WO2018137643A1 - 溶瘤病毒作为用于***和/或癌症的免疫刺激剂的应用 - Google Patents

溶瘤病毒作为用于***和/或癌症的免疫刺激剂的应用 Download PDF

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WO2018137643A1
WO2018137643A1 PCT/CN2018/073954 CN2018073954W WO2018137643A1 WO 2018137643 A1 WO2018137643 A1 WO 2018137643A1 CN 2018073954 W CN2018073954 W CN 2018073954W WO 2018137643 A1 WO2018137643 A1 WO 2018137643A1
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tumor
cancer
cells
oncolytic virus
day
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赵荣华
郑艳军
胡放
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杭州康万达医药科技有限公司
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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
    • A61K35/761—Adenovirus
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
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    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55588—Adjuvants of undefined constitution
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N2710/10333—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory

Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to the use of oncolytic viruses as immunostimulants for treating tumors and/or cancers.
  • Tumor biotherapy is the fourth model for treating cancer/cancer after surgery, radiotherapy, and chemotherapy.
  • Tumor immunotherapy is one of the most effective methods for tumor biotherapy. Due to the heterogeneity of tumors, tumor cell variability, and immune escape caused by tumor tissue microenvironment, it is not effective to rely solely on a single tumor-specific antigen to stimulate immunity and thereby induce specific T cells to kill tumors (Fridman) Et al., 2012, The immune contexture in human tumours: impact on clinical outcome, Nature reviews Cancer, 12, 298-306). These obstacles are highlighted in the immune cell therapy of solid tumors.
  • Tumor immunotherapy is achieved primarily in vivo by tumor-specific T cell immune responses (Palucka and Banchereau, 2012, Cancer immunotherapy via dendritic cells, Nature reviews Cancer, 12, 265-277).
  • the core anti-tumor immune cycle includes the following seven steps:
  • Tumor tissue induces the release of new antigens (neoantigens) that induce specific cellular immunity under the combined action of internal and external factors (step 1).
  • new antigens that induce specific cellular immunity under the combined action of internal and external factors (step 1).
  • the body should be accompanied by other immune stimuli. Avoid the occurrence of immune tolerance.
  • Such signals can include pro-inflammatory cytokines as well as factors released by dead cells or gut microbes.
  • DC dendritic cells
  • T cell clones that have received a specific DC antigen signal are activated and amplified (step 3).
  • T effector cells T effector cells to regulatory T cells
  • CTLs cytotoxic T lymphocytes
  • helper T cells break through various barriers and are immersed in tumor entities (step 5).
  • TCR T cell receptor
  • TAA tumor-associated antigen
  • the above anti-tumor immune cycle in many cancer patients has not been optimized by treatment.
  • new tumor antigens are not effectively released; DCs and T cells cannot effectively recognize tumor antigens; too many Treg cells are induced to respond to immune tolerance; activated T cells cannot effectively homing to tumor lesions; more importantly, tumor lesions are long-term Maintaining in an immunosuppressive microenvironment prevents infiltrating effector T cells from effectively killing tumors (Motz and Coukos, 2013, Deciphering and reversing tumor immune suppression, Immunity, 39, 61-73). Therefore, one of the core tasks of tumor immunotherapy is to find effective immune stimulants to activate the body's immune system and inhibit the emergence of negative effects.
  • Immunostimulants in tumor immunotherapy are a class of stimulatory modulators for tumor immunotherapy that stimulate the immune system to destroy tumors, including various recombinant, synthetic, and natural agents.
  • Some randomized controlled clinical studies have shown that in the immunotherapy of different types of cancer, the patient's survival and disease-free period are significantly improved, combined with conventional treatment methods will increase the efficacy of 20% -30% (Fridman et al.
  • Immunostimulants commonly found in the prior art include: interleukin-like cytokines that are partially immunostimulatory, such as IL-1, IL-2, IL-7, IL-12, IL-15, IL-21 (Floros and Tarhini) , 2015, Anticancer Cytokines: Biology and Clinical Effects of Interferon-alpha2, Interleukin (IL)-2, IL-15, IL-21, and IL-12, Seminars in oncology, 42, 539-548; Gao et al., 2015, Mechanism of Action of IL-7 and Its Potential Applications and Limitations in Cancer Immunotherapy, International journal of molecular sciences, 16, 10267-10280; Whittington and Faulds, 1993, Interleukin-2.A review of its pharmacological properties and therapeutic use in patients with Cancer, Drugs, 46, 446-514); interferons, such as IFN-alpha (Galani et al., 2016, IFNs-signaling effects on lung cancer: an up-to
  • Oncolytic viruses selectively replicate and kill tumor cells in tumor cells. Compared with traditional chemoradiotherapy, oncolytic viruses have almost no toxic effects on normal human cells; compared with targeted drugs, they can self-replicate in the tumor, continue to infect and dissolve tumor cells, and produce long-lasting effects. Moreover, oncolytic virus can be used as a gene therapy vector to efficiently express various anti-tumor genes in vivo, including cytokines specific for killing tumor cells and cytokines for stimulating immunity.
  • T-Vec Talimogene laherparepvec
  • Imlygic trade name Imlygic
  • alias OncoVex GM-CSF inserts human GM-CSF into a genetically engineered HSV-I (herpes simplex virus type 1) Granulocyte-macrophage colony-stimulating factor) gene, a tumor immunotherapy preparation that induces anti-tumor immunity by the oncolytic action of HSV-I oncolytic virus combined with the immunostimulatory effect of GM-CSF (Sheridan, 2013, Amgen announces oncolytic virus shrinks) Tumors, Nature biotechnology, 31, 471-472).
  • HSV-I herpes simplex virus type 1
  • Granulocyte-macrophage colony-stimulating factor Granulocyte-macrophage colony-stimulating factor
  • GM-CSF as an immunoregulatory cytokine, exhibits a double-edged sword effect and has been shown to inhibit tumor immunity in some research models (De Henau et al., 2016, Overcoming resistance to checkpoint blockade therapy by targeting) PI3Kgamma in myeloid cells, Nature, 539, 443-447).
  • PI3Kgamma in myeloid cells
  • the present invention provides the use of a selective replication oncolytic virus for the preparation of an immunostimulating agent for treating tumors and/or cancer, and the use of the oncolytic virus as an immunostimulant A method of treating tumors and/or cancer.
  • the present invention provides:
  • a replication-type oncolytic virus for the preparation of an immunostimulatory agent for treating tumors and/or cancer, wherein the oncolytic virus does not carry an exogenous immunomodulatory gene, and wherein administration of the immunostimulatory agent
  • the subject is a tumor and/or a cancer patient whose immune function is sufficient to cause an immune response against a tumor cell, wherein the total number of whole blood leukocytes of the tumor and/or cancer patient is ⁇ 4.0 ⁇ 10 9 /L.
  • the oncolytic virus is capable of directly inducing dendritic cells and T cell activation and amplification, inducing tumors and/or in addition to directly killing tumors and/or cancer cells.
  • up-regulation of expression of chemokines in cancer cell sites and promotion of dendritic cells and lymphocyte infiltration at tumor and/or cancer cell sites, induction of MHC class I and/or MHC class II molecules of tumors and/or cancer cells The expression is up-regulated, thereby fully stimulating the body's anti-tumor immune response in the body.
  • the oncolytic virus is a human type 5 adenovirus, a human 5/3 chimeric adenovirus or a human 5/2 chimeric adenovirus; preferably, The virus is unable to express the E1B-55KD polypeptide or a partial deletion of the E1A protein of the virus; more preferably, the oncolytic virus is H101.
  • the immunostimulatory agent comprises the oncolytic virus at a dose of 0.5 ⁇ 10 12 vp - 1.5 ⁇ 10 12 vp / day.
  • a method for treating tumors and/or cancer which comprises administering an immunostimulating effective amount of a selective replication oncolytic virus as an immunostimulating agent in the treatment of tumors and/or cancer to a tumor and/or a cancer patient, wherein The oncolytic virus does not carry an exogenous immunomodulatory gene, wherein the tumor patient has an immune function sufficient to elicit an anti-tumor cellular immune response, and the total number of whole blood leukocytes of the tumor patient is ⁇ 4.0 ⁇ 10 9 /L.
  • the oncolytic virus is capable of efficiently inducing dendritic cells and T cell activation and amplification, inducing tumors and/or in addition to directly killing tumors and/or cancer cells.
  • Up-regulation of expression of chemokines in cancer cell sites and promotion of dendritic cells and lymphocyte infiltration at tumor and/or cancer cell sites, induction of MHC class I and/or MHC class II molecules of tumors and/or cancer cells The expression is up-regulated, thereby fully stimulating the body's anti-tumor immune response in the body.
  • the oncolytic virus is a human type 5 adenovirus, a human 5/3 chimeric adenovirus, or a human 5/2 chimeric adenovirus; preferably, The virus is unable to express the E1B-55KD polypeptide or a partial deletion of the E1A protein of the virus; more preferably, the oncolytic virus is H101.
  • the invention has the following advantages and positive effects:
  • the invention discovers a new use of oncolytic virus through long-term research and experiments, that is, an oncolytic virus can be used as an immunostimulant in tumor and/or cancer treatment without carrying more genetic modification to carry an exogenous immunoregulatory gene.
  • an oncolytic virus can be used as an immunostimulant in tumor and/or cancer treatment without carrying more genetic modification to carry an exogenous immunoregulatory gene.
  • the present invention develops a new principle and a new usage of oncolytic virus against tumor, that is, the oncolytic virus is applied as an effective immunostimulating agent.
