WO2014063601A1 - 诱导肿瘤特异性免疫的疫苗及其应用 - Google Patents

诱导肿瘤特异性免疫的疫苗及其应用 Download PDF

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WO2014063601A1
WO2014063601A1 PCT/CN2013/085626 CN2013085626W WO2014063601A1 WO 2014063601 A1 WO2014063601 A1 WO 2014063601A1 CN 2013085626 W CN2013085626 W CN 2013085626W WO 2014063601 A1 WO2014063601 A1 WO 2014063601A1
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tumor
vaccine
adenovirus
vaccinia virus
virus
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French (fr)
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王尧河
王鹏举
杜攀
王盛典
黎小珠
高冬玲
袁明
莱蒙•尼克
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郑州大学
玛丽女王伦敦大学
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Definitions

  • i ⁇ BH relates to the field of tumor treatment and prevention, in particular to a vaccine consisting of two viral vectors and its use in the treatment of tumors.
  • Tumor immunotherapy is a potential tumor treatment method.
  • the mechanism of anti-tumor effect of immunotherapy is quite different from conventional chemotherapy, radiotherapy and targeted therapy.
  • Immunotherapy treats tumors from the systemic system without cross-resistance. More importantly, T cells and B cells that exert anti-tumor effects can specifically recognize tumor-associated antigens that are slightly different between tumor cells and normal cells. Provides specific killing effects, but the toxicity is very low.
  • tumor immunity mediated by Cytotoxic T Lymphocytes (CTL) plays the most important role in anti-tumor.
  • CTL Cytotoxic T Lymphocytes
  • the body's natural anti-tumor T cell immune response is severely impaired during tumorigenesis and does not prevent tumor development.
  • tumor vaccines have great potential for tumor therapy by activating tumor-targeted CTLs for tumor treatment and prevention of tumor recurrence.
  • Tumor vaccines can be classified into therapeutic vaccines and preventive vaccines according to the purpose of application. The former targets patients with cancer, and the latter targets prevention for normal and high-risk groups.
  • Tumor vaccines generally include six major categories depending on the method: protein antigens/adjuvant vaccines, DNA vaccines, viral vector-based vaccines, tumor cell vaccines, dendritic cell vaccines, and unique antibody vaccines. Most of the clinical potential of these vaccines has been tested. Although preclinical and early clinical trials have shown that these various methods can produce tumor-specific immune responses, in large sample clinical trials, no tumor vaccines have been shown to be strong enough. Anti-tumor effect.
  • Viral vector-based vaccines have shown many advantages, such as low cost, effectiveness, safety, and convenience. This type of vaccine has shown its potential in human infectious diseases and cancer treatment. Over the past twenty years I It is clear that the use of different vectors for the prime-boost protocol is the most effective way to induce a body-specific immune response. Preclinical and early clinical trials have shown that sequential use of vaccinia virus vectors expressing tumor-associated antigens such as CEA, MUC1 and immunostimulatory molecules and fowlpox virus vectors can produce strong tumor-specific T cell immune responses. However, this heterologous challenge and booster vaccine method did not show significant results in the large-sample phase III clinical trial. Therefore, it is necessary to further study the interaction between the original, the carrier and the immune response of the bridge to develop a new tumor vaccine.
  • tumor-associated antigens such as CEA, MUC1 and immunostimulatory molecules and fowlpox virus vectors
  • Replication of selective tumor-dissolving viruses is a novel method of treating tumors.
  • an adenovirus with deletion of the E1B-55K gene H101, Shanghai 3D Biotechnology Co., Ltd.
  • H101 Chinese adenovirus
  • Shanghai 3D Biotechnology Co., Ltd. an adenovirus with deletion of the E1B-55K gene
  • VV Vaccinia virus
  • the technical problem to be solved by the present invention is to provide a vaccine comprising adenovirus and vaccinia virus sequentially used in a specific order, which can induce tumor-specific immunity, can be used for treating tumors and preventing tumor recurrence.
  • a vaccine for inducing tumor-specific immunity which comprises a tumor-infecting adenovirus and a vaccinia virus, and the two viruses are sequentially used.
  • the oncolytic adenovirus is a replicating adenovirus
  • the vaccinia virus is a vaccinia virus for use in a smallpox vaccine.
  • the oncolytic adenovirus is a replicative human type 5 adenovirus
  • the vaccinia virus is a Listeria vaccinia virus for use in a smallpox vaccine.
  • the oncolytic adenovirus and vaccinia virus can be genetically engineered viruses, thereby making them more tumor selective or causing a stronger immune response. For example, deleting the E1ACR2 or ElB19k gene in the tumor-infected adenovirus, or deleting one or more viral genes in E3B, E3gpl9K, VAI RA; or deleting the TK and/or NIL genes in the vaccinia virus.
  • the tumor-infected adenovirus and vaccinia virus can enhance the vaccine effect by expressing an antibody against a tumor-associated antigen, an immunomodulatory gene, and/or an immunomodulatory protein.
  • the immunoregulatory gene is one, two or more of GM-CSF, Flt3L, IL7, IL12, 41BBL, CCL21, IL24, IL2, IL15, IL15 receptor, IL21 and IL23.
  • a cell vaccine that induces tumor-specific immunity, including a component A tumor-infected adenovirus and a component B vaccinia virus, and the component A infects a tumor cell, an allogeneic tumor cell, or a tumor-associated antigen in vitro.
  • the cells are made into a cell vaccine A' infected with component A, and the component B is made into a cell vaccine B' of component B in the same manner, and then the cell vaccines A' and B' are sequentially used in sequence.
  • the sequential use of the sequence is one week, two weeks, three weeks or four weeks; the cell vaccines A' and B' are administered to the body in different ways, including subcutaneous injection, intramuscular injection, intraperitoneal injection, intracavitary injection, intratumoral injection. Or intratumoral injection of lymph nodes in the tumor.
  • the oncolytic adenovirus or vaccinia virus is any one of the above.
  • the vaccine composed of the adenovirus and the vaccinia virus of the present invention can induce tumor-specific immunity, and the anti-tumor effect produced is first studied in the subcutaneously established golden hamster pancreatic cancer HPD-1NR model.
  • Six doses of oncolytic adenovirus (5xl0 8 PFU, lower than the conventional dose of lxl0 1Q PFU) or vaccinia virus (5xl7 7 PFU, lower than the conventional dose of lxl 9 PFU) were used as a single preparation in the established pancreatic cancer HPD.
  • -1NR tumor model not induce significant tumor inhibition; and with three doses of adenovirus (5xl0 8 PFU) and subsequently with 3 doses of vaccinia virus (5xl0 7 PFU), can result in significant anti-tumor effect.
  • vaccinia virus and adenovirus in reverse, although about 25% of the animals completely disappeared at the same time point, there was no significant difference in the therapeutic effect of the single virus, and the treatment effect was poor.
  • the present inventors have found that the effect of tumor-specific immunity produced by sequential combined virus treatment of tumors is long-term and can be used to prevent tumor recurrence.
