WO2021087851A1 - 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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WO2021087851A1
WO2021087851A1 PCT/CN2019/116197 CN2019116197W WO2021087851A1 WO 2021087851 A1 WO2021087851 A1 WO 2021087851A1 CN 2019116197 W CN2019116197 W CN 2019116197W WO 2021087851 A1 WO2021087851 A1 WO 2021087851A1
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tumor
antigen
viral vector
seq
recombinant viral
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徐建青
黄杨
张晓燕
郜明泉
王璐
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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  • the invention belongs to the field of molecular biology and immunology. Specifically, the present invention relates to a recombinant viral vector, an immune composition containing the same, and use thereof. In particular, the present invention relates to a tumor therapeutic vaccine.
  • tumor immunotherapy has made considerable 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 can kill and control the growth of tumor cells by stimulating a specific immune response against tumors in the body and activating huge immune 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.
  • MHC I 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 APCs.
  • 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 expressed in 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 tumor antigens. In mouse tumor models, radiation-inactivated tumor cells are usually used to immunize mice to protect them 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.
  • Experimental studies in primates have confirmed that the best immune effect requires that the tumor protein is stranded with a strong immunogenic protein.
  • an immune adjuvant 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. Coming in the form of reorganization.
  • the method to enhance the immunogenicity of tumor proteins with recombinant forms 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.
  • the purpose of the present invention is to provide a recombinant virus vector, which can be used as a tumor vaccine and can be used to prevent or treat a variety of tumors.
  • the recombinant viral vector comprises a polynucleotide encoding a tumor antigen.
  • Tumor antigen refers to an antigenic substance that is newly emerged or overexpressed in the process of tumor occurrence and development.
  • 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 antigenic substances that are only expressed in tumor cells and not 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.
  • Tumor-testis antigens refer to antigenic substances expressed only in tumor cells and some germ cells. Such as NY-ESO-1, LAGE-1 and MAGE-A3.
  • the tumor antigen is selected from one or more of lung cancer antigen, testicular cancer antigen, melanoma antigen, liver cancer antigen, breast cancer antigen, or prostate cancer antigen.
  • the tumor antigens include but are not limited to alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 1 (MUC1), melanoma-related antigen (Melanoma-associated antigen, MAGE), NY-ESO-1, LAGE-1, p53 and ras, etc.
  • AFP alpha fetoprotein
  • CEA carcinoembryonic antigen
  • MUC1 mucin 1
  • MAGE melanoma-related antigen
  • NY-ESO-1 LAGE-1
  • p53 and ras etc.
  • the tumor antigen is selected from one or more of LAGE antigen, MAGE antigen or NY-ESO-1 antigen.
  • the LAGE antigen is the LAGE-1 antigen
  • the MAGE antigen is the MAGE-A3 antigen.
  • the tumor antigens comprise LAGE-1 antigen, MAGE-A3 antigen and NY-ESO-1 antigen.
  • the amino acid sequence of the LAGE-1 antigen is shown in SEQ ID NO: 1, and the encoding nucleic acid sequence is shown in SEQ ID NO: 2.
  • the amino acid sequence of the MAGE-A3 antigen is shown in SEQ ID NO: 3, and the encoding nucleic acid sequence is shown in SEQ ID NO: 4.
  • the amino acid sequence of the NY-ESO-1 antigen is shown in SEQ ID NO: 5, and the encoding nucleic acid sequence is shown in SEQ ID NO: 6.
  • the tumor antigen further comprises a cholera toxin B subunit polypeptide.
  • the amino acid sequence of the cholera toxin B subunit polypeptide is shown in SEQ ID NO: 7, and its encoding nucleic acid sequence is shown in SEQ ID NO: 8. Show.
  • the amino acid sequence of the tumor antigen including the LAGE-1 antigen, the MAGE-A3 antigen and the NY-ESO-1 antigen is shown in SEQ ID NO: 9, and the encoding nucleic acid sequence is shown in SEQ ID NO: 10.
  • the recombinant virus is a vaccinia virus, 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 a modified vaccinia virus attenuated 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 a modified vaccinia virus attenuated vaccine Ankara strain (Modified Vaccinia Ankara) , MVA).
