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

表达嵌合抗原受体 (CAR) 的工程化 T 细胞已显示出显着的抗癌功效 血液系统恶性肿瘤。然而，CAR T 细胞疗法的广泛实施受到长期的限制。 （3-5 周）且成本高昂（每次治疗 35 万至 45 万美元）离体生产流程。该提案旨在 开发抗原呈递纳米颗粒 (APN)，用于病毒特异性 T 细胞的原位编程，以实现快速、准确的结果 具有成本效益的 CAR 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，因为它们提高了持久性和增殖潜力，并允许病毒疫苗接种 通过其内源性受体增强 CAR 疗法。该提案将重点关注甲型流感病毒（IAV）—— 特定的 T 细胞利用现有的季节性流感疫苗来增强 CAR 活性。将 CAR 交付至 IAV 特异性 T 细胞 APN 将包含脂质纳米颗粒 (LNP)，其中封装了 CAR 编码的 mRNA 和 装饰有 HLA-A 肽主要组织相容性复合物 (pMHC)，展示流感肽 表位。该提案将在以下背景下使用 APN 传递人类 B 细胞成熟抗原 (BCMA) CAR 多发性骨髓瘤，未来目标是扩展到其他 CAR 特异性和适应症，包括 CD19 阳性 癌症。目标 1 的目标是开发 APN，用于用 αBCMA 转染人类流感特异性 T 细胞 体内 CAR，并表征目标 IAV 特异性 T 细胞相对于其他细胞的 CAR 转染特异性 主要细胞群。目标2将重点验证αBCMA CAR T细胞的抗癌功效 使用重现人类多发性骨髓瘤的小鼠模型进行原位转染。疫苗接种策略 将使用灭活的流感病毒粒子来测试扩增 IAV 特异性 T 细胞并增强其效应功能 对表达 CAR 的 IAV 特异性 T 细胞进行疫苗接种，并将所得的抗癌效力与 未接种疫苗的队列。在目标 3 中，CRISPR/Cas9 将与 APN 一起实施，用于 T 细胞的体内基因编辑 具有 CAR，可实现持久的 CAR 表达，并通过延迟 T 细胞分化和增强抗癌效力 精疲力尽。该提案的成功将挑战 T 细胞工程的现有范式，降低成本 CAR T 细胞疗法，并通过流感疫苗接种增强抗癌活性，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： 杰出顾问委员会的指导：(1) Gabe Kwong, Ph.D. （CAR T 细胞工程），(2) Phil 桑坦杰洛博士（mRNA 疗法和 CRISPR/Cas），(3) Rafi Ahmed 博士。 （抗病毒 T 细胞免疫和 记忆/疲劳 T 细胞生物学），以及 (4) Madhav Dhodapkar，医学博士（血液学/肿瘤学和骨髓瘤） 癌症模型）。强大的导师团队以及佐治亚理工学院和埃默里大学提供的丰富资源 大学为候选人实现领导他人的职业目标提供了一个肥沃的指导环境。 独立研究计划专注于开发新技术以改善患者获取和治疗的机会 T细胞免疫疗法对抗癌症的治疗结果。

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.