  • the original pancreatic cancer cell line HPD-1NR or renal cancer cell HaK was re-inoculated into the treated animals, respectively, using the animals in which the HPD-1NR tumor completely disappeared after sequential combination therapy (after one month of tumor disappearance). It was found that sequential combined tumor-forming adenovirus and vaccinia virus can lead to tumor eradication and long-term tumor-specific immunity, resulting in tumor eradication.
  • the present invention Based on the significant anti-tumor effects produced by the above sequential intratumoral infections of adenovirus and vaccinia virus, in particular to induce tumor-specific, long-term anti-tumor T cell immune responses, the present invention also describes a novel cytovirus tumor immunity method. That is, infecting tumor cells with adenovirus or vaccinia virus in vitro, and then infecting the tumor-bearing body with tumor cells infected with two viruses, and found that the tumor cell components infected with the adenovirus are initially immunized, and the tumor cell components infected with the vaccinia virus are used. Enhanced immunity can produce significant tumor protection immunity.
  • the present invention is the first to use a combination of adenovirus and vaccinia virus as a vaccine for the treatment of tumors in vivo, mainly based on different mechanisms of the host's response to the virus and the possibility of utilizing the host's immune response to the infected tumor cells, due to the first virus
  • the induced specific immune response does not affect the second therapeutic virus, and the second virus lyses the tumor-associated antigen and cytokines released by the tumor cells, which greatly enhances the first virus-induced tumor immunity.
  • the present invention combines a tumor-dissected adenovirus and a vaccinia virus into a vaccine to increase the anti-tumor activity, and to produce a stronger cellular immune response to a combined or separate virus, and the effect is better.
  • sequential H The effect of seedlings on HPD-1NR tumors and HAK tumors is better.
  • the survival time of golden hamsters treating HPD-1NR and HAk tumors is longer. It provides sequential anti-tumor adenovirus and vaccinia virus as a new anti-tumor treatment model. There is strong evidence.
  • the humoral immune response and tumor-specific immune response induced by sequential combination vaccines indicate the levels of anti-adenovirus total antibodies or anti-vaccinia virus total antibodies in the golden hamster serum of the sequential combination of lytic septic virus group There was no significant difference from the other groups; the cured golden hamster re-inoculated HPD-1NR cells with long tumors and showed long-term tumor-specific immunity.
  • the tumor vaccine of the present invention can eradicate tumors and produce long-term tumor-specific immunity, can be used for treatment of various tumors of human or animals, and can effectively prevent tumor recurrence, and the vaccine has broad application prospects.
  • the present invention provides a new technical means and method for designing a viral immunotherapy vaccine, and will be a promising vaccine for treating human tumors.
  • the cell virus vaccine method of the present invention can be used for tumor prevention, postoperative adjuvant therapy for tumor patients, thereby preventing tumor recurrence and metastasis.
  • FIG. 1 The growth curve of HPD-1NR tumors treated with different vaccines shows that the sequential combination of adenovirus and vaccinia virus can significantly inhibit tumor growth.
  • FIG. 1 Survival curves of animals treated with different vaccines for HPD-1NR tumors. It can be seen that the sequential combination treatment of adenovirus and vaccinia virus significantly prolonged the survival time of golden hamsters.
  • FIG. 4 Survival curves of animals treated with different vaccines for HAK tumors. It can be seen that the sequential combination treatment of adenovirus and vaccinia virus significantly prolonged the survival time of golden hamsters.
  • Figure 5 shows a comparison of the number of infiltrating lymphocytes after treatment with different vaccines. It can be seen that the sequential adenovirus and vaccinia virus experimental groups can significantly induce lymphocyte infiltration in tumor cells.
  • FIG. 6 Comparison of the number of caspase-positive cells in tumors treated with different vaccines. It can be seen that the sequential combined adenovirus and vaccinia virus experimental groups can induce high levels of tumor cell apoptosis.
  • FIG. 7 Total antibody against anti-adenovirus in golden hamster serum on day 10 after different vaccine treatments.
  • the total anti-adenoviral antibody levels in the hamster serum of the tumor-bearing adenovirus and vaccinia virus groups were not significantly different from those of the other groups.
  • FIG. 8 Total antibody map of anti-vaccinia virus in golden hamster serum on day 10 after different vaccine treatments. This figure shows the total antibody level of anti-vaccinia virus in golden hamster serum of sequential combined tumor-infected adenovirus and vaccinia virus groups. Compared with other groups! ! ⁇ ⁇ .
  • FIG. 9 Schematic diagram of the killing effect of spleen cells of golden hamsters on HPD-1 NR cells on day 10 after treatment with different vaccines. This figure shows the spleen cell pairs of golden hamsters in sequential co-injection tumor adenovirus and vaccinia virus groups. The killing effect of HPD-1NR cells was significantly higher than that of other groups, indicating that the sequential vaccine can cause high levels of tumor-specific immune cells.
  • Fig. 10 is a schematic diagram showing the killing effect of spleen cells of golden hamsters on HAK cells on the 10th day after treatment with different vaccines, showing that the spleen cells of each group of golden hamsters have no obvious killing effect on non-targeting HAK cells, and no Significant differences indicate that tumor immunity caused by sequential use of adenovirus and vaccinia virus is tumor type specific.
  • FIG. 11 Tumor growth curve of re-inoculated tumor cells from golden hamsters with completely cleared tumor after sequential immunotherapy with adenovirus and vaccinia virus. It can be seen that the tumor no longer grows after inoculation with HPD-1NR, and the tumor is inoculated after HAK cells. It then grows normally and exhibits specific immunity against HPD-1NR tumors.
  • FIG. 12 Tumor growth curves of untreated golden hamsters re-inoculated with tumor cells. It can be seen that tumors can grow normally after inoculation of HPD-1NR and HAK cells in untreated golden hamsters.
  • FIG. 13 Tumor growth curves of DT3316 cells immunized with dl331 and dl309. It can be seen that dl331 adenovirus with VAI R A gene deleted significantly inhibited tumor growth compared with dl309 adenovirus without VAI gene.
  • FIG. 14 Tumor growth curve after sequential immunization with modified adenovirus and vaccinia virus. It can be seen that adenovirus expressing IL12 therapeutic gene as a vaccine component for sequential immunotherapy can significantly inhibit large-load planting. The growth of the tumor.
  • FIG. 15 Tumor clearance after sequential immunization with modified adenovirus and vaccinia virus. It can be seen that adenovirus expressing IL12 gene as a vaccine component for sequential immunotherapy can significantly increase heavy load planting. The complete clearance rate of the tumor.
  • FIG 16. Comparison of the interferon gamma (IFN ⁇ ) produced by the modified vaccinia virus-inducing animals. It can be seen that the vaccinia cells of the vaccinia virus (VVLATKNI L) treated group have spleen cells stimulated by tumor-specific antigens. Production 3 Figure 17. Comparison of immune effects of different tumor cell vaccines. It can be seen that the sequential immunoassay group of HPD-1NR immunized with adenovirus-infected HPD-1NR and infected with vaccinia virus was inoculated with HPD- After 1 NR tumor cells, tumors of 80% of experimental animals no longer grew, and the protective ability was superior to other groups.
