WO2021163874A1 - 一种重组病毒载体、包含其的免疫组合物以及用途 - Google Patents

一种重组病毒载体、包含其的免疫组合物以及用途 Download PDF

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WO2021163874A1
WO2021163874A1 PCT/CN2020/075677 CN2020075677W WO2021163874A1 WO 2021163874 A1 WO2021163874 A1 WO 2021163874A1 CN 2020075677 W CN2020075677 W CN 2020075677W WO 2021163874 A1 WO2021163874 A1 WO 2021163874A1
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tumor
human
viral vector
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cells
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徐建青
黄杨
孙甲浩
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苏州工业园区唯可达生物科技有限公司
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Priority to JP2022549503A priority Critical patent/JP7543419B2/ja
Priority to EP20920014.6A priority patent/EP4108246A4/en
Priority to US17/800,637 priority patent/US20230270847A1/en
Priority to PCT/CN2020/075677 priority patent/WO2021163874A1/zh
Priority to CN202080096622.2A priority patent/CN115103681B/zh
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Definitions

  • the invention belongs to the field of molecular biology and immunology. Specifically, the present invention relates to a recombinant virus vector, an immune composition containing the same, and uses. In particular, the present invention relates to a recombinant virus that can be used to prepare tumor vaccines for the prevention and/or treatment of various tumors. Carrier.
  • tumor immunotherapy has made great progress, and has gradually become an important development direction of current tumor treatment.
  • PD-1/PD-L1 immunotherapy the development of tumor immunotherapy has become an emerging hot spot in current tumor treatment.
  • Tumor vaccines stimulate the body's specific immune response against tumors and activate huge immune cells to kill tumor cells and control the growth of tumor cells, thereby achieving the effect of reducing or controlling tumor growth.
  • the development of tumor vaccines is also the key research direction of current tumor immunotherapy.
  • cytotoxic (CD8+) and helper (CD4+) T cells play a key role in tumor rejection. Therefore, the goal of most tumor vaccines is to induce cell-specific T cell responses.
  • the decomposed polypeptides are presented through MHC class I molecules to activate CD8+ T cells on the surface of tumor cells.
  • MHC class II molecules are recognized by CD4+ T cells and are mainly located on the surface of special antigen-presenting cells (APC), including dendritic cells, B cells and macrophages. Exogenous proteins secreted by tumor cells or released by tumor lysis are captured by APC.
  • APC antigens are processed into polypeptide fragments and presented to CD4+ cells by MHC class II. The activated antigen-specific CD8+ cells eventually become cytotoxic T cells and lyse tumor cells.
  • the ideal tumor-specific antigen should have strong immunogenicity and be expressed by tumor cells but not on normal cells. Unfortunately, most tumor antigens are not sufficiently immunogenic to induce an effective immune response, and many tumor antigens are expressed in normal tissues to some extent, leading to immune tolerance in the body. Therefore, these tumor antigens naturally have the characteristics of weak immunogenicity, and the designed tumor vaccine must overcome the body's immune tolerance barriers and activate the immune response against tumor antigens.
  • Tumor vaccines are mainly divided into whole cell vaccines, protein vaccines, peptide vaccines, virus vaccines and dendritic cell vaccines.
  • DNA vaccines and RNA vaccines are still molecular vaccines, but use different expression systems.
  • tumor antigen vaccines In the past, the relationship between tumor antigens and their clinical relevance was not very clear. Therefore, whole cells are used as tumor vaccines so that unknown tumor antigens can be provided to activate the immune system. In mouse tumor models, radiation-inactivated tumor cells are usually used to immunize mice to protect the mice from the inoculated tumor. But when the tumor cell vaccine was postponed to one week after the tumor cell inoculation, the vaccine lost its ability to protect mice. The clinical treatment response of tumor cell vaccine is relatively poor, and it is only suitable for the prevention of recurrence of tumor patients without special tumor antigens. For advanced patients, good results are rarely obtained in clinical research. In recent years, due to the progress in the recognition and analysis of tumor antigens, especially the in-depth understanding of the mechanism of T cell recognition of antigens, tumor antigen vaccines have basically replaced cell vaccines for tumor immunotherapy.
  • Polypeptide and protein vaccines The antigenic polypeptide epitopes on the surface of MHC molecules recognized by T cells are generally 7-12 amino acids. Therefore, antigenic polypeptides can be mixed with immune adjuvants to achieve the purpose of loading empty MHC molecules in the body. So far, almost all peptide-based vaccines are MHC class I antigen-restricted peptides. There are some limitations in the application of peptide vaccines. The peptide vaccine used must match the patient’s MHC class I antigen molecules, which is so-called individualization. However, due to the different subtypes of the MHC class I molecules of different patients, the sequence of the tumor antigen peptides used is also different. This brings great difficulties to the clinical application of tumor antigen peptides.
  • Recombinant molecular vaccines The application of tumor antigen protein vaccines can overcome this difficulty, but the use of protein alone cannot activate the body's immune response. Primate studies have confirmed that the best immune effect requires cross-linking tumor proteins with strong immunogenic proteins. For weak antigens to induce an effective immune response, an immune adjuvant must be used in combination to provide a non-specific signal to activate the immune system. Many immune adjuvants have certain toxicity and cannot be used in clinical practice. Therefore, antigen protein vaccines are mostly used. Appeared in the form of reorganization.
  • the method to enhance the immunogenicity of tumor proteins in a recombinant form is to recombine tumor antigens with cytokines such as GM-CSF and interleukins to form fusion proteins.
  • cytokines such as GM-CSF and interleukins
  • the recombination of weak tumor antigens with bacterial or viral antigens, toxins such as diphtheria toxin, pseudomonas toxin, etc. can significantly improve the antigenicity of tumor antigens, promote the phagocytosis and presentation of tumor antigens by DC, and has achieved certain effects.
  • the method of separate recombination of tumor antigen and toxin has not yet achieved the desired effect so far.
  • Dendritic cell vaccine For an effective T cell-mediated immune response, T cells need antigen to be presented and sensitize the initial T cells, and the sensitized T lymphocytes are re-stimulated. To initiate effective T cell-mediated tumor immunity, tumor antigen polypeptides derived from any part of the body must be recognized by T cells. Therefore, the presentation of antigen is a key step to obtain an effective immune response.
  • the immune response stimulated by the vaccine mainly depends on the initial processing and further presentation of the antigen by the effective APC.