  • the present inventors have found that the oncolytic virus of the present invention can participate in seven steps of total tumor immunization even if it is not combined with other immunotherapy, and produces the following immunopromoting effects in each step:
  • Oncolytic virus oncolytic combined with immune killing, effectively reducing the tumor burden, and releasing the tumor antigen.
  • the oncolytic virus of the present invention can be used as an immunostimulating agent in the treatment of tumors and/or cancer, and the oncolytic effect of the oncolytic virus can be combined with the immunostimulatory effect, thereby increasing the therapeutic effect.
  • FIG. 1 shows the results of MTT assay of H101 in vitro infection of human and murine tumor cell lines in Experimental Example 1 of the present application.
  • 1A is the result of H101 infection of A549 cells;
  • FIG. 1B is the result of H101 infection of B16-F0 cells.
  • FIG. 2 shows the results of H101 promoting apoptosis and tumor antigen release in Experimental Example 2 of the present application.
  • Figure 2A shows the results of immunohistochemistry of tumor antigen release, in which the arrow shows the positive site of Caspase-3 protein immunohistochemistry;
  • Figure 2B shows the results of HE staining of cell lysis, and the left and right images respectively take different parts of the PBS sample.
  • the upper and lower panels of the right side take different parts of the H101 group sample, wherein the arrows show the cell lysis sites.
  • FIG. 3 shows the results of H101 activation of dendritic cells in Experimental Example 3 of the present application.
  • Figure 3A shows the results of flow cytometry of immature DC cells on days 1, 3, and 7 after H101 administration. The ordinate is the percentage (%) of CD11b + CD11c + double positive cells in the spleen, and the abscissa is the number of days after H101 injection.
  • Figure 3B shows the results of flow cytometry of immature DC cells on day 12 after H101 administration. The ordinate is the percentage (%) of CD11b + CD11c + double positive cells in the spleen;
  • Figure 3C-D is the 12th after H101 administration.
  • FIG. 4 shows the results of H101-activated T cells in Experimental Example 4 of the present application.
  • Figure 4A-C shows the results of flow cytometry of CD3 + , CD4 + and CD8 + T cells on day 7 after H101 administration.
  • the ordinate of Figure 4A is the percentage (%) of CD3 + positive cells in the spleen
  • Figure 4B The ordinate is the percentage (%) of CD3 + CD4 + CD8 - cells in the spleen
  • the ordinate on Figure 4C is the percentage (%) of CD3 + CD4 - CD8 + cells in the spleen
  • Figure 4D is the 10th day after administration of H101
  • the T lymphocyte typing flow test results the ordinate is the percentage (%) of CD3 + T cells in the spleen.
  • Fig. 5 shows the results of expression of the chemokine CXCL12 in Experimental Example 5 of the present application, in which the arrow shows the CXCL12 protein immunohistochemical positive site.
  • FIG. 6 shows the results of H101 promoting immersion of NK, DC and CTL into tumor entities in Experimental Example 6 of the present application.
  • FIGS. 6A and B respectively, and CD4 + T cells secreted the ordinate is the active state flow cytometry CD4 + cells results in T cells, as a percentage of FIG. 6A CD3 + CD4 + double positive cells in the H101 tumor after administration ( %), the ordinate of Figure 6B is the percentage (%) of CD4 + CD107 + double positive cells in the tumor, and the abscissa of the two figures is the number of days after H101 injection;
  • Figures 6C and D are CD8 + T cells after H101 administration, respectively.
  • Fig. 6C The results of cell flow assay of CD8+ T cells in secretory activation state, the ordinate of Fig. 6C is the percentage (%) of CD3 + CD8 + double positive cells in the tumor, and the ordinate of Fig. 6D is CD8 + CD107 + double yang in the tumor.
  • FIG 6G is a tumor CD11c + MHCI + percentage of double positive cells (%)
  • the ordinate of FIG 6H is a tumor CD11c + percentage MHCII + double positive cells (%)
  • two The abscissa of the graph is the number of days after H101 injection.
  • Fig. 7 shows the results of H101-stimulated tumor cell MHCI/II expression in Experimental Example 7 of the present application.
  • Fig. 7A shows the flow detection results of cells expressed by MHC class I molecules
  • the ordinate is the percentage (%) of MHCII + positive cells in the tumor
  • the abscissa is the number of days after H101 injection
  • Fig. 7B is the expression of MHC class II molecules.
  • the flow cytometry results of the cells the ordinate is the percentage (%) of MHCII + positive cells in the tumor
  • the abscissa is the number of days after H101 injection.
  • Fig. 8 shows the results of the relative inhibition of tumor growth by H101 in Experimental Example 8 of the present application.
  • 8A shows the result of tumor weight change with time
  • FIG. 8B shows the result of tumor volume change with time
  • FIG. 8C shows a photograph of tumor size change with time.
  • Fig. 9 shows the results of H101 in the experimental example 9 of the present application for improving the overall survival rate of experimental animals.
  • ONYX-015 is a specific oncolytic adenovirus constructed by deleting the E1B-55KD fragment of the human wild type 5 adenovirus DNA sequence. ONYX-015 has no significant effect on normal cells, but can selectively replicate and proliferate in tumor cells, eventually leading to tumor cell lysis.
  • H101 (Accession No. CCTCC No. V98003) is also disclosed in Chinese Patent No. CN1110553C.
  • H101 is an oncolytic adenovirus obtained by genetic engineering of human type 5 adenovirus (Ad5), which is unable to express E1B-55KD polypeptide and lacks the E3 region.
  • Ad5 human type 5 adenovirus
  • H101 can specifically replicate in tumor cells, eventually leading to cell lysis, thereby achieving the purpose of oncolytic.
  • H101 was approved by CFDA in 2006, and the NDA number is: Sinopharm S20060027, and the trade name is Ankerui.
  • oncolytic virus preparations are the same as those of many other targeted specific anti-tumor drugs which have been developed, and are used as drugs which are expected to directly act on the tumor itself and thereby inhibit the same, that is, These preparations utilize the effect that the oncolytic virus can selectively replicate in tumor cells to cause it to dissolve and die.
  • the present invention has discovered a new use of oncolytic viruses through long-term research and experiments, that is, the oncolytic virus of the present invention can be used as an immunostimulating agent in the treatment of tumors and/or cancer, which can be caused individually.
  • the tumor antigen is released in vivo and acts on all seven links of the body's anti-tumor immune system, which activates the body's anti-tumor immune function in an all-round way, achieving a secondary attack on the tumor and producing a sustained therapeutic effect.
  • the inventors of the present invention have found that the oncolytic virus of the present invention can exert various aspects of tumor immunity which stimulates the body anti-tumor without more genetic modification to carry the exogenous immunoregulatory gene, and activates the body anti-tumor immunity in an all-round manner.
  • the present inventors have found that the selective replication oncolytic virus itself (without the exogenous immunoregulatory gene) can be used as an immunostimulant in the treatment of tumors and/or cancer.
  • the inventors of the present invention further found that the subject to be administered should be a tumor and/or cancer patient whose immune function is sufficient to cause an anti-tumor cellular immune response, and/or the tumor and/or cancer.
  • the total number of whole blood leukocytes in cancer patients should be greater than or equal to 4.0 ⁇ 10 9 /L.
  • the present invention provides the use of a selective replication oncolytic virus for the preparation of an immunostimulatory agent for treating tumors and/or cancer, wherein the oncolytic virus does not carry an exogenous immunomodulatory gene, and wherein The immunostimulatory agent is administered to a tumor and/or a cancer patient whose immune function is sufficient to cause an immune response against a tumor cell, wherein the total number of whole blood leukocytes of the tumor and/or cancer patient is ⁇ 4.0 ⁇ 10 9 /L.
  • the oncolytic virus can effectively induce dendritic cells and T cell activation and amplification, induce up-regulation of tumor and/or cancer cell site chemokine expression, and promote Tumor and/or cancer cell locus dendritic cells and lymphocyte infiltration induce up-regulation of MHC class I and/or MHC class II molecules of tumors and/or cancer cells, thereby fully stimulating the body resistance of the patient Tumor immune response.
  • the oncolytic virus is a human type 5 adenovirus, a human 5/3 chimeric adenovirus, or a human 5/2 chimeric adenovirus.
  • the virus is unable to express an E1B-55 KD polypeptide or a partial deletion of the E1A protein of the virus.
  • the oncolytic virus lacks at least a portion of the E3 region gene. It is further preferred that the oncolytic virus is H101.
  • the immunostimulatory agent comprises a clinically administered dose of the oncolytic virus, for example comprising 0.5 x 10 12 vp to 1.5 x 10 12 vp per day of the oncolytic virus.
  • the administration scheme of the oncolytic virus as an immunostimulant may be: intratumor or intravenous injection at a dose of 0.5 ⁇ 10 12 vp-1.5 ⁇ 10 12 vp/day of the oncolytic virus, once a day, continuous administration 4 -5 days; or injecting the oncolytic virus into a cell vector for infusion, for example, loading the oncolytic virus into an autologous or allogeneic immune cell cultured in vitro, and administering it by intravenous infusion or the like (Ilett et al., 2009, Dendritic cells and T cells deliver oncolytic reovirus for tumour killing despite pre-existing anti-viral immunity, Gene therapy, 16, 689-699).
  • the immunostimulating agent according to the present invention may also be administered in combination with other tumor immunological preparations, including but not limited to: some interleukin-like cytokines having immunostimulatory effects, such as IL-1, IL-2, IL-7, IL-12, IL-15, IL-21; interferons, such as IFN-alpha; colony-stimulating factors such as G-CSF, GM-CSF; tumor necrosis factor, such as TNF-alpha; immune cytokines Such as CD40L/CD40; chemokines, such as CCL-3, CCL-5, CCL-26, CX3CL1, CXCL9, CXCL10; Toll-like receptor (TLR) signal-related molecules; and immunological-related preparations Such as anti-PD-1 antibody, anti-CTLA4 antibody.