  • Another object of the present invention is to provide an immune composition comprising a 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 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 viral vector and/or immune composition and/or tumor according to the present invention, and instructions for use thereof.
  • the present invention also provides the use of the recombinant virus vector, immune composition and/or tumor vaccine according to the present invention in the preparation of drugs for treating or preventing tumors.
  • the tumor is a malignant tumor.
  • the malignant tumor is breast cancer or colon cancer.
  • the present invention also provides a method for treating or preventing tumors, the method comprising administering to a subject in need a therapeutically effective amount of the recombinant viral vector, immune composition and/or tumor vaccine according to the present invention; 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 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 pSC65-LMNB with LAGE-1, MAGE-A3 and NY-ESO-1 antigen coding sequences, respectively.
  • FIG. 3 shows the 4T1-hNY-ESO-1 mouse tumor model in Example 4, and the tumor growth of each group of mice.
  • Figure 4 shows the 4T1-hNY-ESO-1 mouse tumor model in Example 4, and the overall survival status of mice in each group.
  • Figure 5 shows the CT26-MAGE-A3 mouse tumor model in Example 5. The tumor growth of each group of mice.
  • Figure 6 shows the CT26-MAGE-A3 mouse tumor model in Example 5, and the overall survival status of mice in each group.
  • Figure 7 shows the CT26-LAGE-1 mouse tumor model in Example 6, and the tumor growth of mice in each group.
  • Figure 8 shows the CT26-LAGE-1 mouse tumor model in Example 6, and the overall survival status of mice in each group.
  • Figure 9 shows the CT26-LAGE-1 mouse tumor model in Example 7, and the overall survival status of mice in each group.
  • Figure 10 shows the 4T1-NY-ESO-1 mouse tumor model in Example 8, and the overall survival status of mice in each group.
  • the eukaryotic expression vector pVKD1.0-LMNB expressing the triple fusion tumor antigens LAGE-1, MAGE-A3, NY-ESO-1 and the B subunit of cholera toxin was provided by Suzhou Industrial Park Viking Biotechnology Co., Ltd. (refer to CN109575142A) , Wherein the amino acid sequence of the LAGE-1 antigen is shown in SEQ ID NO: 1, and the encoding nucleic acid sequence is shown in SEQ ID NO: 2; the amino acid sequence of the MAGE-A3 antigen is shown in SEQ ID NO: 3.
  • the coding nucleic acid sequence is shown in SEQ ID NO: 4; the amino acid sequence of the NY-ESO-1 antigen is shown in SEQ ID NO: 5, and the coding nucleic acid sequence is shown in SEQ ID NO: 6;
  • the amino acid sequence of the cholera toxin B subunit polypeptide is shown in SEQ ID NO: 7, and its coding nucleic acid sequence is shown in SEQ ID NO: 8;
  • the amino acid sequence of the LMNB fusion protein is shown in SEQ ID NO: 9, and its nucleic acid coding sequence is shown in SEQ ID NO: 10.
  • Enzyme digestion system volume Plasmid pSC65-LMNB 3 ⁇ L, about 1 ⁇ g Sal I (Bao Biological, Item No. 1080A) 1 ⁇ L Kpn I (Bao Biological, Item No. 1068A) 1 ⁇ L Enzyme digestion buffer 1 ⁇ L ddH2O 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 on a 6-well cell culture plate (JET, TCP-010-006) ( 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 pSC65-LMNB 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.
  • FBS fetal bovine serum
  • Example 3 Amplification preparation and titration of recombinant vaccinia virus vector rvv-LMNB
  • the recombinant vaccinia virus vector rvv-LMNB constructed in Example 2 and the wild strain of vaccinia virus were respectively placed in Vero cells ( CCL-81) 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 ultrasonic conditions are: 50 watts, 5 seconds ultrasonic / 5 second interval, 15 minutes in total.
  • the amplified vaccinia virus was 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-LMNB 1.0 ⁇ 10 8
  • mice in the control group all developed tumors and grew rapidly on the 12th day after the tumor was attacked (that is, after tumor inoculation).