  • Interleukin 7 is a hematopoietic growth factor secreted by bone marrow and thymic stromal cells. It can also be produced by keratinocytes, dendritic cells, hepatocytes, neuronal cells and epithelial cells, but normal lymphocytes will not secrete IL-7. IL-7 promotes the differentiation of hematopoietic stem cells into lymphoid precursor cells. It can also stimulate the proliferation of all cells of the lymphatic line, such as B cells, T cells and NK cells. It is essential for the maturation of B cells at a specific stage, the survival, development and balance of T cells and Natural Killer Cells (NK cells).
  • NK cells Natural Killer Cells
  • Interleukin 15 is secreted by mononuclear phagocytes after virus infection. Its structure is similar to Interleukin 2 (IL-2). IL-15 transmits signals by binding to the IL-2/IL-15 receptor ⁇ chain (CD122) and the shared receptor ⁇ chain (CD132) complex. This cytokine regulates the activation and proliferation of T cells and NK cells. But in the absence of antigen, IL-15 can provide a signal to maintain the survival of memory T cells. IL-15 has been shown in preclinical studies to enhance the anti-tumor effect of CD8+ T cells. IL-15 can also be used as a vaccine adjuvant to increase vaccine immunogenicity.
  • Interleukin 21 is secreted by activated CD4+ T cells, which can regulate various cells of the immune system. IL-21 can achieve anti-tumor effects by continuously increasing CD8+ T cell responses (Journal of Immunology.173(2):900-9). IL-21 also plays an important role in the control of chronic viral infections. In HIV-infected people, IL-21 can enhance HIV-specific cytotoxic T cell response (Blood.109(9):3873-80.) and NK cell function (Journal of Leukocyte Biology.87(5):857-67) .)
  • Granulocyte-macrophage colony-stimulating factor (Granulocyte-macrophage colony-stimulating factor, GM-CSF), also known as colony-stimulating factor 2 (colony-stimulating factor 2, CSF2) is composed of macrophages, T cells, hypertrophy A monomeric glycoprotein secreted by cells, NK cells, endothelial cells and fibroblasts.
  • GM-CSF has a variety of functions, can stimulate stem cells to produce a variety of granulocytes and monocytes, quickly activate and proliferate a large number of macrophages, so as to achieve the effect of anti-infection.
  • the oncolytic virus therapy with GM-CSF developed by Amgen has been approved by the FDA for the treatment of melanoma.
  • the present invention jointly expresses multiple immune-related cytokines on a recombinant virus vector, in order to improve vaccine immunity Effect and play the anti-tumor effect of cytokines.
  • the purpose of the present invention is to provide a recombinant viral vector, which can be used to prepare tumor vaccines, thereby preventing and/or treating various tumors.
  • the recombinant viral vector contains a polynucleotide encoding a cytokine.
  • Cytokine (CK) refers to immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) Etc.)
  • CK Cytokine
  • Cytokines can be divided into interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors and so on.
  • the cytokine is selected from one or more of IL-7, IL-15, IL-21 and GM-CSF.
  • the IL-15 is human IL-15; preferably, the amino acid sequence of the human IL-15 is shown in SEQ ID NO: 2; preferably, the encoding nucleic acid sequence is shown in SEQ ID NO:1 shows.
  • the GM-CSF is human GM-CSF; preferably, the amino acid sequence of the human GM-CSF is shown in SEQ ID NO: 4; preferably, the coding nucleic acid sequence is shown in SEQ ID NO: shown in 3.
  • the IL-7 is human IL-7; preferably, the amino acid sequence of the human IL-7 is shown in SEQ ID NO: 6; preferably, the encoding nucleic acid sequence is shown in SEQ ID NO: shown in 5.
  • the IL-21 is human IL-21; preferably, the amino acid sequence of the human IL-21 is shown in SEQ ID NO: 8; preferably, the encoding nucleic acid sequence is shown in SEQ ID NO: 7 shows.
  • the cytokine includes human IL-15, human GM-CSF, human IL-7 and human IL-21; preferably, the nucleic acid sequence of the polynucleotide encoding the cytokine It is shown in SEQ ID NO: 15; preferably, the encoded amino acid sequence is shown in SEQ ID NO: 16.
  • Tumor antigen refers to an antigenic substance that is newly emerged or overexpressed during the occurrence and development of tumors.
  • Tumor antigens include, but are not limited to, tumor-specific antigens, tumor-associated antigens, tissue differentiation antigens, proto-oncovirus antigens, and tumor-testis antigens (CT antigens).
  • Tumor-Specific Antigens refers to antigen substances that are only expressed in tumor cells and not expressed in normal cells.
  • mutant antigens especially the mutant products of proto-oncogenes and tumor suppressor genes, including ras and p53.
  • Tumor-Associated Antigens (Tumor-Associated Antigens, TAA) refers to antigenic substances expressed in tumor cells and some normal cells.
  • the viral vector is a vaccinia virus vector, preferably a replicating vaccinia virus vector, such as a vaccinia virus Tiantan strain, such as 752-1 strain, or a non-replicating vaccinia virus vector, such as an attenuated vaccinia virus vaccine Ankara strain ( Modified Vaccinia Ankara, MVA).
  • a replicating vaccinia virus vector such as a vaccinia virus Tiantan strain, such as 752-1 strain
  • a non-replicating vaccinia virus vector such as an attenuated vaccinia virus vaccine Ankara strain ( Modified Vaccinia Ankara, MVA).
  • Another object of the present invention is to provide an immune composition comprising a preventive and/or therapeutically effective amount of the recombinant viral vector according to the present invention, and a pharmaceutically acceptable carrier.
  • Another object of the present invention is to provide a tumor vaccine comprising a preventive and/or therapeutically effective amount of the recombinant viral vector according to the present invention, and a pharmaceutically acceptable carrier.
  • Another object of the present invention is to provide a kit containing the recombinant virus vector, immune composition or tumor vaccine according to the present invention, and instructions for use thereof.
  • the present invention also provides the use of the recombinant viral vector, immune composition or tumor vaccine according to the present invention in the preparation of drugs or vaccines for treating and/or preventing tumors.
  • the tumor is a malignant tumor. More preferably, the malignant tumor is breast cancer or colon cancer.