  • interleukin-like cytokines having immunostimulatory effects such as IL-1, IL-2, IL-7, IL-12, IL-15, IL-21
  • Another aspect of the invention provides a method of treating a tumor and/or cancer, comprising immunostimulating an effective amount of a selective replication oncolytic virus of the invention as an immunostimulatory agent in the treatment of tumors and/or cancer Administration to a tumor and/or cancer patient, wherein the oncolytic virus does not carry an exogenous immunomodulatory gene, the immune function of the tumor patient is sufficient to elicit an anti-tumor cellular immune response, and the total number of whole blood leukocytes of the tumor patient is ⁇ 4.0 ⁇ 10 9 /L.
  • the oncolytic virus can effectively induce dendritic cells and T cell activation and amplification, and induce tumor and/or cancer cell site chemotaxis, in addition to directly killing tumors and/or cancer cells.
  • the administration protocol of the immunostimulant is intratumor injection at a dose of 0.5 ⁇ 10 12 vp-1.5 ⁇ 10 12 vp/day of the oncolytic virus, once a day, continuously Apply for 4-5 days.
  • the immunostimulatory agent can be administered in combination with other tumor immunological agents.
  • the other tumor immunological preparations include, but are not limited to, some interleukin-like cytokines having immunostimulatory effects, such as IL-1, IL-2, IL-7, IL-12, IL-15, IL-21; interferons, Such as IFN-alpha; colony stimulating factors, such as G-CSF, GM-CSF; tumor necrosis factor, such as TNF-alpha; immune cytokines, such as CD40L/CD40; chemokines, such as CCL-3, CCL-5, CCL -26, CX3CL1, CXCL9, CXCL10; Toll-like receptor (TLR) signal-related molecules; and immunologically related preparations, such as anti-PD-1 antibody, anti-CTLA4 antibody.
  • interleukin-like cytokines having immunostimulatory effects such as IL-1, IL-2, IL-7, IL-12, IL-15, IL
  • the immunostimulating agent is administered again for 7-10 days after the end of the first continuous administration of the immunostimulating agent, more preferably, 7 days apart, for enhancing immunity;
  • the administration protocol for enhancing immunity is: intratumoral injection at a dose of 0.5 ⁇ 10 12 vp - 1.5 ⁇ 10 12 vp / day of the oncolytic virus, once a day for 1-2 days.
  • the booster immunization can be performed again, and the dose and administration method are the same as above.
  • 3-4 booster immunizations can be performed.
  • A549 human non-small cell lung cancer cells and B16-F0 mouse melanoma cells were all derived from the cell bank of the Culture Collection Committee of the Chinese Academy of Sciences.
  • the oncolytic adenovirus H101 was derived from Shanghai 3D Biotechnology Co., Ltd.
  • C57BL/6 mice male, 6-8 weeks old were obtained from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • the cell flow meter was obtained from Essen Bio (Hangzhou) Co., Ltd., model D2060R.
  • 3 ⁇ 10 3 A549 cells or 4 ⁇ 10 3 B16-F0 cells were implanted into each well in a 96-well cell culture plate, and cultured in high-sugar DMEM containing 10% FBS at 37° C. and 5% CO 2 . After an hour (to allow the cells to adhere completely), add H101 (AMO cell line is 10MOI, B16-F0 cell line is 100MOI), and fresh medium is replaced after 12 hours. The time from the addition of virus is 0 hours, every 24 hours. MTT assay (MTT reagent was purchased from Sigma-Aldrich, product number M5655-1G). The control group was complete medium without any oncolytic virus.
  • the present inventors developed an animal model of subcutaneous tumorigenesis of murine melanoma, and first verified the in vitro cytotoxic effect of H101 on the melanoma cell line (B16-F0).
  • the results showed that the direct killing effect of H101 on the mouse B16-F0 cell line was extremely weak due to the species specificity of the human type 5 adenovirus (see Experimental Example 1, Figure 1B).
  • the model is in line with the research purpose of studying the immunostimulatory effect of H101 as far as possible to rule out the direct oncolytic effect of H101. Thereafter, the effect of H101 on pro-apoptosis of the cell line (B16-F0) and promotion of whole antigen release in the animal model was investigated.
  • B16-F0 cells were injected with tumors in the right back of C57BL/6 male mice in an amount of 2 ⁇ 10 6 cells / 100 ⁇ l of DMEM, 100 ⁇ l per animal.
  • the tumor volume was about 150 mm 3
  • intratumoral injection of H101 2 ⁇ 10 10 vp/50 ⁇ l pH 7.4 PBS PBS reagent purchased from Bio-Bioengineering (Shanghai) Co., Ltd., item number B548117
  • continuous 4 One day, once a day, this was the experimental group; while the control group received continuous intratumoral injection of 50 ⁇ l PBS for 4 days, once a day.
  • mice On the 0th day of the first day of administration, the mice were sacrificed on the 3rd, 7th, 10th, and 13th day to take the tumor for immunohistochemistry (IHC) detection of Caspase-3 (anti-mouse Caspase-3 antibody was purchased from R&D Systems The company, product number AF-605-NA) and HE staining (hematoxylin and eosin (HE) staining kit were purchased from Bioengineering Engineering (Shanghai) Co., Ltd., item number E607318).
  • IHC immunohistochemistry
  • B16-F0 cells were injected with tumors in the right back of C57BL/6 male mice in an amount of 2 ⁇ 10 6 cells / 100 ⁇ l of DMEM, 100 ⁇ l per animal.
  • the experimental group was intratumorally injected with H101 (1 ⁇ 10 10 vp/60 ⁇ l PBS) for 1 day (1 time), and the control group was intratumorally injected with 60 ⁇ l of PBS for 1 day (1 time).
  • B16-F0 cells were injected with tumors in the right back of C57BL/6 male mice in an amount of 2 ⁇ 10 6 cells / 100 ⁇ l of DMEM, 100 ⁇ l per animal.
  • the experimental group was intratumorally injected with H101 (5 ⁇ 10 9 vp/50 ⁇ l PBS) for 4 days, once a day, while the control group was continuously injected intratumorally with 50 ⁇ l of PBS for 4 days, once a day.
  • B16-F0 cells were injected with tumors in the right back of C57BL/6 male mice at a dose of 1.5 ⁇ 10 6 cells/100 ⁇ l DMEM, 100 ⁇ l per animal.
  • the experimental group was injected intratumorally with H101 (4).
  • B16-F0 cells were injected with tumors in the right back of C57BL/6 male mice in the amount of 2 ⁇ 10 6 cells/100 ⁇ l DMEM, 100 ⁇ l per animal.
  • the experimental group was intratumorally injected with H101 (1 ⁇ 10 10 vp/60 ⁇ l PBS) 1 day (1 time), while the control group was intratumorally injected with 60 ⁇ l PBS for 1 day (1 time).
  • the spleen was sacrificed on the 10th day for T lymphocyte typing flow detection (anti-mouse CD3e PE-Cyanine7 antibody was purchased from eBioscience, Inc., number 35-0031-82). The result is shown in Figure 4D.
  • T cell subtype analysis anti-mouse CD4-FITC and CD8-APC antibodies were purchased from BD Pharmingen, Inc., numbered 553729 and 553035, respectively; anti-mouse CD44-PE and CD62L-APC antibodies were purchased from eBioscience, respectively.
  • Naive T original T, CD44 low CD62L hi
  • Tcm central memory T cells, CD44
  • B16-F0 cells were injected with tumors in the right back of C57BL/6 male mice at a dose of 1 ⁇ 10 6 cells/100 ⁇ l DMEM, 100 ⁇ l per animal.
  • the experimental group was injected intratumorally with H101 (4).
  • day 0 was administered, and on the 13th day, tumors were sacrificed for IHC detection of chemokine CXCL12 (anti-mouse CXCL12 antibody was purchased from Abcam, Cat. No. AB25117). The result is shown in Figure 5.
  • chemokine CXCL12 was up-regulated in the tumor site on the 13th day after H101 administration.
  • the up-regulation of this chemokine can promote vascular proliferation, improve the permeability of tumors, and facilitate the return of immune cells at tumor sites. Nest and gathering.
  • B16-F0 cells were injected with tumors in the right back of C57BL/6 male mice in the amount of 2 ⁇ 10 6 cells/100 ⁇ l DMEM, 100 ⁇ l per animal, tumor-bearing day 7 and tumor volume was about 150 mm 3 .
  • the group was intratumorally injected with H101 (2 ⁇ 10 10 vp/50 ⁇ l PBS) for 4 days, once a day, while the control group was continuously injected intratumorally with 50 ⁇ l of PBS for 4 days, once a day.
  • the tumor was taken for flow cytometry analysis (anti-mouse CD314 (NKG2D)-APC, NK1.1-FITC, CD107a-PE, CD3e PE-Cyanine7, MHC Class I (H-2Kd)-PE and MHC Class II (IA/IE)-PE-Cyanine7 antibodies were purchased from eBioscience under the numbers 17-5882-82, 11-5941-85, 12- 1071-83, 35-0031-82, 12-5957-82, and 25-5321-82; anti-mouse CD4-FITC and CD8-APC antibodies were purchased from BD Pharmingen, Inc., numbered 553729 and 553035, respectively; anti-mouse CD11b-FITC The CD11c-APC antibody was purchased from eBioscience under the numbers 11-0112-85 and 17-0114-81, respectively. The result is shown in Figure 6.
