Understanding and enhancing mechanisms of priming in cancer immunotherapy

描述（由申请人提供）：恶性脑肿瘤是35岁以下人群癌症相关死亡的主要原因。迫切需要选择性靶向手术后残留的迁移性胶质瘤细胞的新疗法。对这些极其异质性的肿瘤有效的疗法应该具有广泛的分子靶点。使用靶向多种肿瘤抗原的自体肿瘤细胞裂解物疫苗的免疫疗法是在许多临床试验中测试的有前途的方法。然而，很少实现完全的肿瘤消退。疫苗生产期间细胞培养条件对免疫应答和临床结果的影响知之甚少。此外，尽管肿瘤反应性T细胞可能与选定患者的生存期延长相关，但反应性T细胞的功能异质性及其与抗体反应和临床结果的关系尚未充分确定。我们发现，在疫苗生产过程中用于培养肿瘤细胞的氧浓度触发了免疫开关，决定了细胞毒性T淋巴细胞（CTL）或抗体产生是否在产生的免疫应答中占主导地位。相对于在常规氧水平（20%O2）下生长的细胞，在生理脑氧（5%O2）下生长的胶质瘤细胞具有增强的佐剂性质，富集癌症干细胞表型，并且增加已知免疫原性胶质瘤抗原的表达。此外，相对于用来自20%O2培养物的裂解物接种的小鼠，用从与作为佐剂的CpG寡脱氧核苷酸（ODN）混合的5%O2培养物制备的神经胶质瘤裂解物接种的携带神经胶质瘤的小鼠表现出肿瘤浸润性T细胞的三倍增加和显著增加的存活。我们建议阐明疫苗生产过程中使用的氧气浓度对神经胶质瘤的大型动物模型的免疫反应和临床反应的影响。我们已经确定，患有自发性神经胶质瘤的宠物狗代表了一种优秀的动物模型，其中可以给予类似于人类患者的手术，类固醇和术后化疗，从而为我们的研究结果提供了很大的翻译相关性。在具体目标1中，我们将确定通过手术、化疗和用从在5%或20%O2中培养的细胞制备的胶质瘤裂解物/ CpG ODN接种治疗的患有胶质瘤的狗之间的存活差异。肿瘤负荷和毒性将与人类患者相似地确定。在具体目标2中，将确定分泌多种效应细胞因子和去甲肾上腺素以杀死肿瘤细胞的“多功能”CTL的频率，并将其与肿瘤反应性抗体应答和存活相关联。本研究的可交付成果将是：i）将疫苗生产中使用的氧张力与T和B细胞反应和患者生存相关的新知识，ii）验证可用于预测哪些患者可能对免疫疗法有反应的免疫监测测定，iii）世界上唯一有用的胶质瘤大型动物模型的疗效和安全性数据，以证明人类胶质瘤患者加速临床试验设计的合理性。 公共卫生相关性：神经胶质瘤是一种侵袭性的脑肿瘤，很难治疗。疫苗已经在神经胶质瘤患者中进行了测试，效果不佳。我们已经开发出一种新型疫苗，在小鼠模型中具有更高的功效。在这个项目中，我们将确定疫苗接种是否可以增加患有神经胶质瘤的宠物狗的存活率，作为人类临床试验的前奏。

DESCRIPTION (provided by applicant): Malignant brain tumors are the leading cause of cancer-related death in people under age 35. Novel therapies that selectively target residual migratory glioma cells after surgery are urgently needed. Therapies that will be effective against these extremely heterogeneous tumors should have broad molecular targets. Immunotherapy using autologous tumor cell lysate vaccines targeting multiple tumor antigens is a promising approach being tested in numerous clinical trials. However, complete tumor regression is rarely achieved. The effect of cell culture conditions during vaccine production on the immune response and clinical outcome is poorly understood. Moreover, although tumor-reactive T cells may be correlated to prolonged survival in select patients, the functional heterogeneity of responding T cells and their relation to antibody response and clinical outcome is inadequately defined. We discovered that the oxygen concentration used to culture tumor cells during vaccine production flips an immunologic switch, dictating whether cytotoxic T lymphocytes (CTL) or antibody production dominate in the resulting immune response. Glioma cells grown in physiologic brain oxygen (5% O2) have enhanced adjuvant properties, are enriched for a cancer stem cell phenotype, and increase expression of known immunogenic glioma antigens relative to cells grown in the conventional oxygen level (20% O2). Furthermore, glioma-bearing mice vaccinated with glioma lysates prepared from 5% O2 cultures mixed with CpG oligodeoxynucleotides (ODN) as an adjuvant exhibit a three- fold increase in tumor infiltrating T cells and significantly increased survival relative to mice vaccinated with lysates from 20% O2 cultures. We propose to elucidate the effect that oxygen concentration used during vaccine production has on the immune response and clinical response in an innovative large animal model of glioma. We have established that pet dogs with spontaneous gliomas represent an outstanding animal model in which surgery, steroids, and postoperative chemotherapy can be given similar to human patients, lending great translational relevance to our findings. In specific aim 1, we will determine the difference in survival between dogs with glioma treated by surgery, chemotherapy, and vaccination with glioma lysate / CpG ODN prepared from cells cultured in 5% or 20% O2. Tumor burden and toxicity will be determined similar to human patients. In specific aim 2, the frequency of "polyfunctional" CTLs that secrete multiple effector cytokines and degranulate to kill tumor cells will be determined and correlated with tumor-reactive antibody response and survival. The deliverables of this study will be: i) new knowledge relating oxygen tension used in vaccine production to T and B cell responses and patient survival, ii) validation of immune monitoring assays useful in predicting which patients may respond to immunotherapy, iii) efficacy and safety data in the only useful large animal model of glioma in the world to justify accelerated clinical trial design in human glioma patients. PUBLIC HEALTH RELEVANCE: Glioma is an aggressive brain tumor that is very difficult to treat. Vaccines have been tested in glioma patients with suboptimal results. We have developed a novel vaccine with increased efficacy in mouse models. In this project we will determine if vaccination can increase the survival of pet dogs with glioma as a prelude to human clinical trials.