Exploiting chemoimmunotherapy strategies with genetically-engineered gd T cells

项目摘要 高危神经母细胞瘤(HR-NB)是一种儿童致死性实体瘤。存活率为50%，幸存下来的人受苦 许多治疗与毒性有关，强调了对新的肿瘤靶向治疗的迫切需要。NB患者的存活率 随着抗GD2抗体迪努昔单抗的加入而得到改进，增加了对其他 免疫疗法，如过继转移细胞疗法治疗NB。到目前为止，细胞治疗主要集中在αβT 细胞和NK细胞，到目前为止，对实体瘤的治疗都是不成功的。伽马三角洲(γδ)T细胞 是一种创新和优越的选择，因为它们不依赖于MHC，对肿瘤细胞直接细胞毒，包括 NB可以识别和靶向肿瘤微环境中的免疫抑制细胞，而缺乏 αβT细胞的同种异体反应。过继的γδT细胞由于EX不足而尚未广泛应用于临床 活体扩增方法。我们开发了一种符合GMP的无血清生产策略来 将γδT细胞扩增到足以供人类使用的水平。扩增的细胞高度表达CD16，这是直接 参与抗体导向的细胞毒作用(ADCC)。我们已经证明γδT细胞从NB扩增而来 患者有效地杀死了NB细胞系，并增强了地诺昔单抗诱导的NB细胞死亡。因此，我们的首要任务是 我们的目标是生成临床前和IND支持的数据，证明我们的γδT细胞产品的有效性。 我们还产生了两个针对GD2的嵌合抗原受体(CAR)，以将γδT细胞定位于NB，以及 表明CAR修饰的免疫活性细胞比未修饰的细胞具有更强的细胞毒性。 我们还开发了使正常免疫细胞产生化疗耐药性的策略。这导致了我们的 创新的“抗药性免疫疗法”平台，可联合应用化学保护的γδT细胞 联合化疗可显著增强抗-NB疗效。因此，我们的第二个目标是从基因上 利用一种新的基于重组病毒载体的cDNAs递送来工程γδT细胞 甲基鸟嘌呤甲基转移酶，MGMT，替莫唑胺(TMZ)耐药基因和我们的抗GD2-CAR。 将分析修饰的γδT细胞的各种遗传和功能特征以及抗肿瘤能力， 无论是单独使用还是与TMZ联合使用，在体外和体内都使用了NB细胞系和患者来源的异种移植。目标 这项建议的目的是1)开发强大的临床前数据，以支持儿科第一次使用自体 扩增的γδT细胞用于复发NB，2)确定耐药/GD2CARγδT细胞是否优于 未修饰的γδT细胞，为未来的临床研究提供信息。这一免疫治疗平台在NB中的成功将 为使用类似的策略治疗其他癌症提供基础。

Project Summary High-risk neuroblastoma (HR NB) is a lethal pediatric solid tumor. Survival is < 50% and those that survive suffer many treatment related toxicities, stressing a critical need for novel tumor- targeted therapies. NB patient survival improved with the addition of the anti-GD2 antibody, dinutuximab, increasing the interest in other immunotherapies like adoptive transfer of cellular therapies for NB. Cellular therapy has so far focused on αβ T cells and NK cells, which to date have been uniformly unsuccessful for solid tumors. Gamma delta (γδ) T cells are an innovative and superior choice as they are MHC independent, directly cytotoxic to tumor cells, including NB, can recognize and target immune-suppressing cells in the tumor microenvironment, and lack the alloreactivity of αβ T cells. Adoptive γδ T cells have not been widely used clinically to date due to inadequate ex vivo expansion methods. We have developed a GMP-compliant serum free manufacturing strategy to expand γδ T cells to sufficient levels for human use. Expanded cells highly express CD16, which is directly involved in antibody directed cellular cytotoxicity (ADCC). We have shown that γδ T cells expanded from NB patients effectively kill NB cell lines and enhance dinutuximab-induced NB cell death. Therefore, our primary objective is to generate preclinical and IND enabling data demonstrating the effectiveness of our γδ T cell product. We have also generated two chimeric antigen receptors (CARs) targeting GD2 to localize γδ T cells to NB, and show that CAR-modified immunocompetent cells have enhanced cytotoxicity compared to non-modified cells. We have also developed strategies to confer chemotherapy resistance to normal immune cells. This led to our innovative “drug resistant immunotherapy” platform whereby chemo-protected γδ T cells can be co-administered with chemotherapy to significantly augment anti-NB efficacy. Our second objective is therefore to genetically engineer γδ T cells using a novel recombinant viral vector-based delivery of cDNA sequences that encode for methylguanine methyltransferase, MGMT, a temozolomide (TMZ) resistance gene and our anti-GD2-CAR. Modified γδ T cells will be analyzed for various genetic and functional characteristics and anti-tumor potency, both alone and with TMZ, using NB cell lines and patient derived xenografts both in vitro and in vivo. The goals of this proposal are to 1) develop robust preclinical data to support the first in pediatrics use of autologous expanded γδ T cells for recurrent NB, and 2) determine whether drug resistant/GD2CAR γδ T cells are superior to unmodified γδ T cells to inform future clinical studies. Success of this immunotherapy platform in NB will provide the foundation to treat other cancers using a similar strategy.