  • CD4 + T cells in tumor tissues were significantly up-regulated with CD4 + T cells (CD4 + CD107a + ) in secretory activation (Fig. 6A, B); whereas CD8 + T cells were in 7 days was significantly upregulated, and on days 7 and 10, CD8 + T cells were up-regulated with CD8 + T cells (CD8 + CD107a + ) in secretory activation (Fig. 6C, D).
  • NK cells were significantly upregulated (Fig. 6E).
  • immature DCs (CD11b + CD11c + ) in the tumor were significantly up-regulated on day 7, while mature DCs (CD11c + MHCI + or CD11c + MHCII + ) were significantly up-regulated on days 3 and 7 (Fig. 6F-H).
  • Experimental Example 7 Injection stimulates MHCI/II expression in tumor cells and enhances immune recognition
  • B16-F0 cells were injected with tumors in the right back of C57BL/6 male mice in the amount of 2 ⁇ 10 6 cells/100 ⁇ l DMEM, 100 ⁇ l per animal, tumor-bearing day 7 and tumor volume was about 150 mm 3 .
  • the group was intratumorally injected with H101 (2 ⁇ 10 10 vp/50 ⁇ l PBS) for 4 days, while the control group was continuously injected intratumorally with 50 ⁇ l of PBS for 4 days.
  • MHC class I molecules On days 3 and 7 after H101 administration, the percentage of cells expressed by MHC class I molecules in the tumor site was significantly up-regulated; on days 7 and 10, the percentage of cells expressed by MHC class II molecules was significantly up-regulated.
  • the up-regulation of MHC-like molecules strongly promotes the tumor immune recognition response and improves the killing efficiency of immune cells.
  • B16-F0 cells were injected with tumors in the right back of C57BL/6 male mice in the amount of 2 ⁇ 10 6 cells/100 ⁇ l DMEM, 100 ⁇ l per animal, tumor-bearing day 7 and tumor volume was about 150 mm 3 .
  • the group was intratumorally injected with H101 (2 ⁇ 10 10 vp/50 ⁇ l PBS) for 4 days, once a day, while the control group was continuously injected intratumorally with 50 ⁇ l of PBS for 4 days, once a day. Tumor size was measured every two days on the first day of dosing on day 0, and tumors were weighed on days 3, 7, and 10. The result is shown in Figure 8.
  • B16-F0 cells were injected with tumors in the right back of C57BL/6 male mice in the amount of 2 ⁇ 10 6 cells/100 ⁇ l DMEM, 100 ⁇ l per animal.
  • the experimental group was injected intratumorally with H101 (4).
  • the survival of the mice was observed daily. The result is shown in Figure 9.
  • H101 as an immunostimulant can effectively improve the antitumor effect of an animal model of immune function. Its efficacy not only depends on the direct oncolytic effect of oncolytic virus, but also promotes all seven steps of anti-tumor immunity, effectively stimulating the body's anti-tumor immunity.

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Abstract

选择复制型溶瘤病毒在制备用于***和/或癌症的免疫刺激剂中的应用，该溶瘤病毒不携带外源免疫调节基因。

Description

溶瘤病毒作为用于***和/或癌症的免疫刺激剂的应用 技术领域
本发明属于生物医药领域，具体涉及溶瘤病毒作为用于***和/或癌症的免疫刺激剂的应用。
背景技术
肿瘤生物治疗是继手术、放疗、化疗之后的第四种***/癌症的模式。肿瘤免疫治疗是肿瘤生物治疗中疗效最切实的方法之一。由于机体肿瘤的异质性、肿瘤细胞变异以及肿瘤组织微环境引起的免疫逃逸现象，使得仅仅依靠单一的肿瘤特异性抗原刺激机体免疫并由此诱导特异性T细胞杀伤肿瘤的效果不佳(Fridman et al.,2012,The immune contexture in human tumours:impact on clinical outcome,Nature reviews Cancer,12,298-306)。这些障碍在实体瘤的免疫细胞治疗中得到突出体现，目前单一靶点的免疫细胞治疗，仍然仅仅在以CD19为靶点的血液性肿瘤上得到良好疗效(Khalil et al.,2016,The future of cancer treatment:immunomodulation,CARs and combination immunotherapy,Nature reviews Clinical oncology,13,273-290)。目前学术界与科研界共同面对的世界性难题就是如何有效地刺激机体抗肿瘤免疫，避免肿瘤免疫逃逸。
肿瘤免疫治疗在体内主要通过肿瘤特异性T细胞免疫反应实现(Palucka and Banchereau,2012,Cancer immunotherapy via dendritic cells,Nature reviews Cancer,12,265-277)。其中核心的抗肿瘤免疫循环包括如下七个步骤：
肿瘤组织在内外因素共同作用下诱发可以诱导特异性细胞免疫的新抗原(neoantigens)的释放(步骤一)，在这些肿瘤特异性新抗原释放的同时，机体应该伴随产生其它的免疫刺激信号，以避免免疫耐受现象的发生。这一类的信号可以包括前炎性细胞因子，以及由死 亡细胞或者肠道微生物释放的因子。而后，树突状细胞(DC)通过表面的MHCI、II类分子捕获新抗原表位信号，DC激活，并将特异性激活信号传递给T细胞(步骤二)。接受了特异性DC抗原信号的T细胞克隆激活，扩增(步骤三)。这里，这样的新抗原被免疫***认为是外源的，或者是内源但是没有经过机体发育阶段免疫耐受过程的抗原。但最终免疫激活的效果还需要通过这时机体的免疫***状态来体现。比如T效应细胞与调节性T细胞(Treg)的比例可以在很大程度上决定免疫效果。这些激活的肿瘤特异性T细胞等免疫细胞被炎症或免疫刺激物诱导产生的趋化因子吸引、归巢、来到肿瘤位点附近(步骤四)。之后特异性细胞毒性T淋巴细胞(CTL)/辅助性T细胞突破各种屏障，浸入肿瘤实体(步骤五)。CTL通过T细胞受体(TCR)和肿瘤特异性抗原与MHCI匹配的肿瘤细胞特异性识别，结合(步骤六)。CTL最终杀伤靶细胞(步骤七)。被杀伤的肿瘤细胞再次释放肿瘤相关抗原(TAA)(步骤一)，并进一步拓展抗原谱，强化抗肿瘤免疫(Chen and Mellman,2013,Oncology meets immunology:the cancer-immunity cycle,Immunity,39,1-10)。
但在临床实践中，很多癌症病人的以上抗肿瘤免疫循环没有通过治疗得到优化。比如肿瘤新抗原没有被有效释放；DC和T细胞不能有效识别肿瘤抗原；诱导了太多Treg细胞响应产生免疫耐受；激活的T细胞无法有效归巢至肿瘤病灶；更重要的，肿瘤病灶长期保持在免疫抑制微环境下，使得浸润的效应T细胞无法有效杀伤肿瘤(Motz and Coukos,2013,Deciphering and reversing tumor immune suppression,Immunity,39,61-73)。因此，肿瘤免疫治疗的核心任务之一，是寻找有效的免疫刺激剂激活机体免疫***，并抑制负面效应的出现。