  • the present invention also provides a method for treating and/or preventing tumors, the method comprising administering to a subject in need a preventive and/or therapeutically effective amount of the recombinant viral vector, immune composition or tumor according to the present invention Vaccine; preferably, the tumor is a malignant tumor; more preferably, the malignant tumor is breast cancer or colon cancer.
  • the recombinant virus vector provided by the present invention can stimulate a tumor-specific immune response, effectively inhibit the growth of tumor cells, and prolong the survival time of tumor patients.
  • Figure 1 and Figure 2 are the plasmid map and double restriction identification map of the shuttle vector vector pSC65CY with cytokine coding sequence, respectively.
  • Figure 3 shows the detection result of human GM-CSF activity in Example 5.
  • Figure 4 shows the detection result of human IL-21 activity in Example 6.
  • the human cytokine amino acids and their nucleic acid sequences are from NCBI database.
  • the human cytokine IL-15 (NM_000585.4, the nucleic acid sequence is shown in SEQ ID NO: 1, the amino acid sequence is shown in SEQ ID NO: 2), and the GM-CSF (NM_000758.3, the nucleic acid sequence is shown in SEQ ID NO: 3, the amino acid sequence is shown in SEQ ID NO: 4), IL-7 (NM_000880.3, the nucleic acid sequence is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6), IL-21 (NM_001207006.2, the nucleic acid sequence is shown in SEQ ID NO: 7, the amino acid sequence is shown in SEQ ID NO: 8)
  • the sequence is connected in series after removing the stop codon, and the interval between the nucleic acid sequences of each cytokine is well known to those of ordinary skill
  • the P2A nucleic acid sequence (nucleic acid sequence is shown
  • Enzyme digestion system volume Plasmid pSC65CY 3 ⁇ L, about 1 ⁇ g EcoRV (Bao Biological, article number 1068A) 1 ⁇ L Enzyme digestion buffer 1 ⁇ L ddH 2 O Make up to 10 ⁇ L
  • the specific method for obtaining the recombinant vaccinia virus vector in 143B cells is as follows. On day 1, plate 143B cells ( CRL-8303), 1 ⁇ 10 6 /well, incubate overnight in a carbon dioxide cell incubator at 37°C. On the second day, add vaccinia virus wild strain 752-1 (provided by Beijing Biological Products) at 0.05 MOI (ie 5 ⁇ 10 4 PFU (plaque forming unit)/well), and then place it in a 37°C carbon dioxide cell incubator Incubate for two hours during which the shuttle vector/transfection reagent complex is prepared.
  • the shuttle vector is pSC65CY obtained in Example 1, the transfection reagent is Turbofect (Thermo Fisher Scientific, R0531), and the transfection dosage and compounding method can be found in the transfection reagent manual.
  • the 143B cell supernatant was changed to 2 mL/well of DMEM maintenance medium containing 2% fetal bovine serum (FBS), and then the shuttle vector/transfection reagent complex was added.
  • Example 3 Amplification preparation and titration of recombinant vaccinia virus vector rvv-CY
  • the recombinant vaccinia virus vector rvv-CY and the wild strain of vaccinia virus (rvv-WT) constructed in Example 2 were respectively placed in Vero cells ( CCL-81) for amplification, the amplification method is as follows:
  • the cells were scraped and collected, centrifuged at 1800g for 5 minutes, and the supernatant was removed.
  • the sonication conditions are: 50 watts, 5 seconds ultrasonic / 5 second interval, 15 minutes in total.
  • the amplified and prepared vaccinia virus is titrated on Vero cells, and the specific method is as follows:
  • Table 2 shows the titration results of the vaccinia virus vector.
  • Vaccinia virus Potency (PFU/mL) Vaccinia virus wild type rvv-wt 1.5 ⁇ 10 8 Recombinant vaccinia virus rvv-CY 1.0 ⁇ 10 8
  • the supernatants of vero cells infected with wild-type vaccinia virus rvv-wt and recombinant vaccinia virus rvv-CY were obtained from Example 3, and the content of cytokines in the infected supernatant was detected by ELISA.
  • ELISA kits for detecting human IL-7 (article number: SEK11821), human IL-15 (article number: SEK10360), and human GM-CSF (article number: SEK10015) were purchased from Beijing Yiqiao Shenzhou.
  • the ELISA kit used to detect human IL-21 (article number: 88-8218) was purchased from Thermo Fisher Scientific. Refer to the kit instructions for the detection method.
  • the content of each cytokine measured in the virus infection supernatant is shown in Table 3.
  • the results show that there is no cytokine expression in the vaccinia virus wild-type rvv-wt infection supernatant, and the detection is less than the detection limit ( ⁇ 0.01ng/mL).
  • the prepared recombinant vaccinia virus rvv-CY with cytokine nucleic acid sequence can detect the expression of various cytokines. Among them, human GM-CSF secretes the strongest, reaching the level of 144.6ng/mL, and the secretion of other cytokines is equivalent, at 1 -6ng/mL level.
  • TF-1 cells ( CRL-2003) was cultured in complete RPMI-1640 medium (10% fetal bovine serum (FBS), 1% penicillin (PS), 2ng/mL human IL-3) to the logarithmic phase, and centrifuged at 125g for 10 minutes Collect the cells, resuspend them in serum-free RPMI-1640 medium, collect the cells by centrifugation again, dilute the cells with complete RPMI-1640 medium to 1 ⁇ 10 5 cells/mL, mix well, add to a 96-well plate, 100 micrometers per well Liter (1 ⁇ 10 4 cells).
  • complete RPMI-1640 medium 10% fetal bovine serum (FBS), 1% penicillin (PS), 2ng/mL human IL-3
  • Human GM-CSF standard (near shore protein, item number: CC79) was diluted step by step in a two-fold gradient, and added to a 96-well plate from low concentration to high concentration, and used as a blank control. Make two replicate wells for each concentration, a total of 12 A gradient. Place it in a 37°C, 5% CO 2 incubator for 96 hours.
  • CCK-8 (MCE, catalog number: HY-K0301) reagent to each well, put it back into the incubator and continue to incubate for 2-4 hours, and measure the OD450 with a microplate reader.
  • test results are shown in Figure 3.
  • the recombinant vaccinia virus rvv-CY with the cytokine nucleic acid sequence prepared in Example 3 secreted human GM-CSF in the supernatant (the half effective concentration (EC 50 ) was 4.2 ng). /mL), and is equivalent to human GM-CSF standard (EC 50 of 2.8 ng/mL).
