TCR engineering of human T cells for immunotherapy of hepatocellular carcinoma

项目摘要 在全球范围内，肝癌是第六大常见癌症，也是第四大常见癌症死亡原因。 世界。根据CDC的数据，在过去的20年里，肝癌的发病率翻了一番，使其成为世界上 美国增长最快的癌症。据估计，将有42,220例新病例和30,200例肝脏死亡 2018年的癌症。因此，我们正在解决美国日益严重的健康问题。虽然肝切除是 治愈后，缺乏辅助治疗成为手术成功的关键障碍，导致~70% 5年复发率。因此，迫切需要新的治疗方法，免疫治疗具有巨大的潜力。 大多数(80%-90%)的肝癌是肝细胞癌，它重新表达甲胎蛋白(AFP)。 用AFP表位特异的T细胞受体(TCR)基因工程患者的自体T细胞提供了一种特异的 肝癌的免疫治疗。目前，一种针对AFP158的高亲和力TCR刚刚获准在 2017年5月。但是，高亲和力的TCR可能会引起严重的靶上和靶外毒性。我们推断，这将需要 一组不同亲和力的TCR寻找使人类T细胞杀伤肝癌的最佳TCR 无毒的细胞。因此，我们建议鉴定多个AFP特异性TCR，并测试它们的抗肿瘤效果 和毒性。我们假设一种有效的体内免疫策略将产生有效的人甲胎蛋白。 不同TCR谱系对人甲胎蛋白(HAFP)特异性CD8反应在HLA-A2小鼠中的作用这大量的CD8细胞 使我们能够直接研究它们的抗肿瘤功能，并鉴定一组具有不同亲和力的TCR， 增加了找到最佳TCR的机会，以产生具有有效抗肿瘤效果且无毒性的TCR-TS。 为了验证这一假设，我们制定了具有三个具体目标的具体研究策略。A1：识别 HAFP158特异的TCR以产生不同功能亲和力的TCR-T；A2：识别hAFP158特异的TCR 没有或具有最小的交叉反应；以及A3：创建可扩展和可拆卸的TCR-T，其茎长度为 达到体内有效的抗肿瘤作用。目标是获得最优的TCR基因，这些基因可以 原代人类T细胞将成为有效的TCR-ts，能够在不损害正常人的情况下杀死人肝癌细胞 并建立产生安全有效的TCR-T的方案，为临床试验做好准备。在 最后，将提交调查性新药和临床试验的申请。这最终将导致新的 肝癌的治疗途径。此外，建立AFP特异性TCR文库的方法学方法和 寻找高效、无毒的最佳TCRs将为其他抗原的研究提供平台 和人类白细胞抗原单倍型，这将极大地影响肿瘤免疫治疗领域。

Project Summary Liver cancer is the 6th most common cancer and the 4th most common cause of cancer death in the world. According to CDC, the incidence rate of liver cancer has doubled over last 20 years, making it the fastest growing cancer in US. It is estimated that there will be 42,220 new cases and 30,200 death of liver cancer in 2018. Thus, we are addressing an increasing health problem in US. Although liver resection is curative, the lack of adjuvant therapy becomes a critical barrier to the success of surgery, resulting in ~70% 5yr recurrence rate. Thus, novel therapies are urgently needed and immunotherapy has a great potential. Majority (80-90%) of liver cancer is hepatocellular carcinoma (HCC), which re-express alpha fetoprotein (AFP). Engineering patient’s autologous T cells with AFP epitope-specific T cell receptor (TCR) genes offers a specific immunotherapy for HCC. Currently, one AFP158-specific high-affinity TCR just got approved for clinical trial in May of 2017. But, high-affinity TCRs may cause severe on- and off-target toxicity. We reason that it will require a panel of TCRs with different affinity to find the optimal TCRs that enable human T cells to kill HCC tumor cells without toxicity. Thus, we propose to identify multiple AFP-specific TCRs and test their antitumor efficacy and toxicity. We hypothesize that an effective in vivo immunization strategy will induce potent human AFP (hAFP)-specific CD8 response with diverse TCR repertoire in HLA-A2 mice. This large number of CD8 cells enables us to directly study their antitumor function and identify a panel of TCRs with different affinity, increasing the chance of finding the optimal TCRs to create TCR-Ts of potent antitumor effect without toxicity. To test this hypothesis, we have developed a concrete research strategy with three specific aims. A1: Identify hAFP158-specific TCRs to create TCR-Ts of different functional avidity; A2: Identify the hAFP158-specific TCRs without or with minimal cross-reactivity; and A3: Create expandable and removable TCR-Ts with stemness to achieve potent in vivo antitumor effect. The goals are to obtain the optimal TCR genes that can engineer primary human T cells to become effective TCR-Ts capable of killing human HCC cells without harming normal cells and to establish protocol for generating safe and effective TCR-Ts that are ready for clinical trials. In the end, applications for an investigative new drug and clinical trial will be filed. This will eventually lead to new therapeutic approach for HCC. In addition, the methodical approach of creating AFP-specific TCR library and identifying optimal TCRs with potent antitumor effect and no toxicity will serve as a platform for other antigens and HLA haplotypes, which will greatly impact the field of tumor immunotherapy.