  • IFN ⁇ interferon gamma
  • Example 1 Effect of in vivo oncolytic virus combination or treatment of HPD-1NR and HAK golden hamster tumor models alone IX 10 6 HPD-1NR or 5 ⁇ 10 6 HAK cells were subcutaneously inoculated into the right of 6-8 week old golden hamsters.
  • Tumors were measured twice weekly with vernier calipers and measured every three days and every four days. The measurements were mean tumor size and standard deviation, and were statistically processed using post-hoc Bonferroni's One-way A OVA. The results are shown in Figures 1 to 4.
  • Figure 1 shows that Sequentially Combined Lymphoma and Vaccinia Viruses Treat HPD-1NR Tumors Best;
  • Figure 2 shows that the golden hamsters treated with sequential tumor-dissociated adenovirus and vaccinia virus for HPD-1NR tumors have longer survival time.
  • Figure 3 shows that sequential co-injection tumor adenovirus and vaccinia virus are better at treating HAK tumors;
  • Figure 4 shows that the golden hamsters treated with sequential lytic adenovirus and vaccinia virus for HAK tumors have a longer survival time.
  • Example 2 Sequential immunolysis tumor adenovirus and vaccinia virus induce tumor-induced lymphocyte infiltration and tumor cell apoptosis.
  • the golden hamster was sacrificed on the 10th day after the final virus treatment, and the tumor tissues were collected, and further histological and immunohistochemical analysis was performed to observe the infiltration of immune cells and tumor cells in different groups of tumor tissues.
  • the apoptotic situation The results showed that in the sequential immunotherapy group of adenovirus and vaccinia virus on the 10th day after treatment, there were a large number of infiltrating lymphocytes in the tumor (Fig. 5), which induced more apoptosis of tumor cells (Fig. 6).
  • Example 3 Sequential immunolysis of tumor adenovirus and vaccinia virus, reverse combination or separate virus treatment induced body 3 ⁇ 4 The response is compared to a tumor-specific immune response.
  • Example 2 As in the treatment of Example 1, the golden hamster was sacrificed and serum and spleen were collected on the 10th day after the virus treatment. Detection of sequential combined oncolytic adenovirus and vaccinia virus, reverse combined or separate viral treatment induces humoral immune responses and tumor-specific immune responses.
  • A549 or CV1 cells were plated in 96-well plates, and the serum was diluted 2 times in the next day, then adenovirus or vaccinia virus (100 PFU) was added, and cultured in the incubator for one week. , Observe the cytopathic condition in each well.
  • Detection of tumor-specific immune response After spleen cells were isolated in vitro, the tumor cells treated with mitomycin C (2 mg/ml) were stimulated with HPD1NR, and after three days, the stimulated spleen cells were used as effector cells, HPD1NR and HAK were used as Target cells, detecting the killing effect of effector cells on target cells. The results are shown in Figures 7 to 10.
  • Figure 7 shows that the levels of anti-adenovirus total antibodies in the golden hamster serum of the sequential immunolysolytic tumor adenovirus and vaccinia virus groups were not significantly different from those of the other groups;
  • Figure 8 shows the sequential combined tumor-forming adenovirus and vaccinia virus groups. The level of anti-vaccinia virus total antibody in the golden hamster serum was not significantly different from the other groups;
  • Figure 9 shows that the spleen cells of the golden hamsters of the sequential immunolysolytic tumor adenovirus and vaccinia virus groups have significantly higher killing effect on HPD-1 NR cells than the other groups, indicating that the sequential vaccine can cause high levels of tumor-specific immune cells;
  • Figure 10 shows that the spleen cells of each group of golden hamsters have no obvious killing effect on non-targeting HAK cells, and there is no significant difference between the groups, indicating that the tumor immunity caused by sequential use of adenovirus and vaccinia virus is tumor type specific.
  • Example 4 Sequential immunolytic tumor adenovirus and vaccinia virus cured golden hamsters exhibit long-term tumor-specific immunity.
  • the golden hamster and the untreated golden hamster with complete tumor removal were taken, and 30 days later, 1 ⁇ 10 6 HPD-1NR or 5 ⁇ 10 6 HAK cells were re-inoculated on the left side of the golden hamster.
  • Tumors were measured twice a week using vernier calipers and measured every three days and every four days. The measured values were mean tumor size standard deviations.
  • Figure 11 and Figure 12 show that the golden hamsters vaccinated with sequential immunosuppressive tumor adenovirus and vaccinia virus inoculated HPE cells with long tumors, showing long-term tumor-specific immunity; tumors inoculated with HAK cells grew normally.
  • the specific immunity against HPD-1NR tumors was obtained; while the untreated golden hamsters were inoculated with HPD-1NR and HA cells, the tumors grew normally.
  • Example 5 Adenovirus deletes the VAI RNA gene and acts as a vaccine to inhibit tumors.
  • Adenovirus dl331 is Ad5 deleted by VAI (virus-associated type I RA) gene, and VAI RNA binds to DAI (double-stranded RNA activation inhibitor) to prevent its activation, thereby completing viral translation and protein expression. It was found that the deletion of the VAIRNA gene can induce more infected tumor cells to form autophagic cell death, which leads to a stronger immune response and better inhibits tumor growth, see Figure 13. Figure 13 shows that deletion of the VAIRNA gene of the adenovirus type 5 enhances the vaccine effect and significantly inhibits tumor growth more than the dl309 which does not delete the VAI gene.
  • Example 6 Adenovirus expressing the IL12 therapeutic gene as a vaccine component for the treatment of large tumors.
  • Ad-TD-hIL12+VVL15 showed stronger anti-tumor effect than PBS and control virus Ad-TD-RFP+VVL15, and could significantly inhibit tumor growth.
  • the total tumor clearance rate was in Ad-TD-hIL12+VVL15 group. Up to 71.4%, while the Ad-TD-RFP+VVL15 group is 0% (see Figure 14, Figure 15).
  • Figure 14 shows that after adenovirus is added to the immune-related gene interleukin 12 (IL12), sequential tumor vaccine treatment has better tumor suppressive effect;
  • Figure 15 shows the complete tumor clearance rate of sequential combined vaccine after adenovirus is added to the immune-related gene IL12. Higher.
  • IL12 immune-related gene interleukin 12
  • Example 7 Tumor-specific immunity following vaccinia virus deletion of the NIL gene.
  • Example 8 Vaccine effect of tumor-infected adenovirus and vaccinia virus in infecting tumor cells in vitro to induce a tumor-specific immune response.
  • the HPD-1 NR tumor cell line was infected with the tumor-bearing adenovirus and vaccinia virus in vitro to prepare an adenovirus FPn-i NR tumor cell vaccine and a vaccinia virus HPD-1 NR tumor cell vaccine.
  • Golden hamsters were first immunized with 5 ⁇ 10 6 adenovirus or vaccinia virus-infected HPD-1 NR tumor vaccine, while golden hamsters were immunized with 5 ⁇ 10 6 mitomycin C-treated HPD-1 NR as control; after 8 weeks, reuse 5> ⁇ 10 6 adenovirus or vaccinia virus HPD-1NR tumor vaccine immunization, the control group still immunized 5x10 6 mitomycin C treated HPD-1NR; 2 weeks later, each group was subcutaneously inoculated 2xl0 on the opposite side of the immune side 6 HDP-1NR tumor cells were observed for tumor growth in each group of animals.