  • Mino cells CRL-3000 was cultured in complete RPMI-1640 medium (10% fetal bovine serum (FBS), 1% penicillin (PS)) to the logarithmic phase, the cells were collected by centrifugation at 125g, and the complete medium was diluted to 2 ⁇ 105 Pieces/mL.
  • FBS fetal bovine serum
  • PS penicillin
  • Human IL-21 standard (Beijing Yiqiao Shenzhou, Item No.: 10584-HNAE) was gradually diluted in a two-fold gradient in a 96-well plate, and used as a blank control. A total of 12 gradients, 100 microliters per well, added after the dilution was completed 50 microliters of mixed cells (1 ⁇ 10 4 cells), 150 microliters of liquid per well; 96-well plates are placed in a 37°C, 5% CO 2 incubator for 6-7 days.
  • CCK-8 reagent MCE, item number: HY-K0301
  • test results are shown in Figure 4.
  • the recombinant vaccinia virus rvv-CY with the cytokine nucleic acid sequence prepared in Example 3 secreted human IL-21 in the supernatant (EC 50 of 1.1 ng/mL), and It is equivalent to human IL-21 standard (EC 50 is 7 ng/mL).
  • Example 5 The results of Example 5 and Example 6 show that the prepared recombinant vaccinia virus rvv-CY with the nucleic acid sequence of the cytokine can correctly express the cytokine with biological activity.
  • mice 20 female BAL B/c mice aged 6-8 weeks were purchased from the Animal Experiment Center of Soochow University and kept in the SPF animal room of the Animal Experiment Center of Soochow University. On day 0, all mice were subcutaneously inoculated with tumor cells CT26 ( CRL-2638), the inoculation dose was 1 ⁇ 10 5 cells/head, and then randomly divided into two groups. On the 1, 14 and 28 days after tumor cell inoculation, mice were respectively inoculated with the recombinant virus vector rvv-CY prepared in Example 3 or the wild strain rvv-WT of the control vaccinia virus vector.
  • mice in the vaccinia group were inoculated with the vaccinia virus vector prepared in Example 3 (see Table 4 for the specific vaccination plan). Continuous observation and measurement of tumor growth after inoculation. Calculate the tumor volume according to the following formula:
  • Tumor volume (mm 3 ) length ⁇ width 2 /2.
  • the tumor volume of the mouse exceeds 2000 mm 3 , the mouse is sacrificed.
  • mice in the vaccinia virus vector vaccine rvv-CY treatment group was significantly better than that in the control group.
  • the results indicate that the vaccinia virus vector vaccine rvv-CY can improve the survival of mice with expressing tumors.

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Abstract

一种重组病毒载体、包含其的免疫组合物以及用途，所述重组病毒载体包含编码细胞因子的多核苷酸，所述细胞因子选自IL-7、IL-15、IL-21或GM-CSF中的一种或多种，该重组病毒载体可用于制备抗肿瘤疫苗。

Description

一种重组病毒载体、包含其的免疫组合物以及用途 技术领域