  • the tumor growth of the mice in the rvv-LMNB treatment group was slower.
  • the average tumor size of the mice in the treatment group was significantly smaller than that in the control group. The results show that the vaccinia virus vector vaccine rvv-LMNB can inhibit the growth of tumors with NY-ESO-1 expression.
  • the survival curve results of the survival status of the mice are shown in Figure 4.
  • the overall survival of the mice in the vaccinia virus vector vaccine rvv-LMNB treatment group (median OS 40 days) is better than that of the control mice (median OS 36 days, p ⁇ 0.05).
  • the results indicate that the vaccinia virus vector vaccine rvv-LMNB can improve the survival of mice with tumors expressing NY-ESO-1.
  • mice in each group The tumor growth of immunized mice in each group is shown in Figure 5.
  • All mice in the control group developed tumors and grew rapidly on the 15th day after tumor inoculation.
  • the average tumor size of the mice in the treatment group was significantly smaller than that in the control group.
  • the results show that the vaccinia virus vector vaccine rvv-LMNB can inhibit the growth of tumors with MAGE-A3 expression.
  • the survival curve results of the survival status of the mice are shown in Figure 6.
  • the overall survival of the mice in the vaccinia virus vector vaccine rvv-LMNB treatment group (median OS 40 days) is better than that of the control mice (median OS 33 days, p ⁇ 0.05).
  • the results indicate that the vaccinia virus vector vaccine rvv-LMNB can improve the survival of mice with tumors expressing MAGE-A3.
  • the survival curve results of the survival status of the mice are shown in Figure 8.
  • the overall survival of the mice in the vaccinia virus vector vaccine rvv-LMNB treatment group (median OS 58 days) is better than that of the control mice (median OS 51 days, p ⁇ 0.05).
  • the results show that the vaccinia virus vector vaccine rvv-LMNB can improve the survival of mice with tumors expressing LAGE-1.
  • 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 expressing LAGE-1 tumor antigen CT26-LAGE-1 stably transfected cell line (provided by Suzhou Industrial Park Weida Biotechnology Co., Ltd.), the inoculation dose was 1 ⁇ 10 5 cells per mouse, then randomly divided into 2 groups.
  • LAGE-1 tumor antigen CT26-LAGE-1 stably transfected cell line provided by Suzhou Industrial Park Weida Biotechnology Co., Ltd.
  • the proteome mouse protein vaccine LMNB was given (the specific preparation method and vaccination method are as described in Example 8 and Example 9 of Chinese Invention Patent Application CN109575141A) After vaccination, protein vaccines are fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), and then injected subcutaneously on the back, 10 ⁇ g/mouse.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • mice in the vaccinia group were inoculated with the vaccinia virus vector prepared in Example 3 (the specific vaccination schedule is shown in Table 6). Continuous observation and measurement of tumor growth after inoculation.
  • the survival curve results of the survival status of the mice are shown in Figure 9.
  • the overall survival of the mice in the vaccinia virus vector vaccine rvv-LMNB treatment group (median OS 58 days) is better than that of the mice in the protein group (median OS 51 days, p ⁇ 0.05).
  • the results show that the vaccinia virus vector vaccine rvv-LMNB can improve the survival of mice with tumors expressing LAGE-1.
  • 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 expressing NY-ESO-1 tumor antigen 4T1-hNY-ESO-1 stably transfected cell line (provided by Suzhou Industrial Park Weike Biotechnology Co., Ltd.), inoculation dose 2 ⁇ 10 5 cells/head, and then randomly divided into 2 groups.
  • tumor cells expressing NY-ESO-1 tumor antigen 4T1-hNY-ESO-1 stably transfected cell line (provided by Suzhou Industrial Park Weike Biotechnology Co., Ltd.), inoculation dose 2 ⁇ 10 5 cells/head, and then randomly divided into 2 groups.
