Democratizing CAR T cell therapy by in situ programming of virus-specific T cells

表达嵌合抗原受体(CARS)的工程化T细胞已显示出显著的治疗效果 恶性血液病。然而，CAR T细胞疗法的广泛实施受到冗长时间的限制 (3-5周)和昂贵(每种治疗35万-45万美元)体外生产流水线。这项建议旨在 开发抗原提呈纳米粒(APN)用于病毒特异性T细胞的原位编程，用于快速和 成本效益高的汽车T细胞制造。病毒特异性T细胞为增强CAR提供了一个有希望的机会 T细胞疗法，因为它们具有改善持久性和增殖潜力，并允许病毒疫苗接种 通过内源性受体加强汽车治疗。这项建议将集中于甲型流感病毒(IAV)- 利用现有的季节性流感疫苗接种特定的T细胞来促进CAR活动。把汽车送到 IAV特异性T细胞，APN将包括脂质纳米粒(LNPs)，它封装CAR编码的mRNA和 用显示流感多肽的人类白细胞抗原-A肽-主要组织相容性复合体(PMHC)装饰 表位。这项提议将使用APN在以下背景下递送人B细胞成熟抗原(BCMA)CAR 多发性骨髓瘤，未来目标是扩大到其他CAR特异性和适应症，包括CD19阳性 癌症。目标1的目标是开发用于用αBCMA转染人流感特异性T细胞的APN CAR在体内的表达，并鉴定CAR在靶向IAV特异性T细胞中的转染特异性 主要细胞群。目标2将重点验证αBCMA CAR T细胞的抗癌效果 利用重现人类多发性骨髓瘤的小鼠模型进行原位转基因。预防接种策略 将使用灭活的流感病毒粒子测试扩增IAV特异性T细胞并增强其效应器功能 接种表达CAR的IAV特异性T细胞，并将所产生的抗癌效力与 未接种疫苗的队列。在AIM 3中，CRISPR/Cas9将与APN一起实施，用于体内T细胞的基因编辑 使用CAR持久表达CAR，并通过延迟T细胞分化和增强抗癌效力 疲惫不堪。这一提议的成功将挑战现有的T细胞工程范式，降低成本 CAR T细胞疗法，并通过接种流感疫苗增强抗癌活性，最终实现民主化 用于癌症治疗的CAR T细胞。通过这项工作，候选人将通过以下方式缩小知识差距 特别顾问委员会的指导：(1)Gabe Kuong，Ph.D.(CAR T细胞工程)，(2)Phil Santangelo博士(信使核糖核酸疗法和CRISPR/CAS)，(3)Rafi Ahmed博士(抗病毒T细胞免疫和 记忆/衰竭T细胞生物学)和(4)Madhav Dhodapkar医学博士(血液学/肿瘤学和骨髓瘤 癌症模型)。这支强大的指导团队以及佐治亚理工学院和埃默里大学提供的丰富资源 大学构成了一个肥沃的指导环境，有助于实现候选人的职业目标，即领导 独立研究计划，专注于开发新技术，以改善患者的接触和 T细胞免疫治疗抗癌的疗效观察

Engineered T cells that express chimeric antigen receptors (CARs) have shown remarkable efficacy against hematological malignancies. However, broad implementation of CAR T cell therapies is limited by the lengthy (3–5 weeks) and costly ($350K–450K per treatment) ex vivo manufacturing pipeline. This proposal seeks to develop antigen-presenting nanoparticles (APNs) for in situ programming of virus-specific T cells for rapid and cost-efficient CAR T cell manufacturing. Virus-specific T cells present a promising opportunity to enhance CAR T cell therapy, as they have improved persistence and proliferation potential, and allow for viral vaccination to augment CAR therapy through their endogenous receptors. This proposal will focus on influenza A virus (IAV)- specific T cells to exploit the existing seasonal influenza vaccination to boost CAR activities. To deliver CAR to IAV-specific T cells, APNs will comprise lipid nanoparticles (LNPs) that encapsulate CAR-encoded mRNA and are decorated with HLA-A peptide-major histocompatibility complex (pMHC) displaying influenza peptide epitopes. This proposal will use APNs to deliver human B-cell maturation antigen (BCMA) CAR in the context of multiple myeloma with future goals to expand to other CAR specificities and indications, including CD19 positive cancers. The goal in Aim 1 is to develop APNs for transfection of human influenza-specific T cells with αBCMA CAR in vivo, and characterize the CAR transfection specificity in the target IAV-specific T cells versus other major cell populations. Aim 2 will be focused on validating the anti-cancer efficacy of αBCMA CAR T cells after in situ transfection using a mouse model recapitulating human multiple myeloma. The vaccination strategy to expand IAV-specific T cells and to boost their effector functions will be tested using inactivated influenza virions to vaccinate the CAR-expressing, IAV-specific T cells and compare the resulting anti-cancer potency with the unvaccinated cohort. In Aim 3, CRISPR/Cas9 will be implemented with APNs for in vivo gene editing of T cells with CAR for durable CAR expression and enhanced anti-cancer potency by delaying T-cell differentiation and exhaustion. The success of this proposal will challenge existing paradigms of T cell engineering, reduce the cost of CAR T cell therapy, and enhance anti-cancer activity through influenza vaccination to ultimately democratize CAR T cells for cancer therapy. Through this work, the candidate will close the knowledge gaps by the mentorship of an exceptional advisory committee: (1) Gabe Kwong, Ph.D. (CAR T cell engineering), (2) Phil Santangelo, Ph.D. (mRNA therapeutics and CRISPR/Cas), (3) Rafi Ahmed, Ph.D. (anti-viral T cell immunity and memory/exhaustion T cell biology), and (4) Madhav Dhodapkar, M.D. (hematology/oncology and myeloma cancer models). This strong mentoring team and the abundant resources provided by Georgia Tech and Emory University constitute a fertile mentoring environment for attaining the candidate's career goal of leading an independent research program focused on developing new technologies to improve patient access and treatment outcome of T-cell immunotherapy against cancer.