本发明属于分子生物学和免疫学领域。具体地，本发明涉及一种重组病毒载体、包含其的免疫组合物以及用途，特别地，本发明涉及一种可以用于制备肿瘤疫苗，从而用于预防和/或治疗多种肿瘤的重组病毒载体。
背景技术
随着肿瘤生物学和免疫学的发展，肿瘤免疫治疗取得了长足的进步，逐步成为当前肿瘤治疗的重要发展方向。特别是伴随PD-1/PD-L1免疫疗法的巨大成功，开发肿瘤免疫疗法成为当前肿瘤治疗新兴的热点。
肿瘤疫苗通过激发体内的针对肿瘤的特异性免疫应答，活化产生庞大的免疫细胞，来杀伤肿瘤细胞和控制肿瘤细胞的生长，从而达到减小或控制肿瘤生长的效果。肿瘤疫苗的开发也是当前肿瘤免疫治疗的重点研究方向。
研究表明细胞毒(CD8+)和辅助(CD4+)T细胞在肿瘤排斥反应中起了关键的作用。因此，大多数肿瘤疫苗的目标都致力于诱导细胞的特异性的T细胞反应。肿瘤细胞在合成肿瘤抗原的过程中，分解的多肽通过MHC I类分子被提呈致肿瘤细胞表面激活CD8+T细胞。MHC II类分子为CD4+T细胞所识别，主要位于特殊的抗原提呈细胞(APC)表面，包括树突状细胞、B细胞和巨噬细胞。肿瘤细胞分泌或肿瘤溶解释放外源性蛋白被APC俘获。在APC内，抗原被加工成多肽片断并由II类MHC提呈给CD4+细胞。激活的抗原特异性CD8+细胞最终成为细胞毒性T细胞并溶解肿瘤细胞。
理想的肿瘤特异性抗原应具有较强的免疫原性，为肿瘤细胞所表达，但不表达于正常的细胞。不幸的是，大多数肿瘤抗原都没有足够的免疫原性以诱导有效的免疫反应，而且，许多肿瘤抗原在某种程度上表达于正常的组织，从而导致体内存在免疫耐受。因此，这些肿瘤抗原天然存在免疫原性弱的特点，设计的肿瘤疫苗必须要克服机体的免疫耐受障碍，激活针对肿瘤抗原的免疫应答产生。
肿瘤疫苗主要分为全细胞疫苗、蛋白疫苗、多肽疫苗、病毒疫苗和树突状细胞疫苗。而DNA疫苗和RNA疫苗实际上仍属于分子疫苗，只是采用了不同的表达系统。
细胞疫苗：过去，对肿瘤抗原与其临床的相关性方面并不十分清楚，因此采用全细胞作为肿瘤疫苗，以便可以提供那些未知的肿瘤抗原来激活 免疫系统。在小鼠肿瘤模型中，通常使用辐射灭活的肿瘤细胞免疫小鼠，以保护小鼠免受接种的肿瘤的侵袭。但当肿瘤细胞疫苗使用的时间推迟到接种肿瘤细胞后一周时，疫苗就失去了保护小鼠的能力。肿瘤细胞疫苗的临床治疗反应比较差，仅仅适用于无特殊肿瘤抗原的肿瘤病人的预防复发。对于进展期病人在临床研究方面很少获得良好效果。近年来，由于在识别分析肿瘤抗原方面的进展，特别是T细胞识别抗原的机制的深入了解，肿瘤抗原疫苗已基本取代细胞疫苗被用于肿瘤的免疫治疗。
多肽和蛋白疫苗：T细胞识别MHC分子表面的抗原多肽表位一般为7-12个氨基酸，因此，抗原多肽可以与免疫佐剂混合应用以达到在体内装载于空虚的MHC分子的目的。到目前为止，几乎所有的以多肽为基础的疫苗都是MHC I类抗原限制性多肽。多肽疫苗应用有一些限制。所应用的多肽疫苗必需与病人的MHC I类抗原分子相匹配，即所谓的个体化，但由于不同的病人MHC I类分子的亚型不同，所使用的肿瘤抗原多肽的序列也不同，因而这给肿瘤抗原多肽的临床应用带来很大的困难。
重组分子疫苗：肿瘤抗原蛋白疫苗的应用可以克服这种困难，但是单纯使用蛋白并不能激活机体的免疫反应。灵长类动物试验研究证实最佳的免疫效果需要将肿瘤蛋白与强免疫原性蛋白相交联。弱抗原要诱导出有效的免疫反应，就必须联合使用免疫佐剂，提供一个非特异性的信号以激活免疫系统，许多免疫佐剂都有一定的毒性而不能应用于临床，所以抗原蛋白疫苗大都是以重组形式出现的。
用重组形式增强肿瘤蛋白的免疫原性的方法就是将肿瘤抗原与细胞因子，如GM-CSF、白细胞介素等重组形成融合蛋白。肿瘤弱抗原与细菌或病毒抗原、毒素如白喉毒素、假单胞菌毒素等的重组可以明显提高肿瘤抗原的抗原性，促进DC对肿瘤抗原的吞噬提呈，取得了一定的效果。但肿瘤抗原与毒素的单独重组的方法到目前为止仍然还没有达到理想的效果。
树突状细胞疫苗：对于有效的T细胞介导的免疫反应，T细胞需要抗原被提呈并致敏初始T细胞，致敏的T淋巴细胞获得再刺激。要启动有效的T细胞介导的肿瘤免疫，来源于体内任何部位的肿瘤抗原多肽必须被T细胞所识别。因此，抗原的提呈是获得有效免疫反应的关键性步骤。疫苗刺激的免疫反应主要依赖于有效的APC对抗原的初加工和进一步的提呈。
白介素7(Interleukin 7，IL-7)是骨髓和胸腺基质细胞分泌的造血生长因子。它也可由角质细胞、树突细胞、肝细胞、神经元细胞和上皮细胞产生，但正常的淋巴细胞不会分泌产生IL-7。IL-7促进造血干细胞分化成淋巴样前体细胞。它也能刺激淋巴系所有细胞的增殖，例如B细胞、T细胞和NK细胞。它对于特定阶段的B细胞成熟，T细胞和天然杀伤细胞(Natural  Killer Cells，NK细胞)的存活、发育和平衡至关重要。
白介素15(Interleukin 15，IL-15)是在病毒感染后由单核吞噬细胞分泌产生。其结构类似白介素2(Interleukin 2，IL-2)，IL-15通过结合IL-2/IL-15受体β链(CD122)和共有受体γ链(CD132)复合体来传递信号。该细胞因子调节T细胞和NK细胞的活化与增值。但抗原不存在时，IL-15可提供维持记忆T细胞的存活信号。IL-15在临床前研究中显示出可以增强CD8+T细胞的抗肿瘤作用。IL-15也可作疫苗佐剂，增加疫苗免疫原性。
白介素21(Interleukin 21，IL-21)由活化的CD4+T细胞分泌产生，可对免疫系统多种细胞起调节作用。IL-21可以通过持续增加CD8+T细胞应答实现抗肿瘤效果(Journal of Immunology.173(2):900–9)。IL-21在控制慢性病毒感染中也发挥重要作用。在HIV感染者中，IL-21可增强HIV特异性细胞毒性T细胞应答(Blood.109(9):3873–80.)和NK细胞功能(Journal of Leukocyte Biology.87(5):857–67.)
粒细胞-巨噬细胞集落刺激因子(Granulocyte-macrophage colony-stimulating factor,GM-CSF)，又被称为集落刺激因子2(colony-stimulating factor 2，CSF2)是由巨噬细胞、T细胞、肥大细胞、NK细胞，内皮细胞和成纤维细胞分泌产生的一种单体糖蛋白。GM-CSF具有多种功能，可刺激干细胞产生多种粒细胞和单核细胞，快速活化增殖大量巨噬细胞，从而实现抗感染的效果。安进(Amgen)公司开发的带有GM-CSF的溶瘤病毒疗法已经被FDA批准用于黑色素瘤治疗。