  • the proteome mouse protein vaccine LMNB was given (the specific preparation method and vaccination method are as described in Example 8 and Example 9 of Chinese Invention Patent Application CN109575141A) After vaccination, protein vaccines are fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), and then injected subcutaneously on the back, 10 ⁇ g/mouse.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • mice in the vaccinia group were inoculated with the vaccinia virus vector prepared in Example 3 (the specific vaccination schedule is shown in Table 7). Continuous observation and measurement of tumor growth after inoculation.
  • the survival curve results of the survival status of the mice are shown in Figure 10.
  • the overall survival of the mice in the vaccinia virus vector vaccine rvv-LMNB treatment group (median OS 40 days) is better than that of the control mice (median OS 36 days, p ⁇ 0.05).
  • the results indicate that the vaccinia virus vector vaccine rvv-LMNB can improve the survival of mice with tumors expressing NY-ESO-1.

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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肿瘤抗原的多核苷酸，所述肿瘤抗原选自LAGE抗原、MAGE抗原或NY-ESO-1抗原中的一种或多种。重组病毒载体疫苗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。

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，本发明涉及一种肿瘤治疗性疫苗。
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MHCI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APCs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，所设计的肿瘤疫苗就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***。
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***肿瘤抗原。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绞联。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***，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。
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，本领域中需要开发出新的肿瘤疫苗。
发明内容
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作为肿瘤疫苗，可以用于预防或治疗多种肿瘤。
在本发明的一个实施方案中，所述重组病毒载体包含编码肿瘤抗原的多核苷酸。
为了本发明的目的以下定义下列术语。
“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。
肿瘤-睾丸抗原是指只在肿瘤细胞和一些生殖细胞中表达的抗原物质。例如NY-ESO-1、LAGE-1和MAGE-A3等。
在本发明的一个实施方案中，所述肿瘤抗原选自肺癌抗原、睾丸癌抗原、黑色素瘤抗原、肝癌抗原、乳腺癌抗原或***癌抗原中的一种或多种。