  • HPD-1NR tumor cell vaccine pre-infected with adenovirus was first immunized with vaccinia virus pre-infection-HPD-1NR tumor cell vaccine.
  • This sequential sequential immunization method can make 80% of the animals produce better tumor specificity.
  • sexual immunization no tumor growth was observed 3 months after inoculation;
  • other immunization methods eg adenovirus-HPD-1NR, adenovirus-HPD-1NR sequential immunization, vaccinia adenovirus-HPD-1NR, adenovirus-HPD- 1NR sequential immunization, vaccinia virus-HPD-1NR, vaccinia virus-HPD-1NR sequential immunization
  • vaccinia virus-HPD-1NR vaccinia virus-HPD-1NR sequential immunization
  • the tumor cell vaccine infected with tumor-infected adenovirus is used for boosting immunity, and the tumor cell vaccine infected with vaccinia virus is boosted, and it can protect about 80% of the animals from growing the same tumor;
  • the tumor cell vaccine is stimulated and boosted to protect only about 40% of the animals from growing the same tumor;
  • the tumor cell vaccine infected with the vaccinia virus alone is used to stimulate and boost the immunity, or the tumor cell vaccine infected with the vaccinia virus is used to stimulate
  • the adenovirus-infected tumor cell vaccine did not produce a strong protective effect, indicating that the tumor vaccine prepared by the tumor cells infected with the tumor-infected adenovirus and vaccinia virus in vitro had the best immune effect.
  • the vaccine is used in a specific sequential combination and has the potential to prevent tumor or postoperative prevention of tumor recurrence and metastasis.

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Abstract

本发明公开了一种由腺病毒和痘苗病毒载体组成的疫苗及其在治疗肿瘤中的应用。所述疫苗包括溶瘤腺病毒和痘苗病毒，两种病毒序贯使用，其中溶瘤腺病毒为复制性人5型腺病毒，痘苗病毒为用于天花疫苗的痘苗病毒。

Description

诱导肿瘤特异性免疫的疫苗及其应用
技术领域
, i^ BH涉及肿瘤治疗及预防领域， 特别是涉及一种由两种病毒载体组成的疫苗及其在 治疗肿瘤上的应用。
背景技术
说
当今， 肿瘤已成为威胁人类健康的重大疾病， 每年世界上约有一千万新发肿瘤病人， 尽管我们对肿瘤发生、 发展和转移的分子机理有了较深的理解， 肿瘤外科治疗、 化疗、 放 疗、 靶向治疗及其他治疗方法也取得较大进展， 但书是对多数实体肿瘤， 其整体生存率几十 年来并无显著提高。 因此， 需要开发新的方法来治疗肿瘤和防治治疗后的肿瘤复发。
肿瘤免疫治疗是一种具有潜能的肿瘤治疗方法， 因免疫治疗发挥抗肿瘤作用的机制与常 规的化、 放疗和靶向治疗截然不同。 免疫治疗从全身系统来治疗肿瘤， 不会有交叉抵抗， 更 为重要的是， 发挥抗肿瘤作用的 T细胞和 B细胞能特异地识别肿瘤细胞和正常细胞之间差异 微小的肿瘤相关抗原， 能提供特异杀伤作用， 但所引起的毒性非常低。 在所有的肿瘤免疫治 疗中， 细胞毒性 T细胞 (Cytotoxic T Lymphocytes, CTL) 所介导的肿瘤免疫在抗肿瘤中发挥着 最为重要的作用。 但是， 在肿瘤发生过程中机体自然的抗肿瘤 T细胞免疫反应被严重削弱， 不能阻止肿瘤的发生发展。 为此， 通过激活肿瘤靶向性的 CTL, 肿瘤疫苗对肿瘤治疗和防止 肿瘤复发是具有很大潜力的肿瘤治疗方法。
肿瘤疫苗根据应用目的可分为治疗性疫苗和预防性疫苗。 前者针对已发癌症病人， 后者 针对正常人群和高危人群的预防。 肿瘤疫苗根据方法不同大致包括六大类别： 蛋白抗原 /佐剂 疫苗、 DNA疫苗、病毒载体为基础的疫苗、肿瘤细胞疫苗、树突状细胞疫苗和独特抗体疫苗。 以上这些疫苗在临床中的应用潜能多数已被检测， 尽管临床前和早期临床试验显示以上各种 方法能产生肿瘤特异免疫反应， 但在大样本临床试验中， 目前未有任何肿瘤疫苗显示足够强 大的抗肿瘤效果。
