Glycolipid-anchored cytokines as breast cancer membrane vaccine adjuvants

摘要：肿瘤的抗原性和免疫抑制能力差是疫苗的主要障碍 发展此外，对抗原的免疫应答的遗传差异导致抗原谱， 因患者而异。尽管在癌症疫苗领域取得了巨大的进展，但一种有效且简单的疫苗仍然存在。 制备和施用针对每个个体患者定制的疫苗的方法高度 但目前缺乏临床环境。在这项资助中，我们建议评估一种 一种新型的膜基乳腺癌疫苗，可以在临床环境中使用 患者的肿瘤细胞膜与糖基磷脂酰肌醇（GPI）锚定的细胞因子结合， 蛋白质转移技术为达致这项研究建议的主要目的，我们 产生了GPI锚定形式的白细胞介素，并成功地表明它们在功能上与 活性为可溶形式。表达膜结合GPI-IL-12或GPI-IL-2的鼠乳腺癌细胞是 当用全细胞攻击小鼠时有效诱导保护性抗肿瘤免疫。在这份赠款中， 应用，我们提出：1）通过蛋白质转移将GPI锚定的细胞因子纯化并掺入到 分离的乳腺癌膜囊泡，并测试它们在乳腺癌中诱导保护性抗肿瘤免疫的功效。 研究肿瘤特异性T细胞和抗体应答，定量抗原特异性CD 8 + T细胞 扩增，并在用GPI-细胞因子掺入的疫苗接种的小鼠中表征记忆T细胞表型 乳腺癌膜疫苗; 3）鉴定与抗肿瘤相关的免疫学机制 由修饰有GPI-细胞因子佐剂的乳腺癌膜引起的免疫和4）研究 GPI-细胞因子膜疫苗接种诱导的肿瘤消退和免疫应答 建立肿瘤。所提出的乳腺癌膜疫苗策略的优点是：1） 大多数鉴定的乳腺癌相关肿瘤抗原是膜相关抗原，2）GPI- 细胞因子可以稳定地掺入到从肿瘤组织获得的膜片段上 需要建立来自患者的细胞系用于疫苗开发，3）疫苗制备可以 在一天内完成，并可应用于100%的乳腺癌患者，从他们的肿瘤组织， 4）由于它们的膜结合性质，GPI-细胞因子将留在疫苗接种部位， 不太可能引起全身毒性，和5）这些姜黄素修饰的肿瘤膜将具有 将肿瘤抗原递送至抗原呈递细胞，例如树突细胞，并且可以同时激活 它们引发有效的抗肿瘤免疫，可以克服肿瘤诱导的免疫抑制， 环境这种疫苗模式的成功示范具有开发 有效的膜疫苗，易于制备，储存和管理的独特肿瘤， 在大多数临床环境下，个体乳腺癌患者。

Abstract: Poor antigenicity and immunosuppressive ability of cancers pose major hurdles to vaccine development. Also, genetic differences in immune responses to an antigen result in an antigenic profile that varies from patient to patient. Despite tremendous progress in the cancer vaccine field, an efficient and easy method for preparing and administering vaccines that are customized to each individual patient is highly desirable, but is currently lacking in clinical settings. In this grant, we propose to evaluate the efficacy of a novel membrane-based breast cancer vaccine that can be rapidly prepared in clinical settings using the patient's tumor cell membranes incorporated with glycosyl phosphatidylinositol (GPI)-anchored cytokines by protein transfer technology. To accomplish the major objectives of the current research proposal, we have generated the GPI-anchored forms of interleukins and have successfully shown that they are as functionally active as soluble forms. Murine breast cancer cells expressing membrane bound GPI-IL-12 or GPI-IL-2, are effective in inducing protective anti-tumor immunity when mice are challenged with whole cells. In this grant application, we propose to: 1) Purify and incorporate GPI-anchored cytokines by protein transfer onto isolated breast cancer membrane vesicles and test their efficacy in inducing protective antitumor immunity in mice; 2) Investigate tumor-specific T cell and antibody responses, quantify antigen-specific CD8+ T cell expansion, and characterize memory T cell phenotype in mice vaccinated with GPI-cytokine incorporated breast cancer membrane vaccines; 3) Identify immunological mechanisms correlating with antitumor immunity elicited by the breast cancer membranes decorated with GPI-cytokine adjuvants and 4) Study tumor regression and immune responses induced by vaccination with GPI-cytokine-membranes in mice with established tumors. The advantages of the proposed membrane vaccine strategy for breast cancers are: 1) most breast cancer associated tumor antigens identified are membrane associated antigens, 2) GPI- cytokines can be stably incorporated onto membrane fragments obtained from tumor tissue thus obviating the need for establishing cell lines from patients for vaccine development, 3) vaccine preparation can be accomplished in a day and can be applied to 100% of breast cancer patients from whom tumor tissues are available, 4) due to their membrane-bound nature, GPI-cytokines will stay in the vaccination site and are unlikely to cause systemic toxicity, and 5) these cytokine-decorated tumor membranes will have the ability to deliver tumor antigens to antigen-presenting cells, such as dendritic cells, and can simultaneously activate them to elicit an effective anti-tumor immunity that can overcome tumor-induced immunosuppressive environment. The successful demonstration of this vaccine modality has the potential for development of effective membrane vaccines that are easy to prepare, store and administer to the unique tumors of individual breast cancer patients under most of the clinical settings.