在本发明的一个实施方案中，所述肿瘤抗原包括但不限于甲胎蛋白(alpha fetoprotein，AFP)、癌胚抗原(carcinoembryonic antigen，CEA)、粘蛋白1(mucins，MUC1)、黑色素瘤相关抗原(Melanoma-associated antigen，MAGE)、NY-ESO-1、LAGE-1、p53和ras等。
在本发明的一个实施方案中，所述肿瘤抗原选自LAGE抗原、MAGE抗原或NY-ESO-1抗原中的一种或多种。优选地，LAGE抗原为LAGE-1抗原，MAGE抗原为MAGE-A3抗原。进一步优选地，所述肿瘤抗原包含LAGE-1抗原、MAGE-A3抗原和NY-ESO-1抗原。优选地，所述LAGE-1抗原的氨基酸序列如SEQ ID NO:1所示，其编码核酸序列如SEQ ID NO:2所示。优选地，所述MAGE-A3抗原的氨基酸序列如SEQ ID NO:3所示，其编码核酸序列如SEQ ID NO:4所示。优选地，所述NY-ESO-1抗原的氨基酸序列如SEQ ID NO:5所示，其编码核酸序列如SEQ ID NO:6所示。优选地，所述肿瘤抗原还包含霍乱毒素B亚单位多肽，优选地，所述霍乱毒素B亚单位多肽的氨基酸序列如SEQ ID NO:7所示，其编码核酸序列如SEQ ID NO:8所示。优选地，包含LAGE-1抗原、MAGE-A3抗原和NY-ESO-1抗原的肿瘤抗原的氨基酸序列如SEQ ID NO:9所示，其编码核酸序列如SEQ ID NO:10所示。
优选地，重组病毒是痘苗病毒，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和/或免疫组合物和/或肿瘤，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、免疫组合物和/或肿瘤疫苗在制备治疗或预防肿瘤的药物中的用途。优选地，所述肿瘤为恶性肿瘤。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或预防肿瘤的方法，所述方法包括给予有需要的受试者治疗有效量的根据本发明的重组病毒载体、免疫组合物和/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LAGE-1、MAGE-A3和NY-ESO-1抗原编码序列的穿梭载体载体pSC65-LMNB的质粒图谱和双酶切鉴定图。
图3为实施例4中的4T1-hNY-ESO-1小鼠肿瘤模型，各组小鼠肿瘤生长情况。
图4为实施例4中的4T1-hNY-ESO-1小鼠肿瘤模型，各组小鼠总体存活状况。
图5为实施例5中的CT26-MAGE-A3小鼠肿瘤模型，各组小鼠肿瘤生长情况。
图6为实施例5中的CT26-MAGE-A3小鼠肿瘤模型，各组小鼠总体存活状况。
图7为实施例6中的CT26-LAGE-1小鼠肿瘤模型，各组小鼠肿瘤生长情况。
图8为实施例6中的CT26-LAGE-1小鼠肿瘤模型，各组小鼠总体存活状况。
图9为实施例7中的CT26-LAGE-1小鼠肿瘤模型，各组小鼠总体存活状况。
图10为实施例8中的4T1-NY-ESO-1小鼠肿瘤模型，各组小鼠总体存活状况。
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pSC65-LMNB构建
表达三联融合肿瘤抗原LAGE-1、MAGE-A3、NY-ESO-1以及霍乱毒素B亚单位的真核表达载体pVKD1.0-LMNB由苏州工业园区唯可达生物科技有限公司提供(参考CN109575142A)，其中，所述LAGE-1抗原的氨基酸序列如SEQ ID NO:1所示，其编码核酸序列如SEQ ID NO:2所示；所述MAGE-A3抗原的氨基酸序列如SEQ ID NO:3所示，其编码核酸序列如SEQ ID NO:4所示；所述NY-ESO-1抗原的氨基酸序列如SEQ ID NO:5所示，其编码核酸序列如SEQ ID NO:6所示；所述霍乱毒素B亚单位多肽的氨基酸序列如SEQ ID NO:7所示，其编码核酸序列如SEQ ID NO:8所示；LMNB融合蛋白氨基酸如SEQ ID NO:9所示，其核酸编码序列如SEQ ID NO:10所示。用Sal I和Kpn I从pVKD1.0-LMNB切下含有LMNB片段，然后转移至穿梭载体pSC65(addgene，货号：30327)上的多克隆位点Sal I与Kpn I之间，构建成可表达融合蛋白抗原的穿梭载体pSC65-LMNB(质粒图谱如图1)，经测序鉴定正确后入库。用限制内切酶Sal I与Kpn I鉴定载体pSC65-LMNB(酶切体系如表1)，其酶切验证图谱如图2所示。
表1 质粒pSC65-LMNB的酶切鉴定体系(37℃酶切2小时)
酶切体系 体积
质粒pSC65-LMNB 3μL，约1μg
Sal I(宝生物，货号1080A) 1μL
Kpn I(宝生物，货号1068A) 1μL
酶切缓冲液 1μL
ddH2O 补至10μL
实施例2 重组痘苗病毒载体rvv-LMNB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 PCTCN2019116197-appb-000001
CRL-8303)，1×10 6/孔，于37℃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pSC65-LMNB，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
