WO2020143221A1 - 增强系统免疫应答的免疫溶瘤病毒组合药物及其应用 - Google Patents

增强系统免疫应答的免疫溶瘤病毒组合药物及其应用 Download PDF

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WO2020143221A1
WO2020143221A1 PCT/CN2019/098798 CN2019098798W WO2020143221A1 WO 2020143221 A1 WO2020143221 A1 WO 2020143221A1 CN 2019098798 W CN2019098798 W CN 2019098798W WO 2020143221 A1 WO2020143221 A1 WO 2020143221A1
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oncolytic virus
oncolytic
immuno
cancer
cells
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French (fr)
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余力
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四川安可康生物医药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
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    • 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 is in the field of biomedicine, and relates to oncolytic virus biomacromolecule drug technology for enhancing the immune response of the body system and its application.
  • Cancer has become one of the major diseases that threaten human health, and China is a major country with cancer incidence and death. According to China’s latest cancer data report (2016) released by the National Cancer Center, in China, 4.29 million new cancer cases occur each year, and 10,000 people are diagnosed with cancer every day, accounting for 20% of new cases worldwide, and China’s cancer deaths account for 27% of global deaths. 7500 people die every day, the mortality rate is higher than the world average.
  • stomach cancer, lung cancer, esophageal cancer, liver cancer and colorectal cancer are the top five cancers. Among them, new lung cancer in China exceeds 30% of new cases worldwide, liver cancer and esophageal cancer exceed 50%, and gastric cancer exceeds 45%.
  • age of 85 a person has a 36% risk of cancer; cancer prevention is not optimistic, and the incidence of cancer will continue to rise in the next decade or two.
  • PD-1 and PD-L1 antibodies are both immune checkpoint blocking drugs, which kill the tumor by overcoming the immune suppression in the patient and reactivating the patient's own immune cells.
  • the current existing methods of treating tumors surgical treatment, radiotherapy, chemotherapy and biological treatment are far from achieving the therapeutic effect of completely eradicating tumor cells. Due to the high complexity, diversity and variability of tumor biological characteristics, finding more effective treatment methods and drugs still faces great challenges.
  • the body's immune system can protect the body from diseases and infections, and it can also help fight cancer.
  • the immune system is essential to fight against the occurrence and development of cancer. Killing cancer cells mainly depends on the activation of a variety of T cells, but some cancers themselves or treatment methods can weaken or inhibit the recognition and attack of T cells on cancer cells. Therefore, it is very important to develop new immunotherapy methods and drugs to treat cancer.
  • T cell responses have na ⁇ ve T cells with homologous T cell receptors (TCR) expressing specific epitopes.
  • T cells require two signals to be fully activated.
  • TCR provides an antigen-specific first signal that interacts with peptide-MHC molecules on the membrane of antigen-presenting cells (APCs).
  • APCs antigen-presenting cells
  • the second signal, the costimulatory signal is non-specific to the antigen and is provided by the interaction between the costimulatory molecule expressed on the APC membrane and T cells.
  • T cell co-stimulation is necessary for T cell proliferation, differentiation and survival. T cell activation without co-stimulation will result in T cell incompetence, T cell loss or immune tolerance.
  • T cells One of the best characterized costimulatory molecules expressed by T cells is CD28, which interacts with CD80 (B7.1) and CD86 (B7.2) on the APC membrane.
  • Another costimulatory receptor expressed by T cells is ICOS (Inducible Costimulatory Factor), which interacts with ICOS-L.
  • Some cancer cells can produce inhibitory molecules to block the interaction between APC and T cells, thereby evading immune surveillance and/or hindering the immune response. For example, studies have shown that almost 80% of tumor cells constitutively produce inhibitory checkpoint CTLA-4 (cytotoxic T lymphocyte associated protein 4) (Contardi, 2005. Int J Cancer. 117:538-550).
  • CTLA-4 cytotoxic T lymphocyte associated protein 4
  • CTLA-4 inhibits T cell function by binding to CD80 and CD86 stimulation checkpoints on APC cells and by neutralizing the function of CD28 receptors on T cells, and mainly affects naive T cells .
  • Another inhibitory checkpoint PD-1/programmed death ligand 1 or 2 (PD-L1 or PD-L2) mainly downregulates the activity of effector T cells in tissues and tumors (Pardoll, 2012. Nat Rev. Cancer. 12:252 -264.).
  • PD-L1 or PD-L2 Another inhibitory checkpoint PD-1/programmed death ligand 1 or 2 mainly downregulates the activity of effector T cells in tissues and tumors.
  • Checkpoint inhibitors Tumors can use checkpoint suppression to protect themselves from the immune system. Immunotherapy with checkpoint antibodies (anti-CTLA-4 therapy or anti-PD-1 and PD-L1) can block inhibitory checkpoints and allow T cells to attack the tumor.
  • T cell therapy for example CAR-T cell therapy.
  • This is a treatment method that attempts to improve the anti-cancer ability of T cells.
  • T cells are derived from the subject's tumor, which is a T cell activation technique in vitro.
  • Monoclonal antibodies Also called therapeutic antibodies, which are proteins of the immune system produced in the laboratory. These antibodies are designed to attach to specific targets found on cancer cells. Some monoclonal antibodies label cancer cells so that they are better recognized and destroyed by the immune system. Other monoclonal antibodies directly prevent cancer cells from growing or causing them to self-destruct. Because therapeutic monoclonal antibodies recognize specific proteins on cancer cells, they are also considered targeted therapy.
  • Oncolytic virus has recently been considered as a promising new treatment for cancer.
  • Oncolytic viruses are defined as genetically engineered or naturally occurring viruses that can preferentially infect and kill cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles, which help to destroy the remaining tumors by repeatedly infecting the tumor cells. Therefore, it is a targeted therapy and a replication biotherapeutic drug.
  • OV may also play a role in cancer immunotherapy.
  • the OV lysis of tumor cells can not only release OV antigens, but also produce or expose cancer-specific antigens, including mutated new antigens and cancer differentiation antigens. These antigens are necessary to elicit APC and T cell responses.
  • OV infection of tumor tissue can also increase T cell infiltration of tumor by inducing inflammatory factors such as tumor necrosis factor (TNF), interleukin-1 ⁇ (IL-1 ⁇ ) and complement.
  • TNF tumor necrosis factor
  • IL-1 ⁇ interleukin-1 ⁇
  • complement a tumor necrosis factor
  • T-Vec talimogene laherparepvec
  • H101 a modified adenovirus
  • the purpose of the present invention is to overcome the shortcomings of the existing treatment technology and the deficiencies of treatment drugs, to provide a new class of oncolytic virus combination drugs that enhance the system immune response and their applications.
  • T cells As mentioned above, the activation of T cells to achieve consistency with cancer (including metastatic cancer), a sustained systemic immune response is the key to eradicating cancer.
  • the response of T cells to the primary antigen requires a second signal in addition to the first signal of histocompatibility complex (MHC).