发明内容
因此，为了提高肿瘤抗原的免疫原性，激发肿瘤特异性免疫应答，从根本上治愈肿瘤，本发明将多个与免疫相关的细胞因子联合表达在一种重组病毒载体上，以期提高疫苗的免疫效果和发挥细胞因子的抗肿瘤作用。
本发明的目的是提供一种重组病毒载体，该重组病毒载体可以用于制备肿瘤疫苗，从而用于预防和/或治疗多种肿瘤。
在本发明的一个实施方案中，所述重组病毒载体包含编码细胞因子的多核苷酸。
为了本发明的目的，以下定义下列术语。
“细胞因子(cytokine，CK)”是指由免疫细胞(如单核细胞、巨噬细胞、T细胞、B细胞、NK细胞等)和某些非免疫细胞(内皮细胞、表皮细胞、纤维母细胞等)经刺激而合成、分泌的一类具有广泛生物学活性的小分子蛋白质。细胞因子一般通过结合相应受体调节细胞生长、分化和效应，调控免疫应答。细胞因子是免疫原、丝裂原或其他刺激剂诱导多种细 胞产生的低分子量可溶性蛋白质，具有调节固有免疫和适应性免疫、血细胞生成、细胞生长、APSC多能细胞以及损伤组织修复等多种功能。细胞因子可被分为白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子、生长因子等。在本发明的实施方案中，所述细胞因子选自IL-7、IL-15，IL-21和GM-CSF中的一种或多种。在本发明的一个实施方案中，所述IL-15为人IL-15；优选地，所述人IL-15的氨基酸序列如SEQ ID NO:2所示；优选地，其编码核酸序列如SEQ ID NO:1所示。在本发明的一个实施方案中，所述GM-CSF为人GM-CSF；优选地，所述人GM-CSF的氨基酸序列如SEQ ID NO:4所示；优选地，其编码核酸序列如SEQ ID NO:3所示。在本发明的一个实施方案中，所述IL-7为人IL-7；优选地，所述人IL-7的氨基酸序列如SEQ ID NO:6所示；优选地，其编码核酸序列如SEQ ID NO:5所示。在本发明的一个实施方案中，所述IL-21为人IL-21；优选地，所述人IL-21的氨基酸序列如SEQ ID NO:8所示；优选地，其编码核酸序列如SEQ ID NO:7所示。在本发明的一个优选实施方案中，所述细胞因子包括人IL-15、人GM-CSF、人IL-7和人IL-21；优选地，所述编码细胞因子的多核苷酸的核酸序列如SEQ ID NO:15所示；优选地，其编码的氨基酸序列如SEQ ID NO:16所示。
“肿瘤抗原”是指在肿瘤发生、发展过程中新出现或过度表达的抗原物质。肿瘤抗原包括但不限于肿瘤特异性抗原、肿瘤相关抗原、组织分化抗原、原癌病毒抗原和肿瘤-睾丸抗原(cancer-testis antigens，CT抗原)等。
“肿瘤特异性抗原”(Tumor-Specific Antigens，TSA)是指仅在肿瘤细胞中表达，不在正常细胞中表达的抗原物质。例如突变的抗原，特别是原癌基因和肿瘤抑制基因的突变产物，包括ras和p53等。
“肿瘤相关抗原”(Tumor-Associated Antigens，TAA)是指在肿瘤细胞中和一些正常细胞中表达的抗原物质。
优选地，所述病毒载体是痘苗病毒载体，优选为复制型痘苗病毒载体，例如痘苗病毒天坛株，例如752-1株，或者为非复制型痘苗病毒载体，例如痘苗病毒减毒疫苗安卡拉株(Modified Vaccinia Ankara，MVA)。
本发明的另一目的是提供一种免疫组合物，所述免疫组合物包含预防和/或治疗有效量的根据本发明的重组病毒载体，以及药学上可接受的载体。
本发明的另一目的是提供一种肿瘤疫苗，所述肿瘤疫苗包含预防和/或治疗有效量的根据本发明的重组病毒载体，以及药学上可接受的载体。
本发明的另一目的是提供一种药盒，所述药盒包含根据本发明的重组病毒载体、免疫组合物或肿瘤疫苗，以及其使用说明。
本发明还提供了根据本发明的重组病毒载体、免疫组合物或肿瘤疫苗 在制备治疗和/或预防肿瘤的药物或疫苗中用途。优选地，所述肿瘤为恶性肿瘤。更优选地，所述恶性肿瘤为乳腺癌或结肠癌。
本发明还提供了一种用于治疗和/或预防肿瘤的方法，所述方法包括给予有需要的受试者预防和/或治疗有效量的根据本发明的重组病毒载体、免疫组合物或肿瘤疫苗；优选地，所述肿瘤为恶性肿瘤；更优选地，所述恶性肿瘤为乳腺癌或结肠癌。
本发明提供的重组病毒载体能够激发肿瘤特异性免疫应答，有效抑制肿瘤细胞生长，延长肿瘤患者的生存时间。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1和图2分别是带有细胞因子编码序列的穿梭载体载体pSC65CY的质粒图谱和双酶切鉴定图。
图3为实施例5中的人GM-CSF活性检测结果。
图4为实施例6中的人IL-21活性检测结果。
实施发明的最佳方式
下面结合实施例进一步说明本发明，应当理解，实施例仅用于进一步说明和阐释本发明，并非用于限制本发明。
除非另外定义，本说明书中有关技术的和科学的术语与本领域内的技术人员所通常理解的意思相同。虽然在实验或实际应用中可以应用与此间所述相似或相同的方法和材料，本文还是在下文中对材料和方法做了描述。在相冲突的情况下，以本说明书包括其中定义为准，另外，材料、方法和例子仅供说明，而不具限制性。
实施例1穿梭载体pSC65CY构建
所有人细胞因子氨基酸及其核酸序列均来自NCBI数据库。将人细胞因子IL-15(NM_000585.4，核酸序列如SEQ ID NO:1所示，氨基酸序列如SEQ ID NO:2所示)，GM-CSF(NM_000758.3，核酸序列如SEQ ID NO:3所示，氨基酸序列如SEQ ID NO:4所示)，IL-7(NM_000880.3，核酸序列如SEQ ID NO:5所示，氨基酸序列如SEQ ID NO:6所示)，IL-21(NM_001207006.2，核酸序列如SEQ ID NO:7所示，氨基酸序列如SEQ ID NO:8所示)序列去掉终止密码子后串联在一起，各个细胞因子核酸序列之间间隔有普通技术人员熟知的P2A核酸序列(核酸序列如SEQ ID NO:9所示，氨基酸序列如SEQ ID NO:10所示，内源性蛋白酶切位点，可以有效切割融合蛋白，形成有活性的细胞因子单体，见参考文献Kim JH,Lee S-R,Li L-H,Park H-J, Park J-H,et al.(2011)High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1in Human Cell Lines,Zebrafish and Mice.PLoS ONE 6(4):e18556.)，最后串联一段普通技术人员熟知的绿色荧光蛋白核酸表达序列EGFP(核酸序列如SEQ ID NO:11所示，氨基酸序列如SEQ ID NO:12所示)用于标记筛查，最终形成四联细胞因子核酸表达序列CY(核酸序列如SEQ ID NO:13所示，氨基酸序列如SEQ ID NO:14所示)。将序列经苏州金唯智公司合成后，通过分子克隆技术插入穿梭载体pSC65(addgene，货号：30327)上的Xho I和Bam HI酶切位点之间，构建成可表达4个细胞因子的穿梭载体pSC65CY(质粒图谱如图1)，经测序鉴定正确后入库。用限制内切酶EcoR V鉴定载体pSC65CY(酶切体系如表1)，其酶切验证图谱如图2所示。
