Glycolipid-anchored cytokines as breast cancer membrane vaccine adjuvants

描述（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，对抗原的免疫应答的遗传差异导致不同患者的抗原谱不同。尽管在癌症疫苗领域取得了巨大的进展，但是非常需要一种用于制备和施用针对每个个体患者定制的疫苗的有效且容易的方法，但是目前在临床环境中缺乏这种方法。在这项研究中，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快速制备，使用患者的肿瘤细胞膜与糖基磷脂酰肌醇（GPI）锚定的细胞因子通过蛋白质转移技术。为了实现当前研究提案的主要目标，我们已经产生了GPI锚定形式的白细胞介素，并成功地表明它们与可溶性形式一样具有功能活性。表达膜结合GPI-IL-12或GPI-IL-2的鼠乳腺癌细胞在用全细胞攻击小鼠时有效诱导保护性抗肿瘤免疫。在本授权申请中，我们提出：1）通过蛋白转移将GPI锚定的细胞因子纯化并整合到分离的乳腺癌膜囊泡上，并测试它们在小鼠中诱导保护性抗肿瘤免疫的功效; 2）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，并表征用GPI-细胞因子掺入的乳腺癌膜疫苗接种的小鼠中的记忆T细胞表型; 3）鉴定与由修饰有GPI-细胞因子佐剂的乳腺癌膜引起的抗肿瘤免疫相关的免疫学机制，以及研究通过在具有已建立肿瘤的小鼠中接种GPI细胞因子膜诱导的肿瘤消退和免疫应答。所提出的乳腺癌膜疫苗策略的优点是：1）鉴定的大多数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，从而避免了建立来自患者的细胞系用于疫苗开发的需要，3）疫苗制备可以在一天内完成，并且可以应用于100%的可获得肿瘤组织的乳腺癌患者，4）由于其膜结合性质，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，以及5）这些经精氨酸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）的能力，并且可以同时激活它们以引发有效的抗肿瘤免疫，该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证明具有开发有效的膜疫苗的潜力，这些疫苗在大多数临床环境下易于制备、储存和施用于个体乳腺癌患者的独特肿瘤。 公共卫生相关性：该拨款提案旨在开发一种有效的基于膜的乳腺癌疫苗，该疫苗可以在大多数临床实验室环境下为个体患者快速定制。所提出的疫苗不仅使用细胞因子的有效抗肿瘤特性，而且还采用了蛋白质转移技术的多功能性，以简化乳腺癌的膜基疫苗的制备。这些研究的结果将为设计基于膜的乳腺癌疫苗提供基础，这些疫苗可以很容易地制备并在临床环境中施用于人类。

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.