  • MHC histocompatibility complex
  • the T-cell co-activator as the second signal usually does not appear in tumor cells, so that the T-cell co-activator appears on the surface of tumor cells, it is likely that T cells recognize cancer cell antigens and mediate systemic immunity to tumors A necessary condition for reaction.
  • the present invention adopts a practical technical design for T cell activation dual signal.
  • T cells mediate systemic immune responses to completely treat cancer.
  • the medicine of the present invention is a group of genetically modified and modified oncolytic viruses (especially including members of the Flaviviridae family), and a group of biological macromolecules carrying a group of human T cell coactivators.
  • the engineered live virus can replicate itself in cancer cells and at the same time express active human T cell activating factors that are not normally produced in cancer cells.
  • Oncolytic viruses expose tumor antigens by repeatedly infecting and lysing tumor cells; co-activators expressed at the same time activate T cells.
  • the activation of specific T cell subsets produces a series of chain reactions that mediate systemic immune responses: including the induction and activation of killer T cells and memory T cells to re-identify, identify, and destroy tumor cells. Therefore, the key innovations of the present invention include: (1) using attenuated RNA virus as an oncolytic virus therapeutic vector; (2) transplanting T cell co-stimulators from APC to tumor cells through the vector for expression; (3) on the surface of cancer cells The expressed T cell activating factor provides a second costimulatory signal to mediate a systemic immune response, thereby providing long-lasting immunity to tumors.
  • Treating cancer with this immune oncolytic virus will not only improve the defects of pure OV treatment that induces an insufficient immune response, but also antagonize the suppressive immune factors produced in cancer cells, such as the expression of B7, which can be used to block CTAL-4- cancer cells The suppression of the immune system, thereby exerting the greatest anti-tumor effect.
  • Immuno-oncolytic virus combination drugs that enhance systemic immune response include oncolytic viruses that carry foreign gene fragments derived from humans or animals.
  • the oncolytic virus genome contains all nucleotide sequences and coding information for producing infectious virus particles, has a new structural composition and new functional components, and does not affect the infectivity and the ability to transcribe and express foreign functionally active proteins in infected cells .
  • the immuno-oncolytic virus combination drug is a plurality of oncolytic viruses of different serotypes.
  • the oncolytic virus is an attenuated or non-attenuated strain, a vaccine or non-vaccine strain, an amino acid mutation or a non-coding region nucleotide sequence mutant strain, and an intergroup membrane protein hybrid strain of different oncolytic viruses.
  • the oncolytic viruses include flaviviruses and/or non-flaviviruses.
  • the genus Flavivirus is selected from West Nile, Zika, Dengue Fever Type 1-4, Yellow Fever, Japanese Encephalitis, St. Louis Encephalitis or Kujing Virus.
  • the oncolytic viruses of different serotypes carry foreign gene fragments expressing the same functional protein or foreign gene fragments expressing functional proteins with different functions.
  • the oncolytic virus is linked to a plasmid with a cDNA structure and contains a promoter nucleic acid sequence that regulates viral gene expression.
  • the promoter can be a promoter from different tissues, different species, or a promoter unique to tissues or cancer cells.
  • the promoter is selected from CMV promoter or SV40 promoter.
  • the foreign gene fragment is integrated into the oncolytic virus genome, and the functionally active T cell co-activator is amplified and transcribed as the virus replicates.
  • the protein expressed by the foreign gene fragment may be 50-100% molecular weight of the foreign protein.
  • the foreign gene fragment is a T cell co-activation factor and/or an activation factor that specifically activates other different types of T cell subsets, and has a second signal function for the immune response to the activation system.
  • the T cell co-activator is originally mainly expressed on the surface of immune B cells, especially a class of antigen presenting cells (Antigen Presenting Cells, APC), rather than being expressed in cancer cells.
  • the present invention is an in vivo autoimmune therapy for transplanting immune factors from immune system cells to non-immune system cells.
  • the T-cell co-activator produced by transplantation into tumor cells has the necessary second signal functional activity and specific targeting effect to activate the systemic immune response to the tumor.
  • the human T cell co-activator is selected from CD80/86, ICOSL, OX40L, CD40, 4-1BBL, CD70, CD30L, B7-H7, or other activators that specifically activate other subpopulations of different types of T cells .
  • the T cell subset is CD4, CD8, NK cells, cytotoxic T cells, lymphokine T cells, induced T cells or helper T cells.
  • the viral vector carries human T cell co-activation genes for in vivo immunotherapy of cancer.
  • the second feature of the present invention provides the application of the combined oncolytic virus of any of the above for the treatment of tumors.
  • the method for administering the combination of immuno-oncolytic virus drugs described above is administered in the form of biomacromolecule DNA/RNA or infectious virus particles.
  • the immune oncolytic virus drug combination is used for two or more alternate treatment courses to avoid drug resistance caused by oncolytic virus treatment.
  • the tumor is a solid tumor such as melanoma, lung cancer, cervical cancer, lung epithelial cell carcinoma, prostate cancer, breast cancer, kidney cancer, colon cancer, or epithelial cancer.
  • RNA virus drugs have more advantages than DNA viruses: they have fewer viral proteins and the viral genes are not integrated into the host chromosome.
  • Flavivirus is a family of RNA viruses with members of more than 70 kinds of viruses.
  • members of the virus that infect humans include West Nile virus, Cushing virus, Yellow fever virus, Japanese encephalitis virus, Dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4), Montana Bat leukoencephalitis virus, Yusuta virus, St. Louis encephalitis virus and Alkhurma virus, Zika virus and tick-borne encephalitis virus.
  • viruses have many common structural features: they are small enveloped viruses (approximately 50nm in diameter), with glycoprotein envelope (E) and membrane protein (M) on the outer membrane, which encapsulates core protein (C) and approximately 11,000 nuclei Single positive strand RNA genome of glucuronide (abstract figure). The genome is conserved, with infectious single-stranded RNA, m7G at the 5'end, but no polyadenylic acid at the 3'end. Flaviviruses can infect many vertebrates, and many can be spread by arthropods such as ticks and mosquitoes. Their genome mimics cellular mRNA molecules and translates polyprotein precursors before viral replication begins in the cytoplasm of infected cells.
  • Non-structural proteins are involved in viral RNA replication and viral particle assembly, and new viral particles are assembled on the host cell membrane.
  • new viral particles are assembled on the host cell membrane.
  • tumor cells are lysed.
  • flaviviruses such as West Nile and Dengue to carry human genes as cancer immunotherapy.
  • the use of this virus as a gene therapy carrier has never been reported at home and abroad. Therefore, the immuno-oncolytic virus drug will be a new drug and new technology for immunotherapy of cancer from the treatment mechanism to clinical administration.
  • oncolytic viruses have two most adverse side effects as therapeutic drugs: (1) the pathogenicity of the virus to humans, especially neurotoxicity; (2) the antigenicity of the virus itself.