肿瘤免疫治疗中的免疫刺激剂是一类用于肿瘤免疫疗法的刺激型调节剂，可以刺激免疫***来摧毁肿瘤，包括各种重组、合成和天然的制剂。在具体的实践、研究和实验中有一系列各异的策略方法。一些随机对照临床研究报告显示，在不同类型癌症的免疫治疗中，患者的生存期和无病期都有显著提高，与常规治疗方法联合更会增加 20％-30％的疗效(Fridman et al.,2012,The immune contexture in human tumours:impact on clinical outcome,Nature reviews Cancer,12,298-306；Khalil et al.,2016,The future of cancer treatment:immunomodulation,CARs and combination immunotherapy,Nature reviews Clinical oncology,13,273-290；Kono et al.,2002,Prognostic significance of adoptive immunotherapy with tumor-associated lymphocytes in patients with advanced gastric cancer:a randomized trial,Clinical cancer research:an official journal of the American Association for Cancer Research,8,1767-1771；Takayama et al.,2000,Adoptive immunotherapy to lower postsurgical recurrence rates of hepatocellular carcinoma:a randomised trial,Lancet,356,802-807)。现有技术中常见的免疫刺激剂包括：部分有免疫刺激作用的白介素类细胞因子，如IL-1、IL-2、IL-7、IL-12、IL-15、IL-21(Floros and Tarhini,2015,Anticancer Cytokines:Biology and Clinical Effects of Interferon-alpha2,Interleukin(IL)-2,IL-15,IL-21,and IL-12,Seminars in oncology,42,539-548；Gao et al.,2015,Mechanism of Action of IL-7and Its Potential Applications and Limitations in Cancer Immunotherapy,International journal of molecular sciences,16,10267-10280；Whittington and Faulds,1993,Interleukin-2.A review of its pharmacological properties and therapeutic use in patients with cancer,Drugs,46,446-514)；干扰素类，如IFN-alpha(Galani et al.,2016,IFNs-signaling effects on lung cancer:an up-to-date pathways-specific review,Clinical and experimental medicine)；集落刺激因子，如G-CSF、GM-CSF(Hoeller et al.,2016,Systematic review of the use of granulocyte-macrophage colony-stimulating factor in patients with advanced melanoma,Cancer immunology,immunotherapy:CII,65,1015-1034；Li et al.,2005,Preclinical ex vivo expansion of G-CSF-mobilized peripheral blood stem cells:effects of serum-free media,cytokine combinations and chemotherapy,European journal of haematology,74,128-135)；肿瘤坏死因子，如TNF-alpha； 免疫细胞因子，如CD40L/CD40(Lippitz,2013,Cytokine patterns in patients with cancer:a systematic review,The Lancet Oncology,14,e218-228)；趋化因子，如CCL-3、CCL-5、CCL-26、CX3CL1、CXCL9、CXCL10(Elbaz et al.,2015,Modulation of the tumor microenvironment and inhibition of EGF/EGFR pathway:novel anti-tumor mechanisms of Cannabidiol in breast cancer,Molecular oncology,9,906-919；Franciszkiewicz et al.,2012,Role of chemokines and chemokine receptors in shaping the effector phase of the antitumor immune response,Cancer research,72,6325-6332；Fridman et al.,2012,The immune contexture in human tumours:impact on clinical outcome,Nature reviews Cancer,12,298-306；Khalid et al.,2015,Recent Advances in Discovering the Role of CCL5in Metastatic Breast Cancer,Mini reviews in medicinal chemistry,15,1063-1072；Mellman et al.,2011,Cancer immunotherapy comes of age,Nature,480,480-489；Peng et al.,2012,PD-1blockade enhances T-cell migration to tumors by elevating IFN-gamma inducible chemokines,Cancer research,72,5209-5218；Tudoran et al.,2015,Baseline blood immunological profiling differentiates between Her2-breast cancer molecular subtypes:implications for immunomediated mechanisms of treatment response,OncoTargets and therapy,8,3415-3423)；Toll样受体(TLR，Toll-like receptor)信号相关分子(Mellman et al.,2011,Cancer immunotherapy comes of age,Nature,480,480-489)；以及免疫点相关制剂，如抗PD-1抗体、抗CTLA4抗体。
溶瘤病毒可选择性地在肿瘤细胞内复制并杀死肿瘤细胞。与传统放化疗相比，溶瘤病毒对人体正常细胞几乎无毒性作用；与靶向药物相比，其可在瘤内自我复制，持续感染和溶解肿瘤细胞，产生持久疗效。并且，溶瘤病毒可以作为基因治疗载体，体内高效表达各种抗肿瘤基因，包括特异性杀伤肿瘤细胞有关的毒性基因以及刺激机体免疫的细胞因子等。例如，国际上已经上市的Talimogene laherparepvec(T-Vec,商品名Imlygic,别名OncoVex GM-CSF)就是在一种经过 基因改造的HSV-I(单纯疱疹病毒1型)中***人源GM-CSF(粒细胞巨噬细胞集落刺激因子)基因，通过HSV-I溶瘤病毒的溶瘤作用配合GM-CSF的免疫刺激作用而诱导抗肿瘤免疫的肿瘤免疫治疗制剂(Sheridan,2013,Amgen announces oncolytic virus shrinks tumors,Nature biotechnology,31,471-472)。因该制剂已经***外源基因，并不能明确HSV-1溶瘤病毒本身的免疫刺激剂作用，其药理学作用，很多是通过其***基因GM-CSF来体现的。而GM-CSF作为一种免疫调节细胞因子，表现出一种双刃剑作用，在一些研究模型中显示出抑制肿瘤免疫的效果(De Henau et al.,2016,Overcoming resistance to checkpoint blockade therapy by targeting PI3Kgamma in myeloid cells,Nature,539,443-447)。然而，在肿瘤和/或癌症的免疫治疗中，仍然需要寻找有效的免疫刺激剂来激活机体的免疫***。
发明内容
为解决上述现有技术中所存在的问题，本发明提供了选择复制型溶瘤病毒在制备用于***和/或癌症的免疫刺激剂中的应用、以及用该溶瘤病毒作为免疫刺激剂***和/或癌症的方法。
具体而言，本发明提供了：
(1)选择复制型溶瘤病毒在制备用于***和/或癌症的免疫刺激剂中的应用，其中所述溶瘤病毒不携带外源免疫调节基因，并且其中所述免疫刺激剂的施用对象为，其机体的免疫功能足以引起抗肿瘤细胞免疫应答的肿瘤和/或癌症患者，其中所述肿瘤和/或癌症患者的全血白细胞总数≥4.0×10 9/L。