以病毒载体为基础的疫苗已显示许多优越性， 如成本低、 有效、 安全， 能方便 j 更多人群， 该类疫苗已在人感染性疾病和肿瘤治疗中显示出其潜能。 过去二十多年 I 明，使用不同载体进行激发、增强（prime-boost) 方案是诱导机体特异免疫反应的最有效方法。 临床前和早期临床试验结果显示， 序贯使用表达肿瘤相关抗原如 CEA、 MUC1及免疫共刺激 分子的痘苗病毒载体和禽痘病毒载体能产生较强的肿瘤特异 T细胞免疫反应。 但是这种异源 性的激发、 增强疫苗方法在大样品三期临床实验中并没有显示出显著效果。 因此， 需要深入 研 疏桥.原、 载体和机体免疫反应之间的相互作用， 从而研制新的肿瘤疫苗。
复制选择性溶肿瘤病毒是一种新的治疗肿瘤方法。在 2005年，具有 E1B-55K基因缺失的 腺病毒 （H101 , 上海三维生物技术有限公司） 作为世界上第一个与化疗药物联合治疗头颈部 肿瘤的药物被批准。 尽管其在临床上较为安全， 但溶肿瘤腺病毒作为单一制剂的药效尚有限， 因而进一步开发更加有效的病毒来治疗肿瘤显得十分迫切。
痘苗病毒 （Vaccinia virus, VV) 的许多内在优点使得其非常适合作为溶肿瘤制剂治疗肿 瘤。 近几年来， 我们对溶肿瘤痘苗病毒作为治疗人类肿瘤的药物进行了全面研究， 发现作为 预防天花的痘苗病毒 VVLister株在体外具有杀伤肿瘤细胞的特性， 尤其是对溶肿瘤腺病毒不 敏感的肿瘤， 痘苗病毒在体内也显示出强大的抗肿瘤效果。 尽管单独利用复制选择性溶肿瘤 病毒的安全性好， 但是其临床效果较差， 改善这些溶肿瘤病毒的治疗效果已迫在眉睫。
发明内容
本发明要解决的技术问题是： 提供一种按照特定顺序序贯使用的包含腺病毒和痘苗病 毒的疫苗， 该疫苗能诱导肿瘤产生特异性免疫， 可用于治疗肿瘤和防止肿瘤的复发。
本发明的技术方案：
一种诱导肿瘤特异性免疫的疫苗， 所述疫苗包括溶肿瘤腺病毒和痘苗病毒， 两种病毒 按先后序贯使用。
所述溶肿瘤腺病毒为复制性腺病毒， 痘苗病毒为用于天花疫苗的痘苗病毒。
所述溶肿瘤腺病毒为复制性人 5型腺病毒， 痘苗病毒为用于天花疫苗的李斯特株痘苗 病毒。
所述溶肿瘤腺病毒和痘苗病毒可为基因改造的病毒， 从而使其更有肿瘤选择性， 或引 起更强的机体免疫反应。 如将所述溶肿瘤腺病毒中的 E1ACR2或 ElB19k基因删除， 或者 将 E3B、 E3gpl9K、 VAI R A中的一个或多个病毒基因删除； 或将痘苗病毒中的 TK 和 /或 NIL基因删除。 所述溶肿瘤腺病毒和痘苗病毒可通过表达肿瘤相关抗原、免疫调节基因和 /或免疫调节 蛋白的抗体， 以增强疫苗效果。
所述免疫调节基因为 GM-CSF、 Flt3L、 IL7、 IL12、 41BBL、 CCL21、 IL24、 IL2、 IL15、 IL15受体、 IL21和 IL23的一种、 两种或两种以上。
, 一^诱导肿瘤特异性免疫的细胞疫苗， 所述细胞疫苗包括 A组份溶肿瘤腺病毒和 B 组份痘苗病毒， 将 A组份在体外感染自身肿瘤细胞、异体肿瘤细胞或表达肿瘤相关抗原的 细胞， 制成感染 A组份的细胞疫苗 A' ， 将 B组份按同样方法制成感染 B组份的细胞疫 苗 B'， 然后将细胞疫苗 A' 、 B' 按先后顺序序贯使用。
所述顺序序贯使用的间隔时间为一周、 两周、 三周或四周； 细胞疫苗 A' 、 B' 采取 不同的途径给入机体， 包括皮下注射、 肌肉注射、 腹腔、 体腔注射、 瘤体内注射或肿瘤附 属淋巴结内注射。
所述的溶肿瘤腺病毒或痘苗病毒为上述的任何一种。
所述的疫苗或细胞疫苗在制备用于治疗肿瘤的药物中的应用。
所述的疫苗或细胞疫苗在制备用于治疗肿瘤的药物中的应用。
本发明中腺病毒和痘苗病毒组成的疫苗能诱导肿瘤产生特异性免疫， 产生的抗肿瘤效 果首次在皮下建立的金黄地鼠胰腺癌 HPD-1NR模型中被研究。 实验中采用 6个剂量的溶 肿瘤腺病毒 （5xl08PFU， 低于常规剂量 lxl01QPFU)或痘苗病毒 （5xl07PFU， 低于常规剂 量 lxl09PFU)作为单一制剂在已建立的胰腺癌 HPD-1NR肿瘤模型中不能诱导明显的肿瘤 抑制； 而用 3个剂量的腺病毒（5xl08PFU)并随之采用 3个剂量的痘苗病毒（5xl07PFU)， 可导致明显的抗肿瘤效果。 然而， 反向联合使用痘苗病毒和腺病毒尽管有 25%左右的动物 在同一时间点肿瘤完全消失， 但与单个病毒的治疗效果并无显著差异， 治疗效果较差。
利用腺病毒和痘苗病毒这种特定的序贯免疫方法， 肿瘤治愈率和荷瘤动物的生存期明 显提高， 大约 75%的动物在研究的 4个月结束前仍然存活； 在建立的肾癌 HAK模型中也 表现出同样的治疗效果。 结果显示， 序贯使用腺病毒和痘苗病毒比反向联合或单独使用的 效果更佳。 上述研究提示这种序贯联合的治疗方案能适用于多种肿瘤。
为了证实序贯联合两种不同的溶肿瘤病毒是否能降低宿主对每一种病毒的体 ¾ 反应， 不同治疗方案后在动物血清中检测了所有抗腺病毒和痘苗病毒的循环抗体和 的中和抗体水平。 结果显示， 抗腺病毒和痘苗病毒的总抗体在各个不同组之间无差异
(P>0.05 ) o事实上，腺病毒和痘苗病毒疫苗的序贯联合治疗诱导的抗腺病毒的抗体水平稍 高于单独腺病毒组， 中和抗体水平和总抗体水平一致， 各组之间无明显差异。 这些结果表 明， 序贯联合腺病毒和痘苗病毒介导的强大抗肿瘤效果不是由于对每个病毒体液免疫的降 ί¥ί¾¾Γ的。
为了进一步证实序贯向肿瘤组织中注射复制性腺病毒和痘苗病毒是一种有效的肿瘤 疫苗方法， 进行动态检测金黄地鼠脾脏内肿瘤特异 CTL 的水平。 结果发现， 在腺病毒 3 次治疗而后痘苗病毒 3次治疗的组与其它治疗组比较， 能产生更高水平的肿瘤特异 CTL, 提示病毒能诱导肿瘤特异性免疫， 可见， 该序贯联合病毒疫苗是体内诱导肿瘤特异性免疫 的有效方法。
本发明发现由序贯联合病毒治疗肿瘤产生的肿瘤特异性免疫作用效果是长期的， 可用 于防止肿瘤复发。 为了证明此效果， 利用序贯联合治疗后 HPD-1NR肿瘤完全消失的动物 (肿瘤消失一个月后）， 将原始的胰腺癌细胞 HPD-1NR或肾癌细胞 HaK分别重新接种上 述治疗后的动物。 结果发现， 序贯联合溶肿瘤腺病毒和痘苗病毒能导致肿瘤的根除和产生 长期的肿瘤特异性免疫， 致使肿瘤的根除。