  • Viral infection leads to the body's immune response, which mainly produces antiviral antibodies to inhibit viral amplification. This is the same mechanism as modern human vaccination to prevent infectious diseases. Due to the inhibition of viral replication by the neutralizing antibodies raised, the oncolytic virus's efficacy will decrease with more than one administration.
  • oncolytic viruses of different serotypes are used selectively. For example, the use of attenuated West Nile and dengue strains in Japan and China, etc., do not use oncolytic JEV vaccine strains.
  • the present invention provides a new combined tumor drug treatment system for immune oncolytic viruses, which relates to oncolytic viruses containing artificially inserted foreign gene fragments. It aims to enhance the immune response of the body's own system to immunotherapy tumors. Because the modified oncolytic virus genome forms a new molecular structure and carries new therapeutic functional components, the system is different from the existing pure oncolytic virus treatment or purely in terms of medicinal molecular structure and therapeutic principle. Oncolytic viruses carry non-specific lymphokine drugs; they are also different from other existing immunotherapy tumor biological drugs.
  • the present invention exposes tumor antigens by cleavage by oncolytic viruses, and mediates the immune response of the body system by T cell co-activation factors. It is a unique new type of immunotherapy tumor technology method in order to achieve the goal of eradicating cancer.
  • the biopharmaceutical dual-treatment tumor pharmacology of the present invention includes: (I) the oncolytic effect of the virus; (II) the systemic immune response of the activated specific T cells to the tumor in vivo.
  • FIG. 1 is the experimental results of 3-week-old mice in Example 1.
  • FIG. 1 is the experimental results of 3-week-old mice in Example 1.
  • FIG. 2 is the experimental results of 2-day-old mice in Example 1.
  • Fig. 3 shows the replication results of oncolytic viruses carrying foreign genes in cells in Example 3.
  • Attenuated flaviviruses have been used as vaccines to prevent human flavivirus infections.
  • live attenuated vaccines including the yellow fever virus 17D strain and the Japanese encephalitis virus 14-14-2 strain, have been vaccinated in a wide population in China and the world, and have a very good history of safety records .
  • Another attenuated flavivirus is the enveloped hybrid flavivirus.
  • the enveloped hybrid flavivirus consists of the flavivirus genome with heterogeneous envelope protein gene segments.
  • the recombinant attenuated rWN/DEN4 ⁇ 30 virus is an enveloped hybrid flavivirus of the wild-type West Nile virus (NY99) genome and the attenuated live dengue fever (4rDEN4) ⁇ 30 strain.
  • the gene encoding the envelope protein of dengue fever-4 was replaced by the West Nile virus (WNV) gene.
  • rWN/DEN4 ⁇ 30 was confirmed to be highly attenuated, with no evidence of neuroinvasive disease, and all monkeys vaccinated with a single dose of rWN/DEN4 ⁇ 30 exhibited moderate to high levels of WNV specificity Neutralizes antibodies and completely protects against WNV NY99 infection.
  • results of a comprehensive study on the neuropathology of vaccines in the central nervous system of rhesus monkeys show that, compared with the 17D reference vaccine for yellow fever, rWN/DEN4 ⁇ 30 has a higher degree of nerve attenuation.
  • Example 1 Attenuated mutations of wild-type viruses: including point mutations in the amino acids of the envelope protein or mutations at the 3'end of the non-coding region, all can produce neuroattenuated flaviviruses.
  • the inventor modified the infectious WNV cDNA and replaced 5 amino acids related to neurotoxicity of the WNV envelope protein.
  • the partial nucleotide sequence of the 3'terminal stem-loop of dengue fever type 2 replaced the wild-type WNV 3'terminal stem-loop sequence, and there were one or more mutations in the nucleotide of the WNV 3'terminal stem-loop secondary structure.
  • the modified WNV showed attenuated characteristics.
  • Example 2 Construction of attenuated membrane hybrid flavivirus (ZIKA/WNV): The present inventors linked the entire WNV genome with a cDNA structure on the pBR322 plasmid vector containing the CMV promoter.
  • the Zika virus (ZIKA) envelope gene fragment was synthesized by PCR. The two ends of the fragment have restriction enzyme sites. After cutting with restriction enzymes, the fragment is ligated to the same site of WNV cDNA cleaved by the same enzyme.
  • the recombinant plasmid was transformed into E. coli cells, and allowed to grow in vitro for proliferation, extraction and purification.
  • the purified recombinant plasmid was transfected into animal or mosquito cells cultured in vitro, and the recombinant plasmid containing the CMV promoter transcribed infectious viral RNA in the cells. These viral RNAs replicate and reproduce, resulting in a hybrid flavivirus (ZIKA/WNV) with a Zika virus envelope using the WNV RNA genome as the backbone. Animal experiment data shows that the hybrid flavivirus with Zika virus envelope has lost the original WNV neurovirulence.
  • ZIKA/WNV hybrid flavivirus
  • Attenuated flaviviruses that have undergone point mutations or envelope replacement have lost their neuropathogenicity, but they maintain the ability to infect and replicate live viruses, so they can be used as gene carrier drugs.
  • enveloped hybrid flavivirus in addition to the benefits of attenuation and non-pathogenic, can also increase the diversity of the same virus (backbone) antigen, avoiding the resistance that is easily produced by treatment with a serotype oncolytic flavivirus .
  • the present invention relates to an oncolytic virus containing artificially inserted foreign gene fragments.
  • These foreign gene fragments are mainly human T cell co-activators.
  • These non-viral exogenous gene fragments are inserted into different types of flavivirus genomes by conventional and commonly used genetic engineering methods.
  • flaviviruses integrating foreign genes without affecting their viral replication refer to the construction of yellow fever virus (YF) 17D/GFP.
  • YF yellow fever virus
  • GFP green fluorescent protein
  • This method takes into account the existence of functional motifs and amino acid sequence flanking between the E and NS1 gene regions, incorporates exogenous genes of green fluorescent protein, allows the correct processing of viral polyprotein precursors, and produces infectious yellow Hot virus.
  • Immunoprecipitation and confocal laser scanning microscopy showed the expression of GFP, which remains in the endoplasmic reticulum and is not secreted from infected cells. Binding to the ER compartment does not interfere with YF assembly, because the recombinant virus is fully capable of replicating and leaving the cell.
  • the virus has genetic stability in the tenth consecutive passage in Vero cells. As demonstrated by the ELISA test, the recombinant virus was able to elicit a neutralizing antibody response against YF and an antibody against GFP.
  • Example 3 The attenuated WNV and hybrid ZIKA/WNV flaviviruses obtained in Example 1 and Example 2, as well as WNV wild virus strains are further genetically modified by conventional genetic engineering methods to make them into carriers carrying foreign gene fragments ,
  • the method is: PCR synthesis of various human or murine T cell co-activator gene fragments or GFP gene fragments, these fragments are connected to the same enzyme cut attenuated WNV and hybrid ZIKA/WNV cDNA, the insertion site and the above Yellow fever virus (YF) 17D/GFP construction is the same.