(2)根据(1)所述的应用，其中所述溶瘤病毒除能够直接杀伤肿瘤和/或癌细胞外，也能有效诱导树突状细胞以及T细胞激活扩增，诱导肿瘤和/或癌细胞位点趋化因子的表达上调，并促进肿瘤和/或癌细胞位点树突状细胞以及淋巴细胞浸润，诱导肿瘤和/或癌细胞的MHC-I类和/或MHC-II类分子表达上调，从而能够全面刺激所述患者的机体抗肿瘤免疫反应。
(3)根据(1)所述的应用，其中所述溶瘤病毒是人5型腺病毒、 人5/3嵌合型腺病毒或者人5/2嵌合型腺病毒；优选的是，所述病毒不能表达E1B-55KD多肽或所述病毒的E1A蛋白部分缺失；更优选的是，所述溶瘤病毒是H101。
(4)根据(1)所述的应用，其中所述免疫刺激剂与其它肿瘤免疫制剂联用。
(5)根据(1)所述的应用，其中所述免疫刺激剂包含0.5×10 12vp-1.5×10 12vp/天剂量的所述溶瘤病毒。
(6)一种***和/或癌症的方法，该方法以免疫刺激有效量的选择复制型溶瘤病毒作为肿瘤和/或癌症治疗中的免疫刺激剂对肿瘤和/或癌症患者施用，其中所述溶瘤病毒不携带外源免疫调节基因，其中所述肿瘤患者的免疫功能足以引起抗肿瘤细胞免疫应答，并且所述肿瘤患者的全血白细胞总数≥4.0×10 9/L。
(7)根据(6)所述的方法，其中所述溶瘤病毒除能够直接杀伤肿瘤和/或癌细胞外，也能有效诱导树突状细胞以及T细胞激活扩增，诱导肿瘤和/或癌细胞位点趋化因子的表达上调，并促进肿瘤和/或癌细胞位点树突状细胞以及淋巴细胞浸润，诱导肿瘤和/或癌细胞的MHC-I类和/或MHC-II类分子表达上调，从而能够全面刺激所述患者的机体抗肿瘤免疫反应。
(8)根据(6)所述的方法，其中所述溶瘤病毒是人5型腺病毒、人5/3嵌合型腺病毒或者人5/2嵌合型腺病毒；优选的是，所述病毒不能表达E1B-55KD多肽或所述病毒的E1A蛋白部分缺失；更优选的是，所述溶瘤病毒是H101。
(9)根据(6)所述的方法，其中所述免疫刺激剂的施用方案为，以0.5×10 12vp-1.5×10 12vp/天的所述溶瘤病毒的剂量瘤内注射，每天1次，连续施用4-5天。
(10)根据(6)所述的方法，其中将所述免疫刺激剂与其它肿瘤免疫制剂联合施用。
(11)根(9)所述的方法，其中首次连续施用所述免疫刺激剂结束后间隔7-10天，再次施用所述免疫刺激剂，以进行增强免疫；其中所述增强免疫的施用方案为：以0.5×10 12vp-1.5×10 12vp/天的所述 溶瘤病毒的剂量瘤内注射，每天1次，连续施用1-2天。
(12)根据(11)所述的方法，其中所述增强免疫再重复进行2-3次，并且后次增强免疫的开始时间与前次增强免疫的结束时间间隔7-10天。
本发明与现有技术相比具有以下优点和积极效果：
本发明通过长期研究和实验发现了溶瘤病毒的新用途，即溶瘤病毒可以不经更多基因改造以携带外源免疫调节基因，就能作为肿瘤和/或癌症治疗中的免疫刺激剂，个体化地引起肿瘤抗原体内释放，并作用于机体抗肿瘤免疫***的全部七个环节，全方位激活机体抗肿瘤免疫功能，实现对肿瘤的二次打击，产生持续疗效。
相对于现有的溶瘤病毒(例如H101)抗肿瘤机理与治疗理念，本发明开发了溶瘤病毒抗肿瘤的新原理和新用法，即将溶瘤病毒作为有效的免疫刺激剂来施药。
本发明发现，本发明所述的溶瘤病毒即使不配合其它免疫疗法，单独施药，也可以参与全部肿瘤免疫的七个步骤，在各步骤中产生以下免疫促进作用：
1)溶瘤，促进肿瘤抗原释放；
2)上调外周血DC含量，促进APC(抗原提呈细胞)激活；
3)上调外周血T细胞含量；
4)刺激肿瘤位点趋化因子释放，有助于免疫细胞在肿瘤位点的归巢聚集；
5)促进DC与CD8 +CTL浸入肿瘤实体；
6)刺激肿瘤细胞MHCI/II表达，促进免疫识别；
7)溶瘤病毒溶瘤配合免疫杀伤，有效减小肿瘤负担，肿瘤抗原再释放。
本发明发现本发明所述的溶瘤病毒作为肿瘤和/或癌症治疗中的免疫刺激剂单独给药即可以使溶瘤病毒的溶瘤效应与免疫刺激效应共同发挥作用，从而使得疗效增高。
附图说明
图1示出本申请实验例1中H101体外感染人源和鼠源肿瘤细胞系的MTT检测结果。其中图1A为H101感染A549细胞的结果；图1B为H101感染B16-F0细胞的结果。
图2示出本申请实验例2中H101促进细胞凋亡和肿瘤抗原释放的结果。其中图2A为肿瘤抗原释放的免疫组化结果，其中箭头示出Caspase-3蛋白免疫组化阳性位点；图2B为细胞溶解的HE染色结果，左侧上下图分别取PBS组样本的不同部分，右侧上下图分别取H101组样本的不同部位，其中箭头示出细胞溶解位点。
图3示出本申请实验例3中H101激活树突状细胞的结果。其中图3A为H101给药后第1、3、7天的未成熟DC细胞流式检测结果，纵坐标为脾脏中CD11b +CD11c +双阳性细胞的百分比(％)，横坐标为H101注射后天数；图3B为H101给药后第12天的未成熟DC细胞流式检测结果，纵坐标为脾脏中CD11b +CD11c +双阳性细胞的百分比(％)；图3C-D为H101给药后第12天的成熟DC细胞流式检测结果，图3C的纵坐标为脾脏中CD11c +CD80 +双阳性细胞的百分比(％)，图3D的纵坐标为脾脏中CD11c +CD86 +双阳性细胞的百分比(％)。
图4示出本申请实验例4中H101激活T细胞的结果。其中图4A-C分别为H101给药后第7天的CD3 +、CD4 +和CD8 +T细胞流式检测结果，图4A的纵坐标为脾脏中CD3 +阳性细胞的百分比(％)，图4B的纵坐标为脾脏中CD3 +CD4 +CD8 -细胞的百分比(％)，图4C的纵坐标为脾脏中CD3 +CD4 -CD8 +细胞的百分比(％)；图4D为H101给药后第10天的T淋巴细胞分型流式检测结果，纵坐标为脾脏中CD3 +T细胞的百分比(％)。
图5示出本申请实验例5中趋化因子CXCL12表达的结果，其中箭头示出CXCL12蛋白免疫组化阳性位点。
图6示出本申请实验例6中H101促进NK、DC与CTL浸入肿瘤实体的结果。其中图6A和B分别为H101给药后CD4 +T细胞和处于分泌激活状态的CD4 +T细胞的细胞流式检测结果，图6A的纵 坐标为肿瘤中CD3 +CD4 +双阳性细胞的百分比(％)，图6B的纵坐标为肿瘤中CD4 +CD107 +双阳性细胞的百分比(％)，两图的横坐标为H101注射后天数；图6C和D分别为H101给药后CD8 +T细胞和处于分泌激活状态的CD8+T细胞的细胞流式检测结果，图6C的纵坐标为肿瘤中CD3 +CD8 +双阳性细胞的百分比(％)，图6D的纵坐标为肿瘤中CD8 +CD107 +双阳性细胞的百分比(％)，两图的横坐标为H101注射后天数；图6E为H101给药后NK细胞的细胞流式检测结果，纵坐标为肿瘤中CD3 -NK1.1 +NKG2D +细胞的百分比(％)，横坐标为H101注射后天数；图6F为H101给药后未成熟DC细胞的细胞流式检测结果，纵坐标为肿瘤中CD11b +CD11c +双阳性细胞的百分比(％)，横坐标为H101注射后天数；图6G和H为H101给药后成熟DC细胞的细胞流式检测结果，图6G的纵坐标为肿瘤中CD11c +MHCI +双阳性细胞的百分比(％)，图6H的纵坐标为肿瘤中CD11c +MHCII +双阳性细胞的百分比(％)，两图的横坐标为H101注射后天数。
图7示出本申请实验例7中H101刺激肿瘤细胞MHCI/II表达的结果。其中图7A为MHC-I类分子表达的细胞的流式检测结果，纵坐标为肿瘤中MHCI +阳性细胞的百分比(％)，横坐标为H101注射后天数；图7B为MHC-II类分子表达的细胞的流式检测结果，纵坐标为肿瘤中MHCII +阳性细胞的百分比(％)，横坐标为H101注射后天数。
图8示出本申请实验例8中H101相对抑制肿瘤生长的结果。其中图8A示出肿瘤重量随时间变化的结果；图8B示出肿瘤体积随时间变化的结果；图8C示出肿瘤大小随时间变化的照片。
图9示出本申请实验例9中H101提高实验动物总生存率的结果。
具体实施方式
以下通过具体实施方式的描述并参照附图对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本 思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。
目前采用溶瘤病毒来***和或/癌症的方法主要是利用溶瘤病毒可以特异性地在肿瘤细胞内复制并导致细胞溶解死亡的机理。专利号为US 5,677,178的美国专利公开了一种溶瘤腺病毒ONYX-015。ONYX-015是一种删除了人野生5型腺病毒DNA序列中的E1B-55KD片段之后构建而成的特异性溶瘤腺病毒。ONYX-015对正常细胞无明显影响，但是可以选择性地在肿瘤细胞中复制、增殖，最后导致瘤细胞溶解。1996年人们已经开始利用ONYX-015进行头颈部肿瘤、脑胶质瘤、胰腺癌、原发肝胆肿瘤、大肠癌肝转移灶、非小细胞肺癌和***等恶性肿瘤治疗的临床试验，试验结果显示出该方法具有一定疗效和安全性。授权公告号为CN1110553C的中国专利也公开了一种溶瘤腺病毒H101(保藏号为CCTCC No.V98003)。H101是利用基因工程技术对人5型腺病毒(Ad5)进行基因重组而得的一种溶瘤腺病毒，其不能表达E1B-55KD多肽并缺失了E3区。H101可以在肿瘤细胞中特异性复制，最终导致细胞溶解，从而达到溶瘤的目的。H101于2006年由CFDA批准上市，NDA号为：国药准字S20060027，商品名为安柯瑞。
以上这些溶瘤病毒制剂与其它很多已开发的靶向特异性抗肿瘤药的治疗理念和用药目的相同，均作为期望直接作用于肿瘤本身并由此对其起到抑制效果的药物被使用，即，这些制剂利用了溶瘤病毒能够选择性地在肿瘤细胞中复制从而使其溶解死亡的作用。
然而，本发明通过长期研究和实验，发现了溶瘤病毒的新用途，即，本发明所述的溶瘤病毒可以作为肿瘤和/或癌症治疗中的免疫刺激剂使用，其能够个体化地引起肿瘤抗原体内释放，并作用于机体抗肿瘤免疫***的全部七个环节，全方位激活机体抗肿瘤免疫功能，实现对肿瘤的二次打击，产生持续疗效。
在本发明的发明人发现，本发明所述的溶瘤病毒不经更多基因改造以携带外源免疫调节基因，就可以发挥刺激机体抗的肿瘤免疫的各个环节，全方位激活机体抗肿瘤免疫功能。因此与T-Vec产品不 同，本发明发现选择复制型溶瘤病毒本身(不携带外源免疫调节基因)即可作为肿瘤和/或癌症治疗中的免疫刺激剂。
