Understanding and enhancing mechanisms of priming in cancer immunotherapy

描述(申请人提供)：恶性脑瘤是35岁以下人群癌症相关死亡的主要原因。迫切需要选择性地针对手术后残留的移行胶质瘤细胞的新的治疗方法。对这些极端异质性肿瘤有效的治疗方法应该有广泛的分子靶点。使用针对多种肿瘤抗原的自体肿瘤细胞裂解疫苗进行免疫治疗是一种很有前途的方法，正在进行大量的临床试验。然而，肿瘤的完全消退很少实现。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还知之甚少。此外，尽管肿瘤反应性T细胞可能与特定患者的生存时间延长有关，但反应性T细胞的功能异质性及其与抗体反应和临床结局的关系还没有得到充分的界定。我们发现，在疫苗生产过程中用于培养肿瘤细胞的氧气浓度翻转了免疫开关，决定了细胞毒性T淋巴细胞(CTL)或抗体产生在结果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佐剂特性，富含癌症干细胞表型，并且相对于在常规氧气水平(20%O2)中生长的细胞，已知的免疫原性胶质瘤抗原的表达增加。此外，接种由5%O2培养物制备的胶质瘤裂解物与CpG寡核苷酸(ODN)混合作为佐剂的胶质瘤荷瘤小鼠，与接种20%O2培养物的小鼠相比，肿瘤浸润性T细胞增加了三倍，存活率显著提高。我们建议阐明在疫苗生产过程中使用的氧气浓度对创新的大动物胶质瘤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，在该模型中，手术、类固醇和术后化疗可以给予类似于人类患者的治疗，这与我们的发现具有很大的翻译相关性。在具体目标1中，我们将确定手术、化疗和接种从5%或20%O2中培养的细胞制备的胶质瘤裂解物/CpG ODN疫苗治疗的狗脑胶质瘤之间的存活率差异。肿瘤负担和毒性的测定将类似于人类患者。在特定目标2中，将测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期相关联。这项研究的成果将是：i)疫苗生产中使用的氧分与T和B细胞反应和患者生存有关的新知识，ii)免疫监测分析的有效性，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，以证明加速对人类胶质瘤患者的临床试验设计是合理的。 与公共卫生相关：胶质瘤是一种侵袭性的脑瘤，非常难以治疗。疫苗已经在胶质瘤患者身上进行了测试，结果并不理想。我们已经开发出一种新的疫苗，在小鼠模型中具有更高的效力。在这个项目中，我们将确定接种疫苗是否可以提高患有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.