  • the cloned recombinant plasmid produced infectious virus in Vero cells. Fluorescence microscopy showed the expression of GFP. Immunofluorescence detected the production of flavivirus.
  • the ZIKV strain was directly injected into mouse gliomas. Histological examination showed that the size of the ZIKV-treated tumor was significantly reduced compared to the control saline-treated tumor. As the amount of virus inoculated increased, ZIKV therapy successfully prolonged the survival rate of tumor-bearing mice in a dose-dependent manner.
  • flaviviruses can be used for cancer treatment as oncolytic viruses. But it also shows that simple flaviviruses for cancer treatment, similar to other oncolytic viruses, lack an immune response to the body system and cannot achieve the therapeutic effect of eradicating cancer (Zhu, JEM, 2017.214: 2843).
  • MFC anterior gastric cancer MFC tumor cells (supplied by Shanghai Fuxiang Biotechnology Co., Ltd.) were cultured (RPMI1640 medium containing 5% fetal bovine serum); take logarithm MFC cells (number of 5 million) grown at the stage of growth were injected subcutaneously in the dorsal side of 6-8 week old mice (C57BL/6). About 20 days later, when the tumor mass of mice grew to an average diameter of 6.5 ⁇ 1 mm, it was used for treatment experiments.
  • MFC tumor mice of anterior gastric cancer were divided into groups of 3 mice and injected intratumorally (i.t.).
  • Control group 100 ⁇ l of MEM medium containing 5% fetal bovine serum.
  • Experimental Group A WE 100ul containing 105 titer / Hc86- (WNV source virus carrying human B7 gene fragment).
  • Experimental Group B 100ul WE titer containing 105 / Mc86- (WNV virus carrying the murine B7 gene fragment).
  • the diameter of the tumor was measured: the average tumor diameter of the control group was 9.5 mm, the experimental group A was 5.0 mm, and the experimental group B was 3.5 mm.
  • the diameter of the tumor was measured: the average tumor diameter of the control group was 11.5 mm, the experimental group A was 5.5 mm, and the experimental group B was 2.5 mm.
  • the immune oncolytic virus (WE/Mc86) has a better effect on cancer treatment than the non-specific simple oncolytic virus (WE/Hc86).
  • WNV oncolytic viruses carrying foreign gene fragments are safe as therapeutic drugs.
  • the present invention is not limited to the foregoing specific embodiments.
  • the invention extends to any new feature or any new combination disclosed in this specification, as well as any new method or process step disclosed or any new combination.

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Abstract

本发明公开了一类增强系统免疫反应的免疫溶瘤病毒系列组合药物，包括携带了来源于人或动物外源基因片段的溶瘤病毒。本发明通过由溶瘤病毒裂解而暴露肿瘤本身抗原，并由同时表达的T细胞共激活因子介导免疫治疗肿瘤。

Description

增强系统免疫应答的免疫溶瘤病毒组合药物及其应用 技术领域
本发明为生物医药领域，涉及以增强机体系统免疫应答的溶瘤病毒生物大分子药物技术及其应用。
背景技术
癌症已经成为威胁人类健康的重大疾病之一，中国是癌症发病和死亡大国。根据国家癌症中心发布的中国最新癌症数据报告(2018)，在中国，每年新发癌症病例达429万，每天1万人确诊癌症，占全球新发病例的20％，中国癌症死亡人数占全球27％，每天7500人死亡，死亡率高于世界平均水平。报告称，胃癌、肺癌、食管癌、肝癌和结直肠癌是发病率前五的癌症。其中，中国新增肺癌超过全球新增病例的30％，肝癌和食道癌超过50％，胃癌超过45％。到85岁，一个人患癌风险36％；癌症防治不容乐观，未来一二十年，癌症发病率还会持续走高。
近十年来，免疫治疗在晚期肿瘤取得了快速发展。目前中国已有多家公司的治疗癌症的PDI单抗药物研究进入临床试验阶段。2018年6月15日，国家药品监督管理局，批准了首个抗PD-1受体纳武利尤单抗注射液(Opdivo)在中国上市。然而必须承认，即使被誉为抗癌明星PD-1抑制剂，单独使用PD-1抑制剂对实体瘤治疗的有效率并不高，只有10％-16％左右。唯一的例外，是经典型霍奇金淋巴瘤，有效率突破60％以上。PD-1和PD-L1抗体同属免疫检查点阻断药物，该药通过克服患者体内的免疫抑制，重新激活患者自身的免疫细胞来杀伤肿瘤。然而目前现有治疗肿瘤方法：手术治疗、放射治疗、化学治疗和生物治疗远不能达到彻底根除肿瘤细胞的治疗效果。由于肿瘤生物学特征的高度复杂性、多样性和可变性，寻找更有效的治疗肿瘤方法和药物仍面临着巨大挑战。
机体免疫系统可以保护身体免受疾病和感染，它也可以帮助对抗癌症。免疫系统对抗癌症的发生和发展至关重要，杀死癌细胞主要靠的是多种T细胞的激活，但是一些癌症本身或治疗方法会削弱或抑制T细胞对癌细胞的识别和攻击。因此，开发新的免疫治疗方法和治疗癌症的药物非常重要。
所有的T细胞反应都具有同源T细胞受体(TCR)的幼稚T细胞表达特异性表位。T细胞需要两个信号才能完全激活，通过TCR提供抗原特异性的第一信号，其 与抗原呈递细胞(APC)膜上的肽-MHC分子相互作用。第二信号即共刺激信号是抗原非特异性的，并且由APC膜上表达的共刺激分子与T细胞之间的相互作用提供。T细胞共刺激对于T细胞增殖，分化和存活是必需的。没有共同刺激的T细胞活化会导致T细胞无能，T细胞缺失或产生免疫耐受。由T细胞表达的最好表征的共刺激分子之一是CD28，其与APC膜上的CD80(B7.1)和CD86(B7.2)相互作用。由T细胞表达的另一种共刺激受体是ICOS(诱导型共刺激因子)，其与ICOS-L相互作用。而一些癌细胞可以产生抑制性分子来阻断APC与T细胞的相互作用，从而逃避免疫监视和/或阻碍免疫反应。例如，研究已经证明几乎80％的肿瘤细胞组成性地产生了抑制性检查点CTLA-4(细胞毒性T淋巴细胞相关蛋白4)(Contardi,2005.Int J Cancer.117：538-550)。与调节淋巴器官中的T细胞活化相反，CTLA-4通过结合APC细胞上的刺激检查点CD80和CD86并通过中和T细胞上CD28受体的功能，抑制T细胞功能，且主要影响幼稚T细胞。另一个抑制性检查点PD-1/程序性死亡配体1或2(PD-L1或PD-L2)主要下调组织和肿瘤内的效应T细胞活性(Pardoll,2012.Nat Rev Cancer.12:252-264.)。近年来，已有几种类型的免疫疗法成功用于治疗癌症，包括：
·检查点抑制剂.肿瘤可以使用抑制检查点来保护自己免受免疫系统攻击。使用检查点抗体(抗CTLA-4疗法或抗PD-1和PD-L1)的免疫治疗可以阻断抑制性检查点,并允许T细胞攻击肿瘤。
·T细胞疗法(例如CAR-T细胞疗法).这是一种试图提高T细胞抗癌能力的治疗方法。在这种治疗中，T细胞来自受试者肿瘤,是一种T细胞体外活化技术。
·单克隆抗体.也称为治疗性抗体，其是实验室中产生的免疫系统蛋白质。这些抗体被设计成附着于癌细胞上发现的特定靶标。一些单克隆抗体标记癌细胞，以便它们更好地被免疫系统识别和破坏。其他单克隆抗体直接阻止癌细胞生长或导致其自毁。由于治疗性单克隆抗体识别癌细胞上的特定蛋白质，因此它们也被认为是靶向治疗。
·治疗疫苗和白细胞淋巴因子.