另外，为了更好地发挥免疫刺激剂的作用，本发明的发明人进一步发现其施用对象应为机体的免疫功能足以引起抗肿瘤细胞免疫应答的肿瘤和/或癌症患者，所述肿瘤和/或癌症患者的全血白细胞总数应大于或等于4.0×10 9/L。
基于以上发现，本发明提供了选择复制型溶瘤病毒在制备用于***和/或癌症的免疫刺激剂中的应用，其中所述溶瘤病毒不携带外源免疫调节基因，并且其中所述免疫刺激剂的施用对象为，其机体的免疫功能足以引起抗肿瘤细胞免疫应答的肿瘤和/或癌症患者，其中所述肿瘤和/或癌症患者的全血白细胞总数≥4.0×10 9/L。
所述溶瘤病毒除能够直接杀伤肿瘤和/或癌细胞外，也能有效诱导树突状细胞以及T细胞激活扩增，诱导肿瘤和/或癌细胞位点趋化因子的表达上调，并促进肿瘤和/或癌细胞位点树突状细胞以及淋巴细胞浸润，诱导肿瘤和/或癌细胞的MHC-I类和/或MHC-II类分子表达上调，从而能够全面刺激所述患者的机体抗肿瘤免疫反应。
在本发明中，优选地，所述溶瘤病毒是人5型腺病毒、人5/3嵌合型腺病毒或者人5/2嵌合型腺病毒。优选的是，所述病毒不能表达E1B-55KD多肽或所述病毒的E1A蛋白部分缺失。更优选的是，所述溶瘤病毒至少缺失了E3区部分基因。进一步优选的是，所述溶瘤病毒是H101。
优选地，所述免疫刺激剂包含临床施用剂量的所述溶瘤病毒，例如包含0.5×10 12vp-1.5×10 12vp/天剂量的所述溶瘤病毒。
所述溶瘤病毒作为免疫刺激剂的施用方案可以为：以0.5×10 12vp-1.5×10 12vp/天的所述溶瘤病毒的剂量瘤内或静脉注射，每天1次，连续施用4-5天；或将溶瘤病毒加载于细胞载体输注，例如将所述溶瘤病毒加载于体外培养的自体或异体的免疫细胞，并通过静脉输注等方式给药(Ilett et al.,2009,Dendritic cells and T cells deliver oncolytic reovirus for tumour killing despite pre-existing anti-viral immunity,Gene therapy,16,689-699)。
本发明所述的所述免疫刺激剂还可以与其它肿瘤免疫制剂联合施用，该其它肿瘤免疫制剂包括但不限于：部分有免疫刺激作用的白介素类细胞因子，如IL-1、IL-2、IL-7、IL-12、IL-15、IL-21；干扰素类，如IFN-alpha；集落刺激因子，如G-CSF、GM-CSF；肿瘤坏死因子，如TNF-alpha；免疫细胞因子，如CD40L/CD40；趋化因子，如CCL-3、CCL-5、CCL-26、CX3CL1、CXCL9、CXCL10；Toll样受体(TLR，Toll-like receptor)信号相关分子；以及免疫点相关制剂，如抗PD-1抗体、抗CTLA4抗体。
本发明另一个方面提供了一种***和/或癌症的方法，该方法以免疫刺激有效量的本发明所述的选择复制型溶瘤病毒作为肿瘤和/或癌症治疗中的免疫刺激剂对肿瘤和/或癌症患者施用，其中所述溶瘤病毒不携带外源免疫调节基因，所述肿瘤患者的免疫功能足以引起抗肿瘤细胞免疫应答，并且所述肿瘤患者的全血白细胞总数≥4.0×10 9/L。
在本发明的方法中，所述溶瘤病毒除能够直接杀伤肿瘤和/或癌细胞外，也能有效诱导树突状细胞以及T细胞激活扩增，诱导肿瘤和/或癌细胞位点趋化因子的表达上调，并促进肿瘤和/或癌细胞位点树突状细胞以及淋巴细胞浸润，诱导肿瘤和/或癌细胞的MHC-I类和/或MHC-II类分子表达上调，从而能够全面刺激所述患者的机体抗肿瘤免疫反应。
优选地，在所述方法中，所述免疫刺激剂的施用方案为，以0.5×10 12vp-1.5×10 12vp/天的所述溶瘤病毒的剂量瘤内注射，每天1次，连续施用4-5天。
在本发明的一个实施方案中，可以将所述免疫刺激剂与其它肿瘤免疫制剂联合施用。该其它肿瘤免疫制剂包括但不限于：部分有免疫刺激作用的白介素类细胞因子，如IL-1、IL-2、IL-7、IL-12、IL-15、IL-21；干扰素类，如IFN-alpha；集落刺激因子，如G-CSF、GM-CSF；肿瘤坏死因子，如TNF-alpha；免疫细胞因子，如CD40L/CD40；趋化因子，如CCL-3、CCL-5、CCL-26、CX3CL1、CXCL9、CXCL10；Toll样受体(TLR，Toll-like receptor)信号相关分子；以及免疫点相 关制剂，如抗PD-1抗体、抗CTLA4抗体。
根据本发明，H101给药后在第7天可以显著诱导DC增殖与肿瘤浸润，此时增强免疫，可协同促进免疫效果。所以在本发明的另一个实施方案中，首次连续施用所述免疫刺激剂结束后间隔7-10天，更优选地，间隔7天，再次施用所述免疫刺激剂，以进行增强免疫；其中所述增强免疫的施用方案为：以0.5×10 12vp-1.5×10 12vp/天的所述溶瘤病毒的剂量瘤内注射，每天1次，连续施用1-2天。增强免疫结束后间隔7-10天，可再次进行增强免疫，剂量与施用方式同上。以此类推，可进行3-4次增强免疫。
以下通过例子的方式进一步解释或说明本发明的内容，但这些例子不应被理解为对本发明的保护范围的限制。
例子
在以下例子中，如果没有特别说明，所用方法和技术均采用本领域的常规操作和条件进行。
以下例子中所用的材料如下：
A549人非小细胞肺癌细胞、B16-F0小鼠黑色素瘤细胞均来源于中国科学院典型培养物保藏委员会细胞库。
溶瘤腺病毒H101来源于上海三维生物技术有限公司。
C57BL/6小鼠(雄性，6-8周龄)来源于北京维通利华实验动物技术有限公司。细胞流式仪来源于艾森生物(杭州)有限公司，型号为D2060R。
体外实验至少重复3次，每次重复实验每组至少3个复孔，并进行统计分析。
除非特别说明，体内试验每组至少5只实验动物。
实验例1：体外感染人源和鼠源肿瘤细胞系
分别在96孔细胞培养板中每孔种植3×10 3个A549细胞或4×10 3个B16-F0细胞，37℃、5％CO 2下培养于含10％FBS的高糖DMEM 中，12小时后(待细胞贴壁完全)，加入H101(A549细胞系为10MOI，B16-F0细胞系为100MOI)孵育，12小时后更换新鲜培养基，从加入病毒时间为0小时记，每24小时进行MTT检测(MTT试剂购于Sigma-Aldrich公司，货号M5655-1G)。对照组为完全培养基不加任何溶瘤病毒。
H101感染A549细胞后第3天开始，MTT结果显示活细胞急剧减少，同时镜下观察到细胞大量死亡破溃。表明H101体外感染可以直接杀伤人源肿瘤细胞系并促进肿瘤抗原释放(如图1A所示)。而感染MOI提高10倍后，仍然不能观察到对小鼠源B16-F0细胞的直接抑制增殖作用(如图1B所示)。
实验例2：体内有效促进鼠源肿瘤细胞系肿瘤抗原的释放
为研究H101的免疫学效应，本发明开发了一种鼠源黑色素瘤皮下荷瘤的动物模型，并首先验证了H101对该黑色素瘤细胞系(B16-F0)的体外细胞毒性作用。结果表明，因人5型腺病毒的感染种属特异性，H101对小鼠B16-F0细胞系的直接杀伤作用极其微弱(结果见实验例1，图1B)。该模型符合尽量排除H101的直接溶瘤效应而研究其免疫刺激剂效应的研究目的。之后，研究了H101在所述动物模型体内对该细胞系(B16-F0)的促凋亡和促进全抗原释放的作用。
B16-F0细胞以2×10 6个细胞/100μl DMEM的量在C57BL/6雄性小鼠右侧背部注射荷瘤，每只动物100μl。荷瘤第7天，待肿瘤体积约为150mm 3，瘤内注射H101 2×10 10vp/50μl pH7.4PBS(PBS试剂购于生工生物工程(上海)股份有限公司，货号B548117)，连续4天，每天1次，此为实验组；同时对照组连续瘤内注射50μl PBS，4天，每天1次。以第一天给药时间为第0天，在第3、7、10、13天处死小鼠取肿瘤进行Caspase-3的免疫组化(IHC)检测(抗鼠Caspase-3抗体购于R&D Systems公司，货号AF-605-NA)以及HE染色(苏木素伊红(HE)染色试剂盒购于生工生物工程(上海)股份有限公司，货号E607318)。
IHC结果显示，Caspase-3主要表达于细胞核。在H101组中，第7和第10天的Caspase-3表达量明显高于第3天；在第7和第10天，H101实验组的Caspase-3蛋白阳性细胞数量明显高于PBS对照组(如图2A所示)，并且仅在第13天观察到明显的细胞溶解现象(如图2B所示)。Caspase-3是细胞凋亡的主要指标，而细胞溶解现象是免疫原性细胞死亡的重要表现，因此本实验说明所用溶瘤病毒可以诱导细胞凋亡以及进而诱发免疫原性细胞死亡，从而有效促进肿瘤抗原释放。
实验例3：刺激机体免疫***，激活树突状细胞(DC)
B16-F0细胞以2×10 6个细胞/100μl DMEM的量在C57BL/6雄性小鼠右侧背部注射荷瘤，每只动物100μl。荷瘤第7天，实验组瘤内注射H101(1×10 10vp/60μl PBS)1天(共1次)，同时对照组瘤内注射60μl PBS 1天(共1次)。以第一天给药时间为第0天，在第1、3、7天处死取脾脏进行DC细胞流式检测(抗鼠CD11b-FITC与CD11c-APC抗体购于eBioscience公司，货号分别为11-0112-85和17-0114-81)。结果如图3A所示。
B16-F0细胞以2×10 6个细胞/100μl DMEM的量在C57BL/6雄性小鼠右侧背部注射荷瘤，每只动物100μl。荷瘤第7天，实验组连续瘤内注射H101(5×10 9vp/50μl PBS)4天，每天1次，同时对照组连续瘤内注射50μl PBS 4天，每天1次。以第一天给药时间为第0天，在第12天处死取脾脏进行DC细胞流式检测(抗鼠CD80-APC、CD11b-FITC与CD11c-APC抗体购于eBioscience公司，货号分别为17-0801-82、11-0112-85和17-0114-81；抗鼠CD86-FITC抗体购于BD Pharmingen公司，货号为561962)。结果如图3B/C/D所示。
连续监测H101给药后的实验动物，发现给药后第7天开始，以脾脏为代表的外周血内未成熟DC显著上调(图3A)。到给药后第12天，动物脾脏内未成熟DC(CD11b +CD11c +)与成熟DC(CD11c +CD80 +或CD11c +CD86 +)仍有上调趋势(图3B-D)。
实验例4：刺激机体免疫***，激活T细胞