将2％低熔点琼脂糖微波炉加热(中高火2分钟左右)至沸，转移至45℃水浴降温并防止其凝固。
吸取适量2×维持培养液(1mL/孔)，按体积比1:50加入X-gal储存液，在45℃水浴中预热。
按等体积比例混合低熔点琼脂糖与含X-gal的2×维持培养液，制成铺斑固体培养基，去细胞上清，小心加入铺斑固体培养基，然后转移至4℃冰箱凝固10分钟，期间不要动6孔板，防止凝固不均匀。
待完全凝固后，转移6孔板至37℃细胞培养箱中孵育2至4小时(有时过夜)，直至蓝斑出现。
待蓝斑出现后，用1mL枪头(预先用剪刀将枪头剪平)优先挑取分散较好的，颜色较深的蓝斑，挑取时一定要将固体培养基下面的细胞层挑到，每孔挑取若干个蓝斑，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-LMNB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-LMNB，以及痘苗病毒野生株分别在Vero细胞(
Figure PCTCN2019116197-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(含)以内的蚀斑数目两复孔求平均×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-LMNB 1.0×10 8
实施例4 肿瘤治疗实验1
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，所有小鼠皮下接种表达NY-ESO-1肿瘤抗原的肿瘤细胞4T1-hNY-ESO-1稳定转染细胞系(由苏州工业园区唯可达生物科技有限公司提供)，接种剂量为2×10 5细胞/只，然后随机分成2两组。在肿瘤细胞接种后第1天，第14天和第28天给相应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 如表3)。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3 实验动物分组与疫苗接种规划
Figure PCTCN2019116197-appb-000003
各组免疫小鼠肿瘤生长情况如图3所示。其中，对照组小鼠在攻瘤后(即肿瘤接种后)第12天全部出现肿瘤，并迅速生长。与未治疗的对照组相比，rvv-LMNB治疗组小鼠肿瘤生长较慢。而且，在小鼠攻瘤后第21天，治疗组小鼠肿瘤平均大小显著小于对照组。结果表明痘苗病毒载体疫苗rvv-LMNB能抑制带有NY-ESO-1表达的肿瘤生长。
小鼠存活状况的生存曲线结果如图4所示，痘苗病毒载体疫苗rvv-LMNB治疗组小鼠总体生存(OS中位数40天)优于对照组小鼠(OS中位数36天，p<0.05)。结果表明痘苗病毒载体疫苗rvv-LMNB能提高患有表达NY-ESO-1肿瘤的小鼠生存。
实施例5 肿瘤治疗实验2
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，所有小鼠皮下接种表达MAGE-A3肿瘤抗原的肿瘤细胞CT26-MAGE-A3稳定转染细胞系(由苏州工业园区唯可达生物科技有限公司提供)，接种剂量为2×10 5细胞/只，然后随机分成2两组。在肿瘤细胞接种后第1天，第14天和第28天给相应小鼠小腿胫骨前肌接种实施例3中制备的痘苗病毒载体(具体疫苗接种规划如表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10000mm 3时，对小鼠处死。
表4 实验动物分组与疫苗接种规划
Figure PCTCN2019116197-appb-000004
各组免疫小鼠肿瘤生长情况如图5所示。其中，对照组小鼠在肿瘤接种后第15天全部出现肿瘤，并迅速生长。在小鼠攻瘤后第30天，治疗组小鼠肿瘤平均大小显著小于对照组。结果表明痘苗病毒载体疫苗rvv-LMNB能抑制带有MAGE-A3表达的肿瘤生长。
小鼠存活状况的生存曲线结果如图6所示，痘苗病毒载体疫苗rvv-LMNB治疗组小鼠总体生存(OS中位数40天)优于对照组小鼠(OS中位数33天，p<0.05)。结果表明痘苗病毒载体疫苗rvv-LMNB能提高患有表达MAGE-A3肿瘤的小鼠生存。
实施例6 肿瘤治疗实验3
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，所有小鼠皮下接种表达LAGE-1肿瘤抗原的肿瘤细胞CT26-LAGE-1稳定转染细胞系(由苏州工业园区唯可达生物科技有限公司提供)，接种剂量为1×10 5细胞/只，然后随机分成2两组。在肿瘤细胞接种后第1天，第14天和第28天给相应小鼠小腿胫骨前肌接种实施例3中制备的痘苗病毒载体(具体疫苗接种规划如表5)。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10000mm 3时，对小鼠处死。
表5 实验动物分组与疫苗接种规划
Figure PCTCN2019116197-appb-000005
各组免疫小鼠肿瘤生长情况如图7所示。在攻瘤后第26天，对照组小鼠肿瘤生长迅速，并显著大于治疗组小鼠，一直持续到第50天。结果表明痘苗病毒载体疫苗rvv-LMNB能抑制带有LAGE-1表达的肿瘤生长。