·溶瘤病毒.
溶瘤病毒(OV)最近被认为是有前途的治疗癌症新方法。溶瘤病毒被定义为基因工程或天然存在的病毒，可以优先感染和杀死癌细胞。由于受感染的癌细胞被 溶瘤破坏，它们释放新的感染性病毒颗粒，通过反复感染肿瘤细胞来帮助破坏剩余的肿瘤。因此是一种靶向治疗和一种复制型生物治疗药物。除了溶瘤作用外，OV还可能在癌症免疫疗法中发挥作用。肿瘤细胞的OV溶解不仅可以释放OV抗原，还可以产生或暴露癌症特异性抗原，包括突变的新抗原和癌症分化抗原。这些抗原是引发APC和T细胞应答所必需的。肿瘤组织的OV感染还可以通过诱导炎症因子,如肿瘤坏死因子(TNF)，白细胞介素-1β(IL-1β)和补体，来增加肿瘤的T细胞浸润。目前，已批准两种OV，talimogene laherparepvec(T-Vec，一种改良的疱疹病毒)和H101(一种改良的腺病毒)分别用于治疗黑色素瘤和恶性脑肿瘤。一些OV正在进行临床试验。数据显示单独T-VEC治疗实体瘤的总反应率(ORR)为26％。T-VEC和ipilimumab联合治疗的临床资料显示，在中位随访20个月后，单臂Ib期试验的客观缓解率为50％。44％的患者持续超过6个月的持久反应，18个月后，无进展生存期(PFS)为50％，总生存率为67％(Doepker,2016)。与临床试验的其他数据一样，这些数据表明，虽然单纯溶瘤病毒治疗能间接地激活免疫系统以攻击癌症,但单纯OV治疗未能诱导对癌症的全身免疫应答。因此，必须设计和开发更有效的靶向性免疫药物，以获得对广泛癌症(包括癌症转移)的一致和持久全身免疫应答，来完全根除患者体内的癌细胞，从而不会复发。
发明内容
本发明的目的是克服现有治疗技术的缺陷和治疗药物的不足，提供一类增强系统免疫反应的新型溶瘤病毒组合药物及其应用。
正如上述，激活T细胞来获得对癌症(包括转移癌)一致的，持久的全身性免疫反应是根治癌症的关键所在。T细胞对原发抗原的反应，除了组织相容性复合物(MHC)第一信号外，还需要第二信号。然而作为第二信号的T细胞共激活因子通常是不出现在肿瘤细胞中，让T细胞共激活因子出现在肿瘤细胞表面，很可能是使T细胞识别癌细胞抗原并介导对肿瘤的系统免疫反应的一个必要条件。本发明就是为满足这一条件,对T细胞激活双信号采取了实用性技术设计。以免疫溶瘤病毒治疗方式，通过T细胞介导系统免疫反应来彻底治疗癌症。
基于免疫学第二信号T细胞共激活原理，以及对癌细胞产生抑制T细胞配体的认识和RNA病毒作为基因载体的改造，发明人开发了一类新型免疫治疗肿瘤药物-免疫溶瘤病毒。这与当前单纯溶瘤病毒治疗或其它免疫治疗的方法不同。本发明药物是一组基因改造修饰的溶瘤病毒(尤其包括黄病毒属家族成员),并携带一组人T细胞共激活因子的生物大分子。当药物注入肿瘤组织内，工程化的活病毒可以在癌细胞中自我复制，并且同时表达通常不在癌细胞中产生的活性人T细胞激活因子。溶瘤病毒通过反复感染裂解肿瘤细胞，来暴露肿瘤抗原；而由同时表达的共激活因子,激活T细胞。各特异T细胞亚群的激活，产生一系列连锁反应，介导系统免疫反应:包括诱导和活化杀手T细胞和记忆T细胞，来重新识别，鉴定，和破坏肿瘤细胞。因此,本发明的创新关键包括：(1)用减毒RNA病毒作为溶瘤病毒治疗载体；(2)通过载体将T细胞共刺激物从APC移植到肿瘤细胞表达；(3)癌细胞表面上表达的T细胞激活因子提供第二共刺激信号以介导全身免疫应答，从而提供持久的对肿瘤免疫。用该免疫溶瘤病毒治疗癌症，不仅会改善诱导免疫反应不足的单纯OV治疗缺陷，而且还会拮抗癌细胞中产生的抑制免疫因子，例如表达B7,可以用于阻断CTAL-4-癌细胞对免疫系统的抑制，从而发挥最大的抗肿瘤效应。
为了达到上述目的，本发明采用了下列技术方案：
增强系统免疫反应的免疫溶瘤病毒组合药物，包括携带了来源于人或动物外源基因片段的溶瘤病毒。溶瘤病毒基因组含有产生感染性病毒颗粒的全部核苷酸序列和编码信息，具有新的结构组成和新的功能成分,且不影响感染性和在感染细胞中转录表达外源功能活性蛋白质的能力。
作为优选，所述免疫溶瘤病毒组合药物为多个不同血清型的溶瘤病毒。
进一步优选，所述溶瘤病毒为减毒的或者未减毒的株，疫苗或非疫苗株，氨基酸突变或者非编码区核苷酸序列突变株,不同溶瘤病毒的组间膜蛋白杂交株。
进一步优选，所述溶瘤病毒包括黄病毒属和/或非黄病毒属。
进一步优选，所述黄病毒属选自西尼罗河、寨卡、1‐4型登革热、黄热病、流行性乙型脑炎、圣路易斯脑炎或库京病毒。
更进一步优选，所述不同血清型溶瘤病毒搭载表达同一功能蛋白的外源基因片段或搭载表达功能不相同功能蛋白的外源基因片段。
作为优选，所述溶瘤病毒以cDNA结构连接在质粒上，并含有调节病毒基因 表达的启动子核酸序列。启动子可以是不同组织，不同种属来源启动子，亦可选择组织或癌细胞中特有的起动子。
进一步优选，所述启动子选自CMV启动子或SV40启动子。
作为优选，所述外源基因片段整合于溶瘤病毒基因组中,且随病毒复制而扩增和转录表达功能活性T细胞共激活因子。
进一步优选，所述外源基因片段其表达的蛋白质可以是其外源蛋白的50-100％分子量。
进一步优选，所述外源基因片段是T细胞共激活因子和/或特异激活其它不同类型T细胞亚群的活化因子，具有对激活系统免疫应答的第二信号功能。
更进一步优选，T细胞共激活因子原本主要表达于免疫B细胞表面，尤其是一类抗原呈递细胞(Antigen Presenting Cells,APC)，而不是在癌细胞中表达产生的。本发明是将免疫因子从免疫系统细胞移植到非免疫系统细胞的体内自身免疫治疗。移植到肿瘤细胞中产生的T-细胞共激活因子,对于激活对肿瘤的系统免疫反应，有着必须的第二信号功能活性和特异靶向效应。
更进一步优选，所述人源T细胞共激活因子选自CD80/86、ICOSL、OX40L、CD40、4-1BBL、CD70、CD30L、B7-H7或特异激活其它其不同类型T细胞亚群的活化因子。
