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。迫切需要有选择性地靶向手术后残余迁移胶质瘤细胞的新疗法。治疗这些极端异质性肿瘤的有效方法应该有广泛的分子靶点。自体肿瘤细胞裂解物疫苗靶向多种肿瘤抗原的免疫治疗是一种很有前途的方法，正在许多临床试验中进行测试。然而，完全的肿瘤消退很少实现。疫苗生产过程中细胞培养条件对免疫反应和临床结果的影响尚不清楚。此外，尽管肿瘤反应性T细胞可能与特定患者的生存期延长有关，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界定。我们发现，在疫苗生产过程中用于培养肿瘤细胞的氧浓度打开了一个免疫开关，决定了细胞毒性T淋巴细胞（CTL）还是抗体产生在由此产生的免疫反应中占主导地位。在生理性脑氧（5% O2）中生长的胶质瘤细胞具有增强的辅助特性，丰富了癌症干细胞表型，并且相对于在常规氧水平（20% O2）中生长的细胞，增加了已知免疫原性胶质瘤抗原的表达。此外，与接种20% O2培养液的小鼠相比，接种5% O2培养液混合CpG寡脱氧核苷酸（ODN）制备的胶质瘤裂解液作为佐剂的胶质瘤小鼠，肿瘤浸润T细胞的数量增加了三倍，存活率显著提高。我们建议阐明疫苗生产过程中使用的氧气浓度对胶质瘤创新大动物模型的免疫反应和临床反应的影响。我们已经确定，患有自发性胶质瘤的宠物狗代表了一种杰出的动物模型，在这种动物模型中，手术、类固醇和术后化疗可以与人类患者类似，这为我们的发现提供了很大的转化相关性。在特定目标1中，我们将确定通过手术、化疗和接种胶质瘤裂解液/ CpG ODN治疗胶质瘤犬的存活率差异，这些胶质瘤裂解液/ CpG ODN由5%或20% O2培养的细胞制备。肿瘤负荷和毒性的测定将类似于人类患者。在特定目标2中，将确定分泌多种效应细胞因子并脱颗粒杀死肿瘤细胞的“多功能”ctl的频率，并将其与肿瘤反应性抗体应答和生存相关。这项研究的成果将包括：i)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)免疫监测分析的有效性验证，可用于预测哪些患者可能对免疫治疗有反应，iii)世界上唯一有用的大型胶质瘤动物模型的有效性和安全性数据，以证明加速人类胶质瘤患者的临床试验设计是合理的。

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.