B16-F0细胞以1.5×10 6个细胞/100μl DMEM的量在C57BL/6雄性小鼠右侧背部注射荷瘤，每只动物100μl，荷瘤第7天，实验组连续瘤内注射H101(4×10 9vp/50μl PBS)4天，每天1次，同时对照组连续瘤内注射50μl PBS 4天，每天1次。以第一天给药时间为第0天，在第7天处死取脾脏进行T淋巴细胞流式检测(抗鼠CD3e PE-Cyanine7抗体购于eBioscience公司，货号为35-0031-82；抗鼠CD4-FITC和CD8-APC抗体购于BD Pharmingen公司，货号分别为553729和553035)。结果如图4A/B/C所示。
B16-F0细胞以2×10 6个细胞/100μl DMEM的量在C57BL/6雄性小鼠右侧背部注射荷瘤，每只动物100μl，荷瘤第7天，实验组瘤内注射H101(1×10 10vp/60μl PBS)1天(共1次)，同时对照组瘤内注射60μl PBS 1天(共1次)。以第一天给药时间为第0天，在第10天处死取脾脏进行T淋巴细胞分型流式检测(抗鼠CD3e PE-Cyanine7抗体购于eBioscience公司，货号为35-0031-82)。结果如图4D所示。
H101给药后第7天，脾脏中CD3 +、CD4 +和CD8 +T细胞均显著上调(图4A-C)。第10天，T细胞亚型分析(抗鼠CD4-FITC和CD8-APC抗体购于BD Pharmingen公司，货号分别为553729和553035；抗鼠CD44-PE和CD62L-APC抗体购于eBioscience公司，货号分别为12-0441-83和17-0621-82)表明在CD3 +T细胞总群中，H101给药组实验动物Naive T(原初T，CD44 low CD62L hi)细胞和Tcm(中心记忆T细胞，CD44 hi CD62L hi)下调，而Tem(效应记忆T细胞，CD44 hi CD62L low)上调，表明T细胞免疫动员，机体T细胞免疫激活(图4D)。
实验例5：瘤内给药刺激肿瘤位点趋化因子CXCL12表达，有助于免疫细胞在肿瘤位点归巢与聚集
B16-F0细胞以1×10 6个细胞/100μl DMEM的量在C57BL/6雄性小鼠右侧背部注射荷瘤，每只动物100μl，荷瘤第7天，实验组连续瘤内注射H101(4×10 9vp/50μl PBS)4天，每天1次，同时对照组连续瘤内注射50μl PBS 4天，每天1次。以第一天给药时间为第0天，在 第13天处死取肿瘤进行趋化因子CXCL12的IHC检测(抗鼠CXCL12抗体购于Abcam公司，货号AB25117)。结果如图5所示。
IHC结果显示，H101给药后第13天，肿瘤位点内趋化因子CXCL12表达上调，该趋化因子上调可促进血管增殖，改善肿瘤实体通透性，有利于免疫细胞在肿瘤位点的归巢与聚集。
实验例6：瘤内注射溶瘤病毒促进NK、DC与CTL浸入肿瘤实体
B16-F0细胞以2×10 6个细胞/100μl DMEM的量在C57BL/6雄性小鼠右侧背部注射荷瘤，每只动物100μl，荷瘤第7天，待肿瘤体积约为150mm 3，实验组连续瘤内注射H101(2×10 10vp/50μl PBS)4天，每天1次，同时对照组连续瘤内注射50μl PBS 4天，每天1次。以第一天给药时间为第0天，在第3、7、10天处死，取肿瘤进行流式细胞分析(抗鼠CD314(NKG2D)-APC、NK1.1-FITC、CD107a-PE、CD3e PE-Cyanine7、MHC Class I(H-2Kd)-PE和MHC Class II(I-A/I-E)-PE-Cyanine7抗体购于eBioscience公司，货号分别为17-5882-82、11-5941-85、12-1071-83、35-0031-82、12-5957-82和25-5321-82；抗鼠CD4-FITC和CD8-APC抗体购于BD Pharmingen公司，货号分别为553729和553035；抗鼠CD11b-FITC与CD11c-APC抗体购于eBioscience公司，货号分别为11-0112-85和17-0114-81)。结果如图6所示。
H101给药后第3和第7天，肿瘤组织内CD4 +T细胞与处于分泌激活状态的CD4 +T细胞(CD4 +CD107a +)显著上调(图6A、B)；而CD8 +T细胞在第7天显著上调，并且在第7和第10天，CD8 +T细胞与处于分泌激活状态的CD8 +T细胞(CD8 +CD107a +)有上调趋势(图6C、D)。给药后第7天，NK细胞显著上调(图6E)。同时肿瘤内未成熟DC(CD11b +CD11c +)在第7天显著上调，而成熟DC(CD11c +MHCI +或者CD11c +MHCII +)在第3、7天显著上调(图6F-H)。
实验例7：注射刺激肿瘤细胞MHCI/II表达，增强免疫识别
B16-F0细胞以2×10 6个细胞/100μl DMEM的量在C57BL/6雄性 小鼠右侧背部注射荷瘤，每只动物100μl，荷瘤第7天，待肿瘤体积约为150mm 3，实验组连续瘤内注射H101(2×10 10vp/50μl PBS)4天，同时对照组连续瘤内注射50μl PBS 4天。以第一天给药时间为第0天，在第3、7、10天处死，取肿瘤进行流式细胞分析(抗鼠MHC Class I(H-2Kd)-PE和MHC Class II(I-A/I-E)-PE-Cyanine7抗体购于eBioscience公司，货号分别为12-5957-82和25-5321-82)。结果如图7A和B所示。
H101给药后第3和第7天，肿瘤位点内MHC-I类分子表达的细胞百分比显著上调；第7和第10天，MHC-II类分子表达的细胞百分比显著上调。MHC类分子表达上调有力促进了肿瘤免疫识别反应，提高免疫细胞杀伤效率。
实验例8：相对抑制实验动物肿瘤生长
B16-F0细胞以2×10 6个细胞/100μl DMEM的量在C57BL/6雄性小鼠右侧背部注射荷瘤，每只动物100μl，荷瘤第7天，待肿瘤体积约为150mm 3，实验组连续瘤内注射H101(2×10 10vp/50μl PBS)4天，每天1次，同时对照组连续瘤内注射50μl PBS 4天，每天1次。以第一天给药时间为第0天，每隔两天测量肿瘤尺寸，并在第3、7、10天处死取肿瘤称重。结果如图8所示。
与对照组相比，H101给药7天以后，动物肿瘤体积明显小于PBS注射的对照组，差异显著(图8A-C)。
实验例9：提高实验动物总生存率
B16-F0细胞以2×10 6个细胞/100μl DMEM的量在C57BL/6雄性小鼠右侧背部注射荷瘤，每只动物100μl，荷瘤第7天，实验组连续瘤内注射H101(4×10 9vp/50μl PBS)4天，每天1次，同时对照组连续瘤内注射50μl PBS 4天，每天1次。每天观察小鼠存活状况。结果如图9所示。
存活率实验结果显示，H101给药可显著延长实验动物的总生存率。
虽然小鼠源B16-F0细胞系在体外杀伤实验中，增殖几乎不受H101影响，但体内给药结果显示，H101给药相对对照组，可以显著抑制实验动物肿瘤生长。结合以上各实验的结果，可以相信H101作为免疫刺激剂，可以有效提高免疫功能健全的动物荷瘤模型的抑瘤效果。其药效不仅依靠溶瘤病毒的直接溶瘤效应，更可以在抗肿瘤免疫的全部七个步骤中起到促进作用，有效刺激机体抗肿瘤免疫。

Claims (12)

  1. 选择复制型溶瘤病毒在制备用于***和/或癌症的免疫刺激剂中的应用，其中所述溶瘤病毒不携带外源免疫调节基因，并且其中所述免疫刺激剂的施用对象为，其机体的免疫功能足以引起抗肿瘤细胞免疫应答的肿瘤和/或癌症患者，其中所述肿瘤和/或癌症患者的全血白细胞总数≥4.0×10 9/L。
  2. 根据权利要求1所述的应用，其中所述溶瘤病毒除能够直接杀伤肿瘤和/或癌细胞外，也能有效诱导树突状细胞以及T细胞激活扩增，诱导肿瘤和/或癌细胞位点趋化因子的表达上调，并促进肿瘤和/或癌细胞位点树突状细胞以及淋巴细胞浸润，诱导肿瘤和/或癌细胞的MHC-I类和/或MHC-II类分子表达上调，从而能够全面刺激所述患者的机体抗肿瘤免疫反应。
  3. 根据权利要求1所述的应用，其中所述溶瘤病毒是人5型腺病毒、人5/3嵌合型腺病毒或者人5/2嵌合型腺病毒；优选的是，所述病毒不能表达E1B-55KD多肽或所述病毒的E1A蛋白部分缺失；更优选的是，所述溶瘤病毒是H101。
  4. 根据权利要求1所述的应用，其中所述免疫刺激剂与其它肿瘤免疫制剂联用。
  5. 根据权利要求1所述的应用，其中所述免疫刺激剂包含0.5×10 12vp-1.5×10 12vp/天剂量的所述溶瘤病毒。
  6. 一种***和/或癌症的方法，该方法以免疫刺激有效量的选择复制型溶瘤病毒作为肿瘤和/或癌症治疗中的免疫刺激剂对肿瘤和/或癌症患者施用，其中所述溶瘤病毒不携带外源免疫调节基因， 其中所述肿瘤患者的免疫功能足以引起抗肿瘤细胞免疫应答，并且所述肿瘤患者的全血白细胞总数≥4.0×10 9/L。
  7. 根据权利要求6所述的方法，其中所述溶瘤病毒除能够直接杀伤肿瘤和/或癌细胞外，也能有效诱导树突状细胞以及T细胞激活扩增，诱导肿瘤和/或癌细胞位点趋化因子的表达上调，并促进肿瘤和/或癌细胞位点树突状细胞以及淋巴细胞浸润，诱导肿瘤和/或癌细胞的MHC-I类和/或MHC-II类分子表达上调，从而能够全面刺激所述患者的机体抗肿瘤免疫反应。
  8. 根据权利要求6所述的方法，其中所述溶瘤病毒是人5型腺病毒、人5/3嵌合型腺病毒或者人5/2嵌合型腺病毒；优选的是，所述病毒不能表达E1B-55KD多肽或所述病毒的E1A蛋白部分缺失；更优选的是，所述溶瘤病毒是H101。
  9. 根据权利要求6所述的方法，其中所述免疫刺激剂的施用方案为，以0.5×10 12vp-1.5×10 12vp/天的所述溶瘤病毒的剂量瘤内注射，每天1次，连续施用4-5天。
  10. 根据权利要求6所述的方法，其中将所述免疫刺激剂与其它肿瘤免疫制剂联合施用。
  11. 根据权利要求9所述的方法，其中首次连续施用所述免疫刺激剂结束后间隔7-10天，再次施用所述免疫刺激剂，以进行增强免疫；其中所述增强免疫的施用方案为：以0.5×10 12vp-1.5×10 12vp/天的所述溶瘤病毒的剂量瘤内注射，每天1次，连续施用1-2天。
  12. 根据权利要求11所述的方法，其中所述增强免疫再重复进行2-3次，并且后次增强免疫的开始时间与前次增强免疫的结束时间间隔7-10天。
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