基于以上序贯肿瘤内感染腺病毒和痘苗病毒所产生的显著性抗肿瘤效果， 尤其是诱导 肿瘤特异的、 长期的抗肿瘤 T细胞免疫反应， 本发明还阐述了一种新的细胞病毒肿瘤免疫 方法。 即体外用腺病毒或痘苗病毒感染肿瘤细胞， 然后用两种病毒感染的肿瘤细胞免疫荷 瘤机体， 发现用腺病毒感染的肿瘤细胞组份做初始免疫， 用痘苗病毒感染的肿瘤细胞组份 做免疫加强可产生显著的肿瘤保护免疫能力。
本发明的积极有益效果：
( 1 ) 本发明首次采用序贯联合腺病毒和痘苗病毒作为疫苗治疗体内肿瘤， 主要是基于宿 主对病毒反应的不同机制和利用宿主对感染的肿瘤细胞免疫反应的可能性， 由于第一种病 毒诱导的特异性免疫反应对第二种治疗病毒不会产生影响， 第二种病毒溶解肿瘤细胞所释 放的肿瘤相关抗原和细胞因子， 可大大增强第一种病毒诱导的肿瘤免疫。
(2) 本发明将溶肿瘤腺病毒和痘苗病毒序贯联合制成疫苗， 能提高其抗肿瘤活性， 向联合或单独病毒作用产生更强的细胞免疫反应， 使用效果更佳。 实用表明， 序贯 H 苗治疗 HPD-1NR肿瘤、 HAK肿瘤的效果较好， 治疗 HPD-1NR、 HAk肿瘤的金黄地鼠的 存活时间较长， 为序贯联合溶肿瘤腺病毒和痘苗病毒作为新的抗肿瘤治疗模式提供了有力 证据。
( 3 ) 序贯联合疫苗所诱导的体液免疫反应和肿瘤特异性的免疫反应表明， 序贯联合溶肿 ¾¾ 苗病毒组的金黄地鼠血清中抗腺病毒总抗体或抗痘苗病毒总抗体的水平与其 他组无明显差异； 治愈的金黄地鼠再接种 HPD-1NR细胞不长肿瘤， 表现出长期的肿瘤特 异性免疫。
(4) 本发明的肿瘤疫苗能将肿瘤根除， 并产生长期的肿瘤特异性免疫， 可用于人或动物 各种肿瘤的治疗， 并能有效防止肿瘤复发， 该疫苗将具有广阔的应用前景。 本发明为设计 病毒免疫治疗疫苗提供了新的技术手段和方法， 将是一种十分有前景的治疗人类肿瘤的疫 苗。
( 5 ) 本发明的细胞病毒疫苗方法， 可用于肿瘤预防， 肿瘤病人术后辅助治疗， 从而阻止 肿瘤复发和转移。
附图说明
图 1，采用不同疫苗治疗 HPD-1NR肿瘤的生长曲线，可看出腺病毒和痘苗病毒的序贯联合 治疗组能显著抑制肿瘤的生长。
图 2，采用不同疫苗治疗 HPD-1NR肿瘤的动物的生存曲线，可看出腺病毒和痘苗病毒的序 贯联合治疗组能显著延长金黄地鼠的存活时间。
图 3，采用不同疫苗治疗 HAK肿瘤生长曲线， 可看出腺病毒和痘苗病毒的序贯联合治疗组 能显著抑制肿瘤的生长。
图 4，采用不同疫苗治疗 HAK肿瘤的动物的生存曲线，可看出腺病毒和痘苗病毒的序贯联 合治疗组能显著延长金黄地鼠的存活时间。
图 5， 采用不同疫苗治疗肿瘤后的浸润淋巴细胞数比较图， 可看出序贯联合腺病毒和痘苗 病毒实验组能显著诱导肿瘤细胞内淋巴细胞的浸润。
图 6， 采用不同疫苗治疗后肿瘤的半胱天冬氨酸蛋白酶阳性细胞数比较图， 可看出序贯联 合腺病毒和痘苗病毒实验组能诱导高水平的肿瘤细胞凋亡。
图 7， 不同疫苗治疗后第 10天的金黄地鼠血清中抗腺病毒的总抗体图， 该图显示序， 溶肿瘤腺病毒和痘苗病毒组的金黄地鼠血清中抗腺病毒总抗体水平与其他组比较没有显 著差异。
图 8， 不同疫苗治疗后第 10天的金黄地鼠血清中抗痘苗病毒的总抗体图， 该图显示序贯联 合溶肿瘤腺病毒和痘苗病毒组的金黄地鼠血清中抗痘苗病毒总抗体水平与其他组比较没 有！！茧 ^。
图 9， 采用不同疫苗治疗后第 10天的金黄地鼠的脾细胞对 HPD-1NR细胞的杀伤作用示意 图， 该图显示序贯联合溶肿瘤腺病毒和痘苗病毒组的金黄地鼠的脾细胞对 HPD-1NR细胞 的杀伤作用明显高于其他组， 说明该序贯疫苗能引起高水平肿瘤特异免疫细胞。
图 10， 采用不同疫苗治疗后第 10天的金黄地鼠的脾细胞对 HAK细胞的杀伤作用示意图， 显示各组金黄地鼠的脾细胞对非靶向 HAK细胞没有明显的杀伤作用， 各组无明显差异， 说明序贯使用腺病毒和痘苗病毒所引起肿瘤免疫力是肿瘤类型特异性的。
图 11， 腺病毒和痘苗病毒的序贯免疫治疗后， 肿瘤完全清除的金黄地鼠重新接种肿瘤细胞 的肿瘤生长曲线， 可看出， 接种 HPD-1NR后肿瘤不再生长， 接种 HAK细胞后肿瘤则正常 生长， 表现出针对 HPD-1NR肿瘤的特异性免疫能力。
图 12， 未治疗的金黄地鼠重新接种肿瘤细胞的肿瘤生长曲线， 可看出， 未治疗的金黄地鼠 接种 HPD-1NR和 HAK细胞后， 肿瘤都能正常生长。
图 13， dl331和 dl309感染的 DT6606细胞免疫接种小鼠后的肿瘤生长曲线， 可看出， 删除 VAI R A基因的 dl331腺病毒与未删除 VAI基因的 dl309腺病毒相比，能显著抑制肿瘤生 长。
图 14， 采用修饰过的腺病毒和痘苗病毒序贯免疫后的肿瘤生长曲线， 可看出， 表达 IL12 治疗基因的腺病毒作为疫苗组份之一进行序贯免疫治疗， 能显著抑制大负荷种植瘤的生 长。
图 15， 采用修饰过的腺病毒和痘苗病毒序贯免疫后肿瘤的清除率， 可看出， 表达 IL12治 疗基因的腺病毒作为疫苗组份之一进行序贯免疫治疗， 能显著增加大负荷种植瘤的完全清 除率。
图 16， 修饰过的痘苗病毒诱导动物产生的干扰素 γ (IFN Y) 的比较图， 可看出， 删阁 基因的痘苗病毒 （VVLATKNI L ) 治疗组， 其脾脏细胞在肿瘤特异性抗原刺激下产 3 图 17， 不同肿瘤细胞疫苗的免疫效果比较图， 可看出， 先用腺病毒感染的 HPD-1NR免疫、 后用痘苗病毒感染的 HPD-1NR加强免疫的序贯免疫实验组，在接种 HPD-1NR肿瘤细胞后 80%的实验动物的肿瘤不再生长， 保护能力优于其它组。
具钛 渝古式
