Glycolipid-anchored cytokines as breast cancer membrane vaccine adjuvants

描述(由申请人提供)：癌症的抗原性和免疫抑制能力差是疫苗开发的主要障碍。此外，对抗原的免疫反应的遗传差异会导致不同患者的抗原谱不同。尽管癌症疫苗领域取得了巨大进步，但人们非常希望有一种高效而简单的方法来制备和管理针对每个患者定制的疫苗，但目前在临床环境中还缺乏这种方法。在这项拨款中，我们建议评估一种新的基于膜的乳腺癌疫苗的有效性，该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，通过蛋白质转移技术快速制备。为了实现目前研究方案的主要目标，我们已经产生了GPI锚定形式的白介素类，并成功地表明它们具有与可溶性形式一样的功能活性。表达膜结合的GPI-IL-12或GPI-IL-2的小鼠乳腺癌细胞在全细胞攻击小鼠时，能有效地诱导保护性抗肿瘤免疫。在这项拨款申请中，我们建议：1)通过蛋白质转移将GPI锚定的细胞因子纯化并整合到分离的乳腺癌膜小泡上，以测试其诱导小鼠保护性抗肿瘤免疫的有效性；2)研究肿瘤特异性T细胞和抗体反应，定量检测抗原特异性CD8+T细胞的扩增，并鉴定GPI-细胞因子修饰的乳腺癌膜疫苗免疫小鼠的记忆T细胞表型；3)确定与GPI-细胞因子佐剂修饰的乳腺癌膜诱导的抗肿瘤免疫相关的免疫学机制；4)研究GPI-细胞因子膜疫苗接种小鼠的肿瘤消退和免疫应答。所提出的用于乳腺癌的膜疫苗策略的优点是：1)大多数被识别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结合到从肿瘤组织获得的膜片段上，从而不需要从患者那里建立用于疫苗开发的细胞系，3)疫苗制备可以在一天内完成，并且可以100%地应用于可从其获得肿瘤组织的乳腺癌患者，4)由于其膜结合的性质，GPI-细胞因子将停留在接种部位并且不太可能引起全身毒性，5)这些细胞因子修饰的肿瘤膜将具有将肿瘤抗原运送到抗原提呈细胞(如树突状细胞)的能力，并能同时激活它们以诱导有效的抗肿瘤免疫，从而克服肿瘤诱导的免疫抑制环境。这种疫苗模式的成功示范有可能开发出有效的膜疫苗，这种疫苗在大多数临床环境下易于制备、储存和管理于个体乳腺癌患者的独特肿瘤。与公共卫生相关：赠款提案旨在开发一种有效的乳腺癌膜基疫苗，该疫苗可以在大多数临床实验室环境下为个别患者快速定制制备。该疫苗不仅利用了细胞因子强大的抗肿瘤特性，而且还利用了蛋白质转移技术的多功能性来简化乳腺癌膜基疫苗的制备。这些研究的结果将为设计基于膜的乳腺癌疫苗提供基础，这种疫苗可以容易地制备并在临床环境中用于人类。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The grant proposal aims to develop an effective membrane-based vaccine for breast cancer that can be custom prepared quickly for individual patients under most clinical laboratory settings. The proposed vaccine not only uses the potent antitumor properties of cytokines but also employs the versatility of the protein transfer technique to simplify the preparation of membrane-based vaccines for breast cancer. The results from these studies will provide the foundation for designing membrane-based breast cancer vaccines that can be easily prepared and administered to humans in clinical settings.