更进一步优选，所述T细胞亚群为CD4、CD8、NK细胞、细胞毒性T细胞、淋巴因子T细胞、诱导性T细胞或辅助性T细胞。
基于病毒载体携带人T细胞共激活基因用于体内免疫治疗癌症，是本发明特征之一。本发明特征之二提供了包括以上任一所述的免疫溶瘤病毒组合药物用于治疗肿瘤的应用。
作为优选，包括以上任一所述的免疫溶瘤病毒药物组合的给药方法，以生物大分子DNA/RNA或者感染性病毒颗粒形式在肿瘤内给药。
进一步优选，所述免疫溶瘤病毒药物组合用于二次以上交替治疗疗程，以避免溶瘤病毒治疗产生耐药性。
作为优选，所述肿瘤为黑色素瘤、肺癌、子宫颈癌、肺上皮细胞癌、前列腺癌、乳腺癌、肾癌、结肠癌或上皮癌等实体瘤。
RNA病毒药物比DNA病毒具有更多优势：它们的病毒蛋白数量较少，病毒基 因不会整合到宿主染色体中。黄病毒属(flavivirus)是一RNA病毒家族，超过70种病毒成员。特别地，感染人类的病毒成员包括西尼罗病毒，库京病毒,黄热病毒,日本脑炎病毒,登革热病毒(DEN-1，DEN-2，DEN-3和DEN-4),蒙大拿蝙蝠脑白质炎病毒,尤苏它病毒,圣路易斯脑炎病毒和Alkhurma病毒,寨卡病毒和蜱传脑炎病毒。这些病毒具有许多共同的结构特征：它们是小的包膜病毒(直径大约50nm)，外膜有糖蛋白包膜(E)和膜蛋白(M)，包裹了核心蛋白(C)和约11000个核苷酸的单个正链RNA基因组(摘要附图)。其基因组是保守的，具有感染性单链RNA，5’端有m7G，但3’端没有多聚腺苷酸。黄病毒可感染许多脊椎动物，并且很多可以被节肢动物如扁虱和蚊子传播。他们的基因组模仿细胞mRNA分子，在感染细胞的细胞质中开始病毒复制之前,翻译多聚蛋白前体。翻译后，通过由宿主和病毒蛋白酶介导的加工切割多聚蛋白前体产生三种结构蛋白和七种非结构蛋白。非结构蛋白参与病毒RNA复制和病毒颗粒组装，新的病毒颗粒组装在宿主细胞膜上。在释放(出芽)过程中，导致瘤细胞裂解。如所预期的，当患者免疫系统对溶瘤黄病毒治疗有反应时，受感染的癌细胞不仅可以被感染的裂解病毒裂解，而且还可以暴露癌抗原,被免疫系统重新识别。我们使用西尼罗河和登革热等黄病毒携带人类基因作为癌症免疫治疗，应用这种病毒作为基因治疗载体,国内外从未报道过。因此，该免疫溶瘤病毒药物从治疗机理到临床给药都将是免疫治疗癌症的新型药物和新型技术。
另一方面，应该注意到，溶瘤病毒作为治疗药物存在着两个最不利副作用：(1)病毒对人的致病性,尤其是神经毒性；(2)病毒本身的抗原性。病毒感染导致机体免疫反应，后者主要产生抗病毒抗体来抑制病毒扩增。这与人类的现代疫苗接种来预防传染性疾病是同一机理。由于通过引发的中和抗体抑制病毒复制，溶瘤病毒的药效将随着超过一次以上用药而降低。为了二次以上用药来实现更有效免疫治疗癌症,又避免对单一溶瘤病毒药物产生快速耐药性的不利副作用,鉴于黄病毒多种血清型成员的事实，我们特别地利用了黄病毒属成员中不同血清型组已有的多个减毒病毒株，共同作为该载体药物治疗的组合成分。不同血清型组的多个减毒病毒株的联合交替用药(包括治疗疗程组里不同时间段的给药)，将克服溶瘤病毒治疗不利副作用；同时多次给药，可交替使用所搭载的不同功能活性的T细胞因子。另外，选用多个减毒病毒株，可以在设计治疗方案中有更多的选择:
·在某些黄病毒疫苗接种广泛或流行病发生率高的地区，选择性使用不同血清 型的溶瘤病毒。例如，在日本和中国应用西尼罗河和登革热减毒株等,不要应用溶瘤JEV疫苗株。
·在癌症治疗周期中使用不同血清型的溶瘤病毒作为替代组合，以避免用一种血清型溶瘤病毒持续治疗。
·将几种各携带了功能不同外源基因的溶瘤病毒,制订在一个治疗疗程组里,最大化地激活对肿瘤的系统免疫。
本发明提供了一个新的免疫溶瘤病毒治疗肿瘤组合药物体系，涉及溶瘤病毒包含了人工插入的外源基因片段.它以提升体内自身系统免疫反应为目标来免疫治疗肿瘤。由于改造地溶瘤病毒基因组形成了新的分子结构并携带有新的治疗功能成分,因此该体系在药用分子结构和治疗原理上，都区别于现有的单纯的溶瘤病毒治疗或者单纯的溶瘤病毒搭载非特异淋巴因子药物；也区别于现有的其它免疫治疗肿瘤生物药物。
本发明具有以下优点：
本发明通过由溶瘤病毒裂解而暴露肿瘤本身抗原，并由T细胞共激活因子介导机体系统免疫反应，是一种独特的新型免疫治疗肿瘤技术方法，以期达到根除癌症的治疗目标。本发明生物药物双重治疗肿瘤药理包括：(I)病毒的溶瘤效应；(II)体内激活特异性T细胞对肿瘤的系统免疫反应。
附图说明
摘要附图:黄病毒药物分子结构。1是插入的外源基因片段；2是病毒基因组。
图1是实施例1中3周龄小鼠实验结果。
图2是实施例1中2天龄小鼠实验结果。
图3是实施例3携带有外源基因溶瘤病毒在细胞中的复制结果。
图4是实施例4实验结果。
具体实施方式
下面结合附图，对本发明作详细的说明。
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
构建减毒的感染性黄病毒:
考虑到使用病毒作为人类药物的安全性，必须证明这些黄病毒已经减毒了,不会引起人类疾病。事实上，许多减毒黄病毒已用作预防人类黄病毒属感染的疫苗。这些减毒活疫苗，包括黄热病病毒17D株和流行性乙型脑炎病毒14-14-2株，已经在中国和世界广泛的人口中进行了疫苗接种,并有非常好的安全记录史。另一种减毒黄病毒是包膜杂交黄病毒。大多数情况下，包膜杂交黄病毒由带有异源包膜蛋白基因区段的黄病毒基因组组成。我们的研究和许多已发表的数据表明，杂交黄病毒相对于它们的野生型亲本病毒本质上是减毒的，并且也不太可能恢复到亲本病毒的毒力。例如,重组减毒rWN/DEN4Δ30病毒是野生型西尼罗河病毒(NY99)基因组与减毒活登革热(4rDEN4)Δ30株的包膜杂交黄病毒。编码登革热-4的包膜蛋白的基因被西尼罗河病毒(WNV)的基因取代。在非人灵长类动物和人体试验中，rWN/DEN4Δ30被证实高度减毒，没有神经侵袭性疾病的证据，并且所有接种单剂量rWN/DEN4Δ30的猴子都表现出中到高水平的WNV特异性中和抗体，并且完全抵御WNV NY99的感染。此外，对恒河猴中枢神经系统中疫苗神经病理发生的综合研究结果表明，与黄热病17D参考疫苗相比,rWN/DEN4Δ30的神经减毒程度较高。