表1质粒pSC65CY的酶切鉴定体系(37℃酶切2小时)
酶切体系 体积
质粒pSC65CY 3μL，约1μg
EcoRV(宝生物，货号1068A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例2重组痘苗病毒载体rvv-CY构建
在143B细胞中获得重组痘苗病毒载体，具体方法如下。第1天，在6孔细胞培养板(JET，TCP-010-006)铺143B细胞(
Figure PCTCN2020075677-appb-000001
CRL-8303)，1×10 6/孔，于37℃在二氧化碳细胞培养箱中过夜孵育。第二天，以0.05MOI(即5×10 4PFU(空斑形成单位)/孔)加入痘苗病毒野生株752-1(由北京生物制品所提供)，然后置于37℃二氧化碳细胞培养箱中孵育两个小时，期间准备穿梭载体/转染试剂复合物。其中穿梭载体为实施例1中获得的pSC65CY，转染试剂为Turbofect(Thermo Fisher Scientific，R0531)，转染剂量与复合方法可参见转染试剂说明书。复合体系完成后，将143B细胞上清换为2mL/孔的含2％胎牛血清(FBS)的DMEM维持培养基，然后加入穿梭载体/转染试剂复合物。转染48小时后，去上清，收集细胞，并在0.5mL维持培养基中重悬，反复冻融三次，然后将重组细胞裂解物接入新的143B细胞上(含50μg/mL BrdU)，37℃孵育1到2天。期间观察细胞病变，待病毒噬斑出现合适数量时(低于20空斑/孔)，进行单斑纯化。
单斑纯化：
在荧光显微镜下观察表达绿色荧光的病毒噬斑，做好标记。
去上清，每孔挑取若干个分散较好的绿色荧光噬斑，分别转移至含 0.5mL维持培养液的Ep管中。
振荡混匀含病毒的Ep管，反复冻融三次(-80℃冰箱约5分钟，室温约2分钟)，最后振荡混匀，-80℃冻存。
重复至少六轮单斑纯化，直至纯度至100％。
实施例3重组痘苗病毒载体rvv-CY扩增制备与滴定
将实施例2中构建的重组痘苗病毒载体rvv-CY以及痘苗病毒野生株(rvv-WT)分别在Vero细胞(
Figure PCTCN2020075677-appb-000002
CCL-81)上扩增，扩增方法如下：
前一天，准备汇集度100％的Vero单层细胞(1×10 7细胞/皿)，共10皿。
去上清，换为维持培养基，将待扩增的痘病毒接种到细胞上(0.01PFU/细胞)，37℃培养箱中孵育2-3天，观察可见明显的细胞病变。
将细胞刮下并收集，1800g离心5分钟，去上清。
用5mL维持培养基进行重悬，在冰上用超声波细胞粉粹机超声，超声条件为：50瓦，5秒超声/5秒间隔，共15分钟。
反复冻融两次(-80℃冰箱约5分钟，室温约2分钟)，最后振荡混匀；
在二级生物安全柜中进行分装至1.5mL离心管中，1mL/支，-80℃冻存。
扩增制备好的痘苗病毒在Vero细胞上进行感染效价滴定，具体方法如下：
前一天，在24孔板中，准备汇集度100％的Vero细胞，3×10 5/孔。
去上清，每孔添加200μL维持培养液，以防止细胞干涸。
取100μL待测痘病毒加入900μL维持培养基，十倍稀释，连续稀释10 1，10 2，10 3，……，直到10 9倍。注意：进行稀释时，因为由高浓度向低浓度稀释，每次向低浓度稀释应更换枪头。
从病毒浓度由小到大(10 9，10 8，……10 4)添加到24孔板中，每孔400μL稀释液，两个重复，连续测定6个稀释倍数。将添加完的24孔板放入37℃细胞培养箱中孵育2天。
显微镜下数出病毒蚀斑的数目，多于20的，记为20+。将可以数出的20以内(含20)蚀斑数目的两复孔求平均×2.5(1000μL/400μL)×相应孔的稀释倍数，即为重组病毒滴度(PFU/mL)。
痘苗病毒载体效价滴定结果如表2所示。
表2痘苗病毒载体效价滴定
痘苗病毒 效价(PFU/mL)
痘苗病毒野生型rvv-wt 1.5×10 8
重组痘苗病毒rvv-CY 1.0×10 8
实施例4细胞因子表达检测
从实施例3中分别获取痘苗病毒野生型rvv-wt和重组痘苗病毒rvv-CY感染vero细胞的上清液，并用ELISA法检测感染上清中细胞因子的含量。其中用于检测人IL-7(货号：SEK11821)、人IL-15(货号：SEK10360)、人GM-CSF(货号：SEK10015)的ELISA试剂盒购自北京义翘神州。用于检测人IL-21(货号：88-8218)的ELISA试剂盒在赛默飞世尔科技公司购买。检测方法参考试剂盒说明书。病毒感染上清中所测的各个细胞因子含量如表3所示，结果表明痘苗病毒野生型rvv-wt感染上清中无细胞因子表达，检测均小于检测限(<0.01ng/mL)。而制备的带有细胞因子核酸序列的重组痘苗病毒rvv-CY均能检测到各个细胞因子的表达，其中人GM-CSF分泌最强，达到144.6ng/mL水平，其余细胞因子分泌相当，在1-6ng/mL的水平之间。
表3细胞因子含量检测(ng/mL)
样本 人IL-7 人IL-15 人GM-CSF 人IL-21
rvv-WT感染上清 <0.01 <0.01 <0.01 <0.01
rvv-CY感染上清 3.7 1.9 144.6 5.7
实施例5细胞因子人GM-CSF活性检测
TF-1细胞(
Figure PCTCN2020075677-appb-000003
CRL-2003)在完全RPMI-1640培养基(10％胎牛血清(FBS)、1％青链霉素(PS)、2ng/mL人IL-3)中培养至对数期，125g离心10分钟收集细胞，用无血清RPMI-1640培养基重悬，再次离心收集细胞，用完全RPMI-1640培养基稀释细胞至1×10 5个/mL，充分混匀，加入96孔板，每孔100微升(1×10 4个细胞)。