小鼠存活状况的生存曲线结果如图8所示，痘苗病毒载体疫苗rvv-LMNB治疗组小鼠总体生存(OS中位数58天)优于对照组小鼠(OS中位数51天，p<0.05)。结果表明痘苗病毒载体疫苗rvv-LMNB能提高患有表达LAGE-1肿瘤的小鼠生存。
实施例7 肿瘤治疗实验4
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，所有小鼠皮下接种表达LAGE-1肿瘤抗原的肿瘤细胞CT26-LAGE-1稳定转染细胞系(由苏州工业园区唯可达生物科技有限公司提供)，接种剂量为1×10 5细胞/只，然后随机分成2两组。在肿瘤细胞接种后第1天，第14天和第28天，给蛋白组小鼠蛋白疫苗LMNB(具体制备方式和接种方式如中国发明专利申请CN109575141A的实施例8和实施例9中所述)接种，蛋白疫苗均在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。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(具体疫苗接种规划如表6)。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10000mm 3时，对小鼠处死。
表6 实验动物分组与疫苗接种规划
Figure PCTCN2019116197-appb-000006
小鼠存活状况的生存曲线结果如图9所示，痘苗病毒载体疫苗rvv-LMNB治疗组小鼠总体生存(OS中位数58天)优于蛋白组小鼠(OS中位数51天，p<0.05)。结果表明痘苗病毒载体疫苗rvv-LMNB能提高患有表达LAGE-1肿瘤的小鼠生存。
实施例8 肿瘤治疗实验5
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，所有小鼠皮下接种表达NY-ESO-1肿瘤抗原的肿瘤细胞4T1-hNY-ESO-1稳定转染细胞系(由苏州工业园区唯可达生物科技有限公司提供)，接种剂量为2×10 5细胞/只，然后随机分成2两组。在肿瘤细胞接种后第1天，第14天和第28天，给蛋白组小鼠蛋白疫苗LMNB(具体制备方式和接种方式如中国发明专利申请CN109575141A的实施例8和实施例9中所述)接种，蛋白疫苗均在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。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(具体疫苗接种规划如表7)。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7 实验动物分组与疫苗接种规划
Figure PCTCN2019116197-appb-000007
小鼠存活状况的生存曲线结果如图10所示，痘苗病毒载体疫苗rvv-LMNB治疗组小鼠总体生存(OS中位数40天)优于对照组小鼠(OS中位数36天，p<0.05)。结果表明痘苗病毒载体疫苗rvv-LMNB能提高患有表达NY-ESO-1肿瘤的小鼠生存。
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肿瘤抗原的多核苷酸，所述肿瘤抗原选自LAGE抗原、MAGE抗原或NY-ESO-1抗原中的一种或多种。
  2. 根据权利要求1所述的重组病毒载体，其中，所述LAGE抗原为LAGE-1抗原；优选地，所述LAGE-1抗原的氨基酸序列如SEQ ID NO:1所示；优选地，其编码核酸序列如SEQ ID NO:2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MAGE抗原为MAGE-A3抗原；优选地，所述MAGE-A3抗原的氨基酸序列如SEQ ID NO:3所示；优选地，其编码核酸序列如SEQ ID NO:4所示。
  4. 根据权利要求1至3任一项所述的重组病毒载体，其中，所述NY-ESO-1抗原的氨基酸序列如SEQ ID NO:5所示；优选地，其编码核酸序列如SEQ ID NO:所示6。
  5. 根据权利要求1至4任一项所述的重组病毒载体，其中，所述肿瘤抗原还包含霍乱毒素B亚单位多肽；优选地，所述霍乱毒素B亚单位多肽的氨基酸序列如SEQ ID NO:7所示；优选地，其编码核酸序列如SEQ ID NO:8所示。
  6. 根据权利要求1至5任一项所述的重组病毒载体，其中，所述肿瘤抗原包含LAGE-1抗原、MAGE-A3抗原和NY-ESO-1抗原；优选地，所述肿瘤抗原的氨基酸序列如SEQ ID NO:9所示；优选地，其编码核酸序列如SEQ ID NO:10所示。
  7. 根据权利要求1至6任一项所述的重组病毒载体，其中，所述重组病毒是痘苗病毒载体，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. 一种用于治疗或预防肿瘤的方法，所述方法包括给予有需要的受试者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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