实施例1：野生型病毒的减毒突变:包括包膜蛋白氨基酸点突变或者非编码区3’末端突变,都可以产生神经减毒的黄病毒。本发明人修饰了感染性WNV cDNA，对WNV包膜蛋白5个与神经毒有关的氨基酸作了置换。同时将登革热2型3'末端茎环部分核苷酸序列取代了野生型WNV 3'末端茎环序列，还在WNV 3'末端茎环二级结构中核苷酸产生有一个或多个突变。经改造地WNV表现出减毒特征。敏感3周龄小鼠实验，皮下注射3'末端突变WNV(MutE)，均未引起小鼠死亡和神经疾病。2天幼鼠脑内注射WNV包膜蛋白突变株(WN/Env5和WNmutE-Env5)，显示神经毒降低了1000倍(Yu,2008.Vaccine.26:5981-5988)。
实施例2：构建减毒胞膜杂交黄病毒(ZIKA/WNV):本发明人将全WNV基因组以cDNA结构连结在含有CMV启动子的pBR322质粒载体上。PCR合成了寨卡病毒(ZIKA)包膜基因片段。片段两个末端具有限制性酶位点，用限制性酶切割后,将此片段连接于相同酶切割的WNV cDNA的相同位点。将此重组的质粒转化于大肠杆菌细胞里，并让其体外培养增殖和提取纯化。将纯化的重组质粒转染体外培养的动物或蚊子细胞，含有CMV启动子的重组质粒在细胞中转录了感染性病毒RNA。这些病毒RNA复制繁 殖，产生了以WNV RNA基因组为骨架，具有寨卡病毒包膜的杂交黄病毒(ZIKA/WNV)。动物实验数据显示，带有寨卡病毒包膜的杂交黄病毒丧失了原有的WNV神经毒力。
这些经点突变或包膜置换的减毒黄病毒已失去了神经致病性,但它们保持了活病毒的感染和复制能力，因此可以使用这些减毒黄病毒作为基因载体药物。另外，使用包膜杂交的黄病毒,除了减毒不致病好处外，还可以增加同一病毒(骨架)抗原的多样性，避免了用一种血清型溶瘤黄病毒治疗容易产生地耐药性。
构建携带外源基因片段的感染性黄病毒：
本发明涉及溶瘤病毒包含有人工插入的外源基因片段，这些外源基因片段,主要是人T细胞共激活因子。这些非病毒外源基因片段是以常规的普遍采用的基因工程方法,插入至不同类型的黄病毒基因组里。整合了外源基因的黄病毒，而不影响其病毒复制的例子，可以参考黄热病毒(YF)17D/GFP的构建。Myrna C Bonaldo(2007,Virology Journal,4:115.)构建了表达绿色荧光蛋白(GFP)的活的，重组YF-17D。该方法考虑了E和NS1基因间区域侧翼的功能性基序和氨基酸序列保守性的存在，融入了绿色荧光蛋白外源基因，允许病毒多聚蛋白前体的正确加工，并产生了感染性黄热病毒。免疫沉淀和共聚焦激光扫描显微镜显示GFP的表达，其保留在内质网中并且不从感染的细胞分泌。与ER隔室的结合不会干扰YF组装，因为重组病毒完全能够复制并离开细胞。该病毒在Vero细胞中的第10个连续传代中具有遗传稳定性。如ELISA测试所证明的，重组病毒能够引发针对YF的中和抗体应答和针对GFP的抗体。
实施例3：将实施例1和实施例2得到的减毒WNV和杂交ZIKA/WNV黄病毒,以及WNV野毒株用常规基因工程方法进一步基因改造,使之成为携带有外源基因片段的载体，方法是:PCR合成各个人或鼠源T细胞共激活因子基因片段或者GFP基因片段,将这些片段分别连接于相同酶切割的减毒WNV和杂交ZIKA/WNV cDNA中，插入的位点与上述黄热病毒(YF)17D/GFP的构建相同。克隆的重组质粒在Vero细胞中产生了感染性病毒，荧光显微镜显示GFP的表达,免疫荧光检测到黄病毒产生。用这些重组黄病毒感染肺或宫颈癌肿瘤细胞，三天后就观察到了细胞正死于溶瘤病毒感染。这些含有外源基因片段的黄病毒,与野生型亲本菌株相比，病毒空斑块小,生长缓慢。这些结果表明整合了外源基因的黄病毒，没有影响其复制。通过敏感小鼠实验，没有显示神经侵袭性疾病的证据，也没有出现发病死亡。再次证明重组黄病毒没有增 加病毒致病毒性，也说明减毒重组黄病毒，可以像黄病毒活疫苗一样，以药物给予患者，不会产生严重的致病性。
溶瘤病毒治疗肿瘤药效评估：
对比例：近期Zhu等人报道了应用寨卡病毒(ZIKV),一黄病毒属成员,用于胶质母细胞瘤治疗的进展，该病毒涉及2015年小头畸形爆发。由于ZIKV倾向于感染和破坏神经细胞的干细胞(GSCs)，研究人员假设该病毒优先靶向GSCs而不是正常神经细胞。在该研究中，用ZIKV感染体外人胶质母细胞瘤和人GBM手术标本，通过免疫荧光分析显示ZIKV优先感染表达GSC标记SOX2的细胞。在GSC与分化的神经胶质瘤细胞中观察到GSC增殖减少和细胞凋亡增加。进一步实验将ZIKV毒株直接注射小鼠神经胶质瘤内。组织学检查表明，与对照盐水治疗的肿瘤相比，ZIKV治疗的肿瘤尺寸明显缩小。随着病毒接种量的增加，ZIKV疗法以剂量依赖的方式成功延长了荷瘤小鼠的存活率。这些研究表明黄病毒作为溶瘤病毒可用于癌症治疗。但也表明单纯的黄病毒治疗癌症，类同于其它溶瘤病毒,缺乏对身体系统免疫反应,不能达到根除癌症的治疗效果(Zhu,JEM,2017.214:2843)。
实施例4：
建立了小鼠前胃癌MFC同源性皮下肿瘤模型.将小鼠前胃癌MFC肿瘤细胞(上海复祥生物科技有限公司供应)进行培养(含5％胎牛血清的RPMI1640培养基)；取对数期生长地MFC细胞(500万数量),于6-8周龄小鼠(C57BL/6)的背侧皮下注射。大约20天后，观察小鼠肿瘤肿块生长到平均为6.5±1mm直径时，用于治疗实验。
动物治疗实验：
前胃癌MFC肿瘤小鼠,以每3支鼠为一组，分别于瘤内注射(i.t.)。
1.对照组:100μl含5％胎牛血清的MEM培养基。
2.实验组A:100ul含10 5滴度的WE/Hc86-(WNV病毒携带有人源B7基因片段).
