-12-24 表达干扰素的溶瘤病毒及其应用 WO2020135390A1 (zh)

Priority Applications (7)

Application Number Priority Date Filing Date Title
CN201980007573.8A CN112384614B (zh) 2018-12-26 2019-12-24 表达干扰素的溶瘤病毒及其应用
CA3124877A CA3124877A1 (en) 2018-12-26 2019-12-24 Oncolytic virus expressing interferon and application thereof
KR1020217023174A KR102667433B1 (ko) 2018-12-26 2019-12-24 인터페론을 발현하는 종양 용해성 바이러스 및 그의 적용
EP19902481.1A EP3907281A4 (en) 2018-12-26 2019-12-24 ONCOLYTIC VIRUS EXPRESSING AN INTERFERON AND APPLICATION THEREOF
AU2019412342A AU2019412342B2 (en) 2018-12-26 2019-12-24 Oncolytic virus expressing interferon and application therefor
US17/418,759 US20220064670A1 (en) 2018-12-26 2019-12-24 Oncolytic virus expressing interferon and application therefor
JP2021538122A JP7378840B2 (ja) 2018-12-26 2019-12-24 インターフェロンを発現する腫瘍溶解性ウイルス及びその応用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811603052.2 2018-12-26
CN201811603052.2A CN111363726A (zh) 2018-12-26 2018-12-26 表达干扰素的溶瘤病毒及其应用

Publications (1)

Publication Number Publication Date
WO2020135390A1 true WO2020135390A1 (zh) 2020-07-02

Family

ID=71125692

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/127782 WO2020135390A1 (zh) 2018-12-26 2019-12-24 表达干扰素的溶瘤病毒及其应用

Country Status (8)

Country Link
US (1) US20220064670A1 (zh)
EP (1) EP3907281A4 (zh)
JP (1) JP7378840B2 (zh)
KR (1) KR102667433B1 (zh)
CN (2) CN111363726A (zh)
AU (1) AU2019412342B2 (zh)
CA (1) CA3124877A1 (zh)
WO (1) WO2020135390A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113337572A (zh) * 2021-06-23 2021-09-03 江苏沃兴生物科技有限公司 一种病毒保存液配方及其制作方法

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114262691A (zh) * 2020-09-16 2022-04-01 杭州康万达医药科技有限公司 分离的能够用于表达外源基因的溶瘤腺病毒、载体、治疗剂及其用途
CN114438128A (zh) * 2020-10-30 2022-05-06 上海市公共卫生临床中心 一种增强型溶瘤腺病毒及其应用
CN115418356B (zh) * 2022-09-05 2024-09-10 福建师范大学 一种表达i型干扰素的hsv-1溶瘤病毒
WO2024054040A1 (ko) 2022-09-07 2024-03-14 신라젠(주) 항암 바이러스의 신규 용도
WO2024082263A1 (zh) * 2022-10-21 2024-04-25 上海鑫湾生物科技有限公司 一种响应缺氧环境控制病毒复制的系统及其用途
CN118267408A (zh) * 2022-12-30 2024-07-02 上海元宋生物技术有限公司 药物组合物及其应用
CN118267407A (zh) * 2022-12-30 2024-07-02 上海元宋生物技术有限公司 药物组合物

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4695623A (en) 1982-05-06 1987-09-22 Amgen Consensus human leukocyte interferon
CN1509764A (zh) * 2002-12-23 2004-07-07 中国科学院上海生命科学研究院 一种抗癌靶向基因病毒药物的制备方法
EP2537527A2 (en) * 2005-03-09 2012-12-26 Guangwen Wei Uses of recombinant super-compound interferons
CN105769819A (zh) * 2016-04-05 2016-07-20 上海希元生物技术有限公司 携带抗癌基因–溶瘤腺病毒的纳米靶向递送系统及其构建与应用

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7078030B2 (en) * 1999-11-15 2006-07-18 Onyx Pharmaceuticals, Inc Oncolytic adenovirus
KR20060130009A (ko) 2003-08-28 2006-12-18 휴이양테크 (유에스에이), 인크. 변경된 공간 구조를 갖는 인터페론의 용도
AU2004298991A1 (en) * 2003-12-10 2005-06-30 Canji, Inc Methods and compositions for treatment of interferon-resistant tumors
US8142770B2 (en) 2004-05-25 2012-03-27 Kenichiro Kosai Drug comprising as the active ingredient proliferative vector containing survivin promoter
US8658778B2 (en) * 2005-03-09 2014-02-25 Board Of Regents, The University Of Texas System hTMC promoter and vectors for the tumor-selective and high-efficient expression of cancer therapeutic genes
JP2011201813A (ja) 2010-03-25 2011-10-13 Kagoshima Univ サービビンプロモーターを含む増殖制御型ウイルスベクターによる造血器腫瘍の遺伝子治療
WO2011136400A1 (en) * 2010-04-26 2011-11-03 Green Cross Corporation Tumor-specific promoter and oncolytic virus vector comprising the same
JP5975352B2 (ja) * 2011-03-25 2016-08-23 国立大学法人 鹿児島大学 癌幹細胞を標的とするウイルスベクター
WO2014160475A1 (en) * 2013-03-13 2014-10-02 Aboody Karen S Tropic cell based virotherapy for the treatment of cancer
PL3198009T3 (pl) * 2014-09-24 2022-01-24 Salk Institute For Biological Studies Onkolityczne wirusy nowotworowe i sposoby zastosowania
FI20165814A (fi) * 2016-10-27 2018-04-28 Tilt Biotherapeutics Oy Interleukiini 8 (il-8) prognostisena ja ennustavana biomarkkerina ja koostumukset ja vektorit käytettäväksi onkolyyttisessa immunoterapiassa
CN106755103B (zh) * 2016-12-26 2019-06-28 中国人民解放军军事医学科学院放射与辐射医学研究所 溶瘤腺病毒，用于制备该腺病毒的载体及其应用
CN108338994A (zh) * 2017-01-25 2018-07-31 杭州康万达医药科技有限公司 溶瘤病毒作为用于治疗肿瘤和/或癌症的免疫刺激剂的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4695623A (en) 1982-05-06 1987-09-22 Amgen Consensus human leukocyte interferon
US4897471A (en) 1982-05-06 1990-01-30 Amgen Consensus human leukocyte interferon
CN1509764A (zh) * 2002-12-23 2004-07-07 中国科学院上海生命科学研究院 一种抗癌靶向基因病毒药物的制备方法
EP2537527A2 (en) * 2005-03-09 2012-12-26 Guangwen Wei Uses of recombinant super-compound interferons
CN105769819A (zh) * 2016-04-05 2016-07-20 上海希元生物技术有限公司 携带抗癌基因–溶瘤腺病毒的纳米靶向递送系统及其构建与应用