实例 1: 体内溶肿瘤病毒联合或单独治疗 HPD-1NR和 HAK金黄地鼠肿瘤模型的效果 将 I X 106个 HPD-1NR或 5xl06个 HAK细胞于皮下接种 6-8周龄金黄地鼠的右背侧 面， 十天后， 当肿瘤直径达 6-7mm时， 随机分为五组， 每组 8只金黄地鼠， A组于第 0、 2、 4、 6、 8和 10天肿瘤内注射 PBS; B组于第 0、 2、 4、 6、 8和 10天肿瘤内注射 5xl08 PFU 腺病毒 Ad5; C组于第 0、 2、 4、 6、 8和 10天肿瘤内注射 5xl07 PFU痘苗病毒 VVLister; D组在第 0、 2和 4天于肿瘤内注射 5xl08PFU腺病毒 Ad5， 并接着于第 6、 8和 10天注射 5xl07 PFU痘苗病毒 VVLister; E组在第 0、 2和 4天于肿瘤内注射 5xl07PFU痘苗病毒 VVLister, 并接着于第 6、 8和 10天注射 5xl08 PFU腺病毒 Ad5。 其中 PBS、 腺病毒 Ad5、 痘苗病毒 VVLister每次的注射量均为 100微升。
每周用游标卡尺测量肿瘤两次， 每三天、 每四天各测量一次， 测量值为平均肿瘤大小 士标准差， 采用 post-hoc Bonferroni的 One-way A OVA进行统计学处理。 结果见图 1至 图 4。
图 1表明序贯联合溶肿瘤腺病毒和痘苗病毒治疗 HPD-1NR肿瘤的效果最好； 图 2表明序 贯联合溶肿瘤腺病毒和痘苗病毒治疗 HPD-1NR肿瘤的金黄地鼠的存活时间较长； 图 3显 示序贯联合溶肿瘤腺病毒和痘苗病毒治疗 HAK肿瘤的效果较好； 图 4表明序贯联合溶肿 瘤腺病毒和痘苗病毒治疗 HAK肿瘤的金黄地鼠的存活时间较长。
实例 2: 序贯免疫溶肿瘤腺病毒和痘苗病毒诱导肿瘤产生淋巴细胞浸润及肿瘤细胞凋亡。
如例 1的免疫方法， 在最后病毒治疗后的第 10天金黄地鼠被处死， 并收集肿瘤组织， 进一步进行组织学和免疫组化分析， 观察不同组肿瘤组织中免疫细胞的浸润和肿瘤细胞的 凋亡情况。 结果显示， 在治疗后第 10 天腺病毒和痘苗病毒序贯免疫治疗组， 肿瘤中有大 量浸润淋巴细胞 （图 5)， 诱导更多的肿瘤细胞凋亡 （图 6)。
实例 3: 序贯免疫溶肿瘤腺病毒和痘苗病毒、 反向联合或单独的病毒治疗诱导的体¾ 反应和肿瘤特异性的免疫反应比较。
如例 1 的治疗方法， 在病毒治疗后的第 10天， 将金黄地鼠处死并收集血清和脾脏。 检测序贯联合溶肿瘤腺病毒和痘苗病毒、 反向联合或单独的病毒治疗诱导的体液免疫反应 和肿瘤特异性的免疫反应。
. ^镇由总抗体的检测： 在 96孔板中铺 A549或 CV1细胞， 第二天将血清按 2倍等比 稀释， 再加入腺病毒或痘苗病毒 （100 PFU) ， 在培养箱培养一周后， 观察每个孔内的细 胞病变情况。 肿瘤特异性免疫反应的检测： 将脾脏细胞体外分离后， 加入丝裂霉素 C ( 2 mg/ml) 处理的肿瘤细胞 HPD1NR刺激， 三天后将刺激后的脾脏细胞作为效应细胞， HPD1NR和 HAK作为靶细胞， 检测效应细胞对靶细胞的杀伤作用。 结果见图 7至图 10。
图 7显示序贯免疫溶肿瘤腺病毒和痘苗病毒组的金黄地鼠血清中抗腺病毒总抗体的水 平与其他组比较没有显著性差异； 图 8显示序贯联合溶肿瘤腺病毒和痘苗病毒组的金黄地 鼠血清中抗痘苗病毒总抗体的水平与其他组比较没有显著性差异；
图 9显示序贯免疫溶肿瘤腺病毒和痘苗病毒组的金黄地鼠的脾细胞对 HPD-1NR细胞 的杀伤作用明显高于其他组， 说明该序贯疫苗能引起高水平的肿瘤特异免疫细胞； 图 10 显示各组金黄地鼠的脾细胞对非靶向 HAK细胞没有明显的杀伤作用， 各组无明显差异， 说明序贯使用腺病毒和痘苗病毒所引起肿瘤免疫力是肿瘤类型特异性的。
实例 4: 序贯免疫溶肿瘤腺病毒和痘苗病毒治愈的金黄地鼠， 表现出长期的肿瘤特异性免 疫。
如实例 1中免疫方法， 取肿瘤完全清除的金黄地鼠和未治疗的金黄地鼠， 30天后重新 接种 l x lO6个 HPD-1NR或 5x l06个 HAK细胞于金黄地鼠的左侧面， 每周用游标卡尺测量 肿瘤两次， 每三天、 每四天各测量一次， 测量值为平均肿瘤大小士标准差。
结果发现， 当动物重新接种先前的 HPD-1NR细胞时， 观察到 105天仍没有发现肿瘤 生长， 而利用 HAK接种后发现 4个动物中有 3个动物的肿瘤生长迅速， 一个动物最初利 用 HAK形成的肿瘤在 63天后受到抑制， 利用 HPD-1NR或 HAK细胞接种的对照动物中 其肿瘤的生长迅速。 见图 11、 图 12。
图 11、 图 12 表明序贯免疫溶肿瘤腺病毒和痘苗病毒治愈的金黄地鼠接种 HPE 细胞不长肿瘤， 表现为长期的肿瘤特异性免疫； 接种 HAK细胞的肿瘤则正常生长， 出针对 HPD-1NR肿瘤的特异性免疫能力； 而未治疗的金黄地鼠接种 HPD-1NR和 HA 细胞后， 肿瘤都能正常生长。
实例 5: 腺病毒删除 VAI RNA基因后作为疫苗抑制肿瘤的效果。
在 5-6周龄的 C57小鼠背部右上侧接种 3x l06个分别预先感染 10PFU/细胞腺病毒 dl309 a 的 DT6606细胞， 观察肿瘤生长状况。 腺病毒 dl331是 VAI (病毒相关 I型 R A) 基因删除的 Ad5， VAI RNA能与 DAI (双链 RNA激活抑制剂） 结合阻止其激活， 从而完 成病毒的翻译和蛋白表达。 实验发现， VAIRNA基因删除后能诱导更多感染的肿瘤细胞形 成自噬细胞死亡， 引起更强免疫反应， 更好地抑制肿瘤生长， 参见图 13。 图 13表明 5型 腺病毒 VAIRNA基因的删除， 增强了疫苗效果， 比未删除 VAI基因的 dl309更能显著地 抑制肿瘤生长。
实例 6: 表达 IL12治疗基因的腺病毒作为疫苗组份， 对大体积肿瘤的治疗效果。
在 5-6周龄的金黄地鼠背部右上侧接种 l x lO6的 HPD-1NR细胞，当肿瘤生长至较大荷 瘤 （300 mm3 ) 在肿瘤内分别注射 PBS ( 100微升 /次） 、 Ad-TD-RFP+VVL15 ( 100微升 / 次）和 Ad-TD-hIL12+VVL15( 100微升 /次），间隔一天一次，共 6次，序贯免疫组 Ad-TD-RFP 和 Ad-TD-hIL12为前三次治疗 （5x l08 pfU /次） ， VVL15为后三次治疗 （5x l07 pfii /次） ， 然后观察肿瘤大小和肿瘤全部清除率。 结果显示， Ad-TD-hIL12+VVL15比 PBS和对照病 毒 Ad-TD-RFP+VVL15显示出更强的抗肿瘤效果， 能明显抑制肿瘤生长， 肿瘤全部清除率 在 Ad-TD-hIL12+VVL15组可达 71.4%， 而 Ad-TD-RFP+VVL15组为 0% (参见图 14、 图 15 ) 。