3.实验组B:100ul含10 5滴度的WE/Mc86-(WNV病毒携带有鼠源B7基因片段).
实验结果如下:
1.所有9只测试小鼠,在注射后的10天内均未观察到发病症状，30天内也没有死亡。
2.在接种后的20天,肿块直径测量:对照组的肿瘤直径平均为9.5mm，实验组A为5.0mm，实验组B为3.5mm。
3.在接种后的30天,肿块直径测量:对照组的肿瘤直径平均为11.5mm，实验组A为5.5mm，实验组B为2.5mm。
4.接种后的30天，肿瘤组织切片及组化检查显示，实验组B残留肿瘤周围有大量T细胞浸润，其数量数倍多于实验组A。
可见:
1、抑制肿瘤生长在各组之间有明显差别。与对照组比较，实验组A肿瘤生长明显受到了抑制，而实验组B肿瘤侧几乎接进消失。
2、T细胞浸润数量差异，很可能归功于实验组B鼠源B7对免疫系统的激活反应。
3、免疫溶瘤病毒(WE/Mc86)比非特异单纯溶瘤病毒(WE/Hc86)有更好的治疗癌症效果。
4、携带有外源基因片段的WNV溶瘤病毒作为治疗药物是安全的。
本发明并不局限于前述的具体实施方式。本发明扩展到任何在本说明书中披露的新特征或任何新的组合，以及披露的任一新的方法或过程的步骤或任何新的组合。

Claims (20)

  1. 增强系统免疫反应的免疫溶瘤病毒组合药物，其特征在于:包括携带了来源于人或动物外源基因片段的溶瘤病毒。
  2. 根据权利要求1所述的免疫溶瘤病毒组合药物，其特征在于：所述溶瘤病毒可以为同属内多个不同血清型的溶瘤病毒。
  3. 根据权利要求1或2所述的免疫溶瘤病毒组合药物，其特征在于：所述溶瘤病毒包括减毒的或者未减毒的株，疫苗或非疫苗株，氨基酸突变或者非编码区核苷酸序列突变株，不同溶瘤病毒的组间膜蛋白杂交株。
  4. 根据权利要求1或2所述的免疫溶瘤病毒组合药物，其特征在于：所述溶瘤病毒包括黄病毒属和/或非黄病毒属。
  5. 根据权利要求4所述的免疫溶瘤病毒组合药物，其特征在于：所述黄病毒属溶瘤病毒选自西尼罗河、寨卡、1‐4型登革热、黄热病、流行性乙型脑炎、圣路易斯脑炎或库京病毒。
  6. 根据权利要求2所述的免疫溶瘤病毒组合药物，其特征在于：所述不同血清型溶瘤病毒搭载编码同一功能蛋白的外源基因片段或搭载编码不相同功能蛋白的外源基因片段。
  7. 根据权利要求1所述的免疫溶瘤病毒组合药物，其特征在于：所述溶瘤病毒以cDNA结构连接在质粒上，并含有调节病毒基因表达的启动子核酸序列。
  8. 根据权利要求6所述的免疫溶瘤病毒组合药物，其特征在于：所述启动子选自CMV启动子或SV40启动子。
  9. 根据权利要求1所述的免疫溶瘤病毒组合药物，其特征在于：所述外源基因片段整合于溶瘤病毒基因组中，且随病毒复制而扩增并转录表达功能活性蛋白质。
  10. 根据权利要求9所述的免疫溶瘤病毒组合药物，其特征在于：所述外源基因片段编码的蛋白质可以是该外源蛋白50-100％分子量。
  11. 根据权利要求9所述的免疫溶瘤病毒组合药物，其特征在于：所述外源基因片段是人源T细胞共激活因子和/或特异激活其它不同类型T细胞亚群的活化因子。
  12. 根据权利要求11所述的免疫溶瘤病毒组合药物，其特征在于：所述人源T细胞共激活因子选自CD80/86、ICOSL、OX40L、CD40、4-1BBL、CD70、CD30L、 B7-H7或T细胞亚群的特异活化因子。
  13. 根据权利要求12所述的免疫溶瘤病毒组合药物，其特征在于：所述T细胞亚群为CD4、CD8、NK细胞、细胞毒性T细胞、淋巴因子T细胞、诱导性T细胞或辅助性T细胞。
  14. 包括权利要求1-13任一所述的免疫溶瘤病毒组合药物的免疫技术方法，其特征在于：由溶瘤病毒基因载体方式，体内易位表达T细胞活化因子和体内激活系统免疫。
  15. 根据权利要求14所述的免疫方法，其特征在于：T细胞活化因子是在肿瘤细胞内易位表达。
  16. 根据权利要求14-15所述的免疫方法，其特征在于：体内激活对肿瘤的系统免疫反应。
  17. 包括权利要求1-13任一所述的免疫溶瘤病毒组合药物的给药方法，其特征在于：以生物大分子DNA/RNA或者感染性病毒颗粒形式在肿瘤内给药。
  18. 根据权利要求17所述的给药方法，其特征在于：所述免疫溶瘤病毒组合药物适用于二次以上交替治疗疗程。
  19. 权利要求1-13任一所述的免疫溶瘤病毒组合药物用于肿瘤的免疫治疗。
  20. 根据权利要求19所述的应用，其特征在于：所述肿瘤为黑色素瘤、肺癌、子宫颈癌、肺上皮细胞癌、前列腺癌、乳腺癌、肾癌、结肠癌或上皮癌。
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