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研制的主要障碍。 发展。此外，对一种抗原的免疫反应的遗传差异也会导致一种抗原谱 不同的病人会有所不同。尽管在癌症疫苗领域取得了巨大的进步，但一种高效和容易的 为每个患者定制的疫苗的制备和给药方法高度 可取的，但目前在临床环境中缺乏。在这笔赠款中，我们建议评估一种 可在临床环境下快速制备的新型膜基乳腺癌疫苗 患者肿瘤细胞膜与糖基磷脂酰肌醇(GPI)锚定的细胞因子 蛋白质转移技术。为了达到目前研究方案的主要目标，我们已经 产生了GPI锚定形式的干扰素，并成功地表明它们在功能上 以可溶性形式活跃的。表达膜结合GPI-IL-12或GPI-IL-2的小鼠乳腺癌细胞 当小鼠受到全细胞攻击时，有效地诱导保护性抗肿瘤免疫。在这笔赠款中 应用，我们建议：1)通过蛋白质转移纯化和整合GPI锚定的细胞因子 乳腺癌膜囊泡的分离及其诱导小鼠保护性抗肿瘤免疫的实验研究 小鼠；2)研究肿瘤特异性T细胞和抗体反应，定量抗原特异性CD8+T细胞 GPI-细胞因子介导的小鼠记忆T细胞表型的扩增和鉴定 乳腺癌膜疫苗；3)确定与抗肿瘤相关的免疫学机制 GPI-细胞因子佐剂修饰乳腺癌膜诱导免疫的研究 GPI细胞因子膜疫苗诱导的小鼠肿瘤消退及免疫应答 已确诊的肿瘤。建议的乳腺癌膜疫苗策略的优点是：1) 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结合到从肿瘤组织获得的膜片段上，从而避免了 需要从患者身上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，可100%应用于肿瘤组织来自的乳腺癌患者 可用，4)由于其膜结合的性质，GPI-细胞因子将停留在疫苗接种部位并被 不太可能引起全身毒性，以及5)这些细胞因子修饰的肿瘤膜将有能力 将肿瘤抗原传递给抗原呈递细胞，如树突状细胞，并可以同时激活 它们诱导有效的抗肿瘤免疫，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有可能开发出 有效的膜疫苗，易于制备，储存和管理，以治疗独特的肿瘤 乳腺癌患者个体在大多数临床设置下。

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.