图 14说明腺病毒加入免疫相关基因白介素 12 (IL12) 后， 序贯联合疫苗治疗的肿瘤 抑制效果较好； 图 15说明腺病毒加入免疫相关基因 IL12后， 序贯联合疫苗治疗的肿瘤完 全清除率较高。
实例 7: 痘苗病毒删除 NIL基因后的肿瘤特异性免疫。
在 C57BL/6小鼠右侧皮下接种 4x l06的 DT6606-OVA肿瘤细胞， 当肿瘤体积达到 160mm3时， 分别在第一、三、五天瘤内注射 PBS、 VVLATK或 VVLATKN 1L (1χ108ρίίι), PBS、 VVLATK VVLATKN 1L每次的注射量均为 100微升； 7天后处死小鼠， 取 ί 胞， 在 RPMI完全培养基中稀释到 5xl06 cells/ml， 每孔 lOOul铺圆底 96孔板， 加入 刺激 48小时后， 收集上清， 用 ELISA检测 IFNY。 结果显示 （图 16)， 删除 NIL基因的痘 苗病毒 （VVLATK IL) , 能诱导产生更强的肿瘤特异性免疫。
实例 8: 溶肿瘤腺病毒和痘苗病毒体外感染肿瘤细胞诱导肿瘤特异免疫反应的疫苗效果。
利用溶肿瘤腺病毒和痘苗病毒在体外分别感染 HPD-1NR肿瘤细胞系，制备成腺病毒 FPn- i NR呷瘤细胞疫苗和痘苗病毒 HPD-1NR肿瘤细胞疫苗。 具体方法： 取同种来源于金 黄地鼠的胰腺癌细胞 HPD-1NR,先在体外用溶肿瘤腺病毒（MOI =10pfu/cell)感染 4小时， 或用痘苗病毒 （MOI=lpfU/Cell) 感染 2小时， 然后收获病毒感染的肿瘤细胞作为疫苗。
首先用 5xl06的腺病毒或痘苗病毒感染的 HPD-1NR肿瘤疫苗免疫金黄地鼠， 同时用 5xl06的丝裂霉素 C处理的 HPD-1NR免疫金黄地鼠作为对照； 8周后， 再用 5><106腺病毒 或痘苗病毒 HPD-1NR 肿瘤疫苗免疫， 对照组依然免疫 5xl06的丝裂霉素 C 处理的 HPD-1NR; 2周后， 各组都在免疫对侧的皮下接种 2xl06的 HDP-1NR肿瘤细胞， 观察各 组动物的肿瘤生长情况。
发现先用腺病毒预感染的 HPD-1NR 肿瘤细胞疫苗免疫， 再用痘苗病毒预感染 -HPD-1NR肿瘤细胞疫苗免疫， 这种顺序序贯免疫方法能让 80%的动物产生较好的肿瘤特 异性免疫， 接种后 3个月没有观察到肿瘤的生长； 而其它免疫方法 （如腺病毒 -HPD-1NR、 腺病毒 -HPD-1NR顺序免疫， 痘苗腺病毒 -HPD-1NR、 腺病毒 -HPD-1NR顺序免疫， 痘苗病 毒 -HPD-1NR、 痘苗病毒 -HPD-1NR顺序免疫）都无法达到相似效果。 同时， 作为肿瘤细胞 全蛋白疫苗的丝裂霉素 C处理过 HPD-1NR免疫组， 在多次试验中没有一只动物产生过有 效的肿瘤特异性免疫， 全部在接种后长出肿瘤， 并不再消退。 结果见图 17。
从图 17 可看出， 用溶肿瘤腺病毒感染的肿瘤细胞疫苗做激发免疫， 痘苗病毒感染的 肿瘤细胞疫苗做加强免疫， 能保护约 80%的动物不再生长同样肿瘤； 单纯用腺病毒感染的 肿瘤细胞疫苗做激发和加强免疫， 只能保护约 40%的动物不再生长同样肿瘤； 单纯用痘苗 病毒感染的肿瘤细胞疫苗做激发和加强免疫， 或用痘苗病毒感染的肿瘤细胞疫苗做激发、 腺病毒感染的肿瘤细胞疫苗做加强免疫， 均不能产生很好的保护作用， 表明序贯溶肿瘤腺 病毒和痘苗病毒体外感染的肿瘤细胞制备的肿瘤疫苗的免疫效果最好。 可见， 该疫苗按照 特定的序贯联合使用， 有预防肿瘤或术后预防肿瘤复发转移的潜能。

Claims

权 利 要 求 书
I.一种 导肿瘤特异性免疫的疫苗， 其'特征是： 所建 包括溶胂瘤腺病毒和痘苗病毒.， 两种病毒按先后序贯使用.。
2.根据权 ¾要求 I所述的疫苗， 其特征是； 所述溶 W瘤朦病毒为复制性腺病毒 痘苗病毒为 用于天花疫苗的痘苗痛毒
3.根据权利要求 1所述的疫苗， 其特征是 所述溶舯瘤籐病'毒为复制性人 5型艨病毒， 痘苗 病毒为用于天花 δ苗的亭 Λ特株痕苗病毒。
4.根据权 W要求〗所述的疫苗，其特征是^所述溶肿瘤腺病毒.和痘苗病毒为基 Β改造的病毒, 将溶 W瘤聽病毒中的 E1ACS2或 Emi .基因删除， 或者将 E3B、 E3gpl 9 , ¥M RNA中的 一个或多个病毒基园删除； 或翁瘇苗病毒中的 TK墓园 ¾/或 111墓因删除。
5.根据概利要求 1所述的疫苗， 其特征是： 所述溶肿瘤》病毒和痘苗病毒通过表达磨瘤相关 抗 JS、 兔疫调节基因稀或兔疫 节蛋白的枕体， 以增强疫苗效果。
6.根据权利要求 5所述的疫苗 其特 ¾Έ是： 折述兔疫囊节基 Β为 GM-CSF、 Flt3L、 IL7. IL12. 4 IBBL, CCL21-. IL24 -. IL2 iL15、 IL15 ¾#, IL21. ,和 IL23中酌一种、 两种或两种 上《 7..—种 导肿攛特异 ¾¾痃的媚胞疫苗，其特征 '是： Fr 细舰疫苗包 K A ffi份 勝瘤,腺《« 和 B组份痘苗病毒，将 A组份在律外感染自身肿瘤细遍、异体肿瘸细胞或表达肿瘤相关抗原 的细胞 制成感染 A組份 |¾细胞疫苗' ,将 B组份按同样方法制成感染 B组份 &¾细飽疫苗 B V, 然后将 Λ龜疾苗 As 、 Β' 按先后顺序序贯使用。
9.根据权 W要求 7所述的细'跑疫苗，其特 II是：所述顺序序贯使用的间隔时 Μ为一厕、两周、 三局或四周； 细胞疫苗 A3 、 BJ ¾取不同的途径鎗入视体， 包括皮下注射、 肌 «注射、 腹 腔、 #腔注射、瘤鉢内注射域肿痼險属淋巴结内注射.。
9-裉握教利要求 7所逑的细飽疫苗 其特征是： 所逑的 肿瘤腺病毒或痘苗病毒为权利荽求 2、 3, 4、 5 中的:任意一 '种。
10.权利要求 1-9任一项所述的疫苗或细胞疫苗在制备用于治 tf.酔瘤的 '药^中的应用。
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