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
COFFEY ET AL., SCIENCE, vol. 282, 1998, pages 1332
KIRN ET AL., NAT. MED., vol. 7, 2001, pages 781
LORENCE ET AL., CANCER RES., vol. 54, 1994, pages 6017
PENG ET AL., BLOOD, vol. 98, 2001, pages 2002
PENG LINHUI , ZHOU JIE , HUO FENG , ZHANG SHILIN ,ZHANG QI , SU CHANGQING , QIAN QIJUN , SHI YANG : "Killing effect and mIFN-gamma expression of the replicative oncolytic adenovirus CNHK300-mIFN-gamma on gastrointestinal malignant tumor in vitro", CHONGQING MEDICINE, vol. 41, no. 22, 31 August 2012 (2012-08-31), pages 2233 - 2236, XP055825548, ISSN: 1671-8348, DOI: 10.3969/j.issn.1671-8348.2012.22.001 *
PENG LIN-HUI, ZHOU JIE, HUO FENG, PU MIAO-SHUI, ZHANG QI, SU CHANG-QING, QIAN QI-JUN, WAN YUN-LE: "Killing effect and mIFN-gamma expression of gene-viral therapeutic system CNHK300-mIFN-gamma on malignant tumor cells in vitro", CHINESE JOURNAL OF PATHOPHYSIOLOGY, vol. 28, no. 5, 31 December 2012 (2012-12-31), CN, pages 802 - 806, XP009528809, ISSN: 1000-4718 *
PENG, LINHUI : "The payload of mouse interferon-gamma gene enhances the antitumor activity of the oncolytic adenovirus CNHK300 in hepatocellular carcinoma", CHINESE JOURNAL OF EXPERIMENTAL SURGERY, vol. 28, no. 6, 30 June 2011 (2011-06-30), pages 920 - 922, XP009528795, ISSN: 1001-9030 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113337572A (zh) * 2021-06-23 2021-09-03 江苏沃兴生物科技有限公司 一种病毒保存液配方及其制作方法

Also Published As

Publication number Publication date
JP7378840B2 (ja) 2023-11-14
EP3907281A1 (en) 2021-11-10
CN112384614A (zh) 2021-02-19
AU2019412342A1 (en) 2021-07-15
KR20210108987A (ko) 2021-09-03
CN111363726A (zh) 2020-07-03
US20220064670A1 (en) 2022-03-03
AU2019412342B2 (en) 2023-11-09
CA3124877A1 (en) 2020-07-02
EP3907281A4 (en) 2022-10-19
CN112384614B (zh) 2023-11-24
KR102667433B1 (ko) 2024-05-21
JP2022516129A (ja) 2022-02-24

Similar Documents

Publication Publication Date Title
WO2020135390A1 (zh) 表达干扰素的溶瘤病毒及其应用
JP7159304B2 (ja) 腫瘍および／または癌の処置のための医薬のための単離された組換え体腫瘍溶解性アデノウイルス、医薬組成物、およびそれらの使用
JP7564572B2 (ja) 単離された組換え体腫瘍溶解性ワクシニアウイルスとｎｋ細胞を含む治療薬、その使用およびそれを適用したキット
US20220339220A1 (en) Therapeutic agents comprising oncolytic vaccinia viruses and nk cells, and uses thereof for drugs for treatment of tumors and/or cancers
CN111606999B (zh) 兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用
WO2019149265A1 (zh) 一种用于治疗肿瘤的伪狂犬病毒
Faghihkhorasani et al. The role of oncolytic virotherapy and viral oncogenes in the cancer stem cells: a review of virus in cancer stem cells
CN109568350B (zh) 一种用于治疗肿瘤的柯萨奇病毒
CN110564700B (zh) 携带鲎凝集素基因的溶瘤痘苗病毒、构建方法及应用
WO2020083324A1 (zh) 表达pd-1结合蛋白的溶瘤病毒及其应用
JP2022520220A (ja) 組換えワクシニアウイルスおよびその使用方法
WO2024140113A1 (zh) 药物组合物
CN1970774A (zh) 一种猪α干扰素重组腺病毒及其构建方法和应用
CN111979204A (zh) 携带海绵凝集素基因的溶瘤痘苗病毒、构建方法及用途
CN116370614A (zh) Lhpp蛋白在制备治疗肺癌药物中的应用
JPWO2020166727A1 (ja) ヒト３５型アデノウイルスを基盤とした腫瘍溶解性ウイルス

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: 19902481

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3124877

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2021538122

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2019412342

Country of ref document: AU

Date of ref document: 20191224

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20217023174

Country of ref document: KR

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2019902481

Country of ref document: EP

Effective date: 20210726