人GM-CSF标准品(近岸蛋白，货号：CC79)按两倍梯度逐级稀释，从低浓度到高浓度分别加入96孔板，并做空白对照，每个浓度做两复孔，共12个梯度。放置在37℃、5％CO 2培养箱中培养96小时。
然后在96孔板中，每孔加入10微升CCK-8(MCE，货号：HY-K0301)试剂，放回培养箱继续培养2-4小时，用酶标仪测定OD450。
检测结果如图3所示，实施例3中制备的带有细胞因子核酸序列的重组痘苗病毒rvv-CY感染上清中分泌的人GM-CSF具有活性(半数有效浓度(EC 50)为4.2ng/mL)，且与人GM-CSF标准品(EC 50为2.8ng/mL)相当。
实施例6细胞因子人IL-21活性检测
Mino细胞(
Figure PCTCN2020075677-appb-000004
CRL-3000)在完全RPMI-1640培养基(10％胎牛 血清(FBS)、1％青链霉素(PS))培养至对数期，125g离心收集细胞，完全培养基稀释至2×105个/mL。
人IL-21标准品(北京义翘神州，货号：10584-HNAE)在96孔板中按两倍梯度逐渐稀释，并作空白对照，共12个梯度，每孔100微升，稀释完成后加入50微升混匀的细胞(1×10 4个细胞)，每孔共150微升液体；96孔板放置于37℃、5％CO 2培养箱中培养6-7天。
培养好的96孔板每孔加入10微升CCK-8试剂(MCE，货号：HY-K0301)，放回培养箱继续培养4-8h(随颜色变化确定时间)，用酶标仪测定OD450。
检测结果如图4所示，实施例3中制备的带有细胞因子核酸序列的重组痘苗病毒rvv-CY感染上清中分泌的人IL-21具有活性(EC 50为1.1ng/mL)，且与人IL-21标准品(EC 50为7ng/mL)相当。
实施例5和实施例6的结果表明制备的带有细胞因子核酸序列的重组痘苗病毒rvv-CY能正确表达具有生物学活性的细胞因子。
实施例7肿瘤治疗实验
从苏州大学动物实验中心购买20只6-8周龄的雌性BAL B/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。在第0天，所有小鼠皮下接种肿瘤细胞CT26(
Figure PCTCN2020075677-appb-000005
CRL-2638)，接种剂量为1×10 5细胞/只，然后随机分成两组。在肿瘤细胞接种后第1天、第14天和第28天，给小鼠分别接种实施例3制备的重组病毒载体rvv-CY或对照痘苗病毒载体野生株rvv-WT。相应地，在肿瘤细胞接种后第1天、第14天和第28天，痘苗组小鼠小腿胫骨前肌接种实施例3中制备的痘苗病毒载体(具体疫苗接种规划见表4)。接种后连续观察并测量肿瘤生长情况。按照以下公式计算肿瘤体积：
肿瘤体积(mm 3)＝长×宽 2/2。
当小鼠肿瘤体积超过2000mm 3时，对小鼠处死。
表4实验动物分组与疫苗接种规划
Figure PCTCN2020075677-appb-000006
痘苗病毒载体疫苗rvv-CY治疗组小鼠总体生存期显著优于对照组小鼠。结果表明痘苗病毒载体疫苗rvv-CY能提高患有表达肿瘤的小鼠的生存。
尽管本发明已进行了一定程度的描述，明显地，在不脱离本发明的精神和范围的条件下，可进行各个条件的适当变化。可以理解，本发明不限于所述实施方案，而归于权利要求的范围，其包括所述每个因素的等同替换。

Claims (12)

  1. 一种重组病毒载体，所述重组病毒载体包含编码细胞因子的多核苷酸，所述细胞因子选自IL-7、IL-15、IL-21和GM-CSF中的一种或多种。
  2. 根据权利要求1所述的重组病毒载体，其中，所述IL-15为人IL-15；优选地，所述人IL-15的氨基酸序列如SEQ ID NO:2所示；优选地，其编码核酸序列如SEQ ID NO:1所示。
  3. 根据权利要求1或2所述的重组病毒载体，其中，所述GM-CSF为人GM-CSF；优选地，所述人GM-CSF的氨基酸序列如SEQ ID NO:4所示；优选地，其编码核酸序列如SEQ ID NO:3所示。
  4. 根据权利要求1至3中任一项所述的重组病毒载体，其中，所述IL-7为人IL-7；优选地，所述人IL-7的氨基酸序列如SEQ ID NO:6所示；优选地，其编码核酸序列如SEQ ID NO:5所示。
  5. 根据权利要求1至4中任一项所述的重组病毒载体，其中，所述IL-21为人IL-21；优选地，所述人IL-21的氨基酸序列如SEQ ID NO:8所示；优选地，其编码核酸序列如SEQ ID NO:7所示。
  6. 根据权利要求1至5中任一项所述的重组病毒载体，其中，所述细胞因子包括人IL-15、人GM-CSF、人IL-7和人IL-21；优选地，所述编码细胞因子的多核苷酸的核酸序列如SEQ ID NO:15所示；优选地，其编码的氨基酸序列如SEQ ID NO:16所示。
  7. 根据权利要求1至6中任一项所述的重组病毒载体，其中，所述病毒载体是痘苗病毒载体，优选为复制型痘苗病毒载体，例如痘苗病毒天坛株，例如752-1株，或者为非复制型痘苗病毒载体，例如痘苗病毒减毒疫苗安卡拉株(Modified Vaccinia Ankara，MVA)。
  8. 一种免疫组合物，其包含预防和/或治疗有效量的根据权利要求1-7中任一项所述的重组病毒载体，以及药学上可接受的载体。
  9. 一种肿瘤疫苗，其包含预防和/或治疗有效量的根据权利要求1-7中任一项所述的重组病毒载体，以及药学上可接受的载体。
  10. 一种药盒，其包含根据权利要求1-7中任一项所述的重组病毒载体、根据权利要求8所述的免疫组合物或根据权利要求9所述的肿瘤疫苗，以及其使用说明。
  11. 根据权利要求1-7中任一项所述的重组病毒载体、根据权利要求8所述的免疫组合物或根据权利要求9所述的肿瘤疫苗在制备治疗和/或预防肿瘤的药物中的用途；优选地，所述肿瘤为恶性肿瘤；更优选地，所述恶性肿瘤为乳腺癌或结肠癌。
  12. 一种用于治疗和/或预防肿瘤的方法，所述方法包括给予有需要的受试者预防和/或治疗有效量的根据权利要求1-7中任一项所述的重组病毒载体、根据权利要求8所述的免疫组合物或根据权利要求9所述的肿瘤疫苗；优选地，所述肿瘤为恶性肿瘤；更优选地，所述恶性肿瘤为乳腺癌或结肠癌。
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