Interrogating the in vivo pharmacokinetics of armored CARs with radiohapten capture

项目总结/摘要 靶向CD 19的嵌合抗原受体（CAR）T细胞疗法在B细胞免疫缺陷中显示出显著的治疗效果。 恶性肿瘤，但许多患者患有有限的反应和CD 19阴性肿瘤复发。最近我们 证明了下一代“装甲”CAR T细胞组成性表达免疫刺激因子， 分子CD 40配体（CD 40 L）在临床前研究中表现出上级的抗肿瘤功效，但尚未被证实。 在CAR抗原阴性肿瘤复发的背景下充分探索。 仍然迫切需要对输注的T细胞进行非侵入性体内追踪，以确定它们的免疫应答。 生物分布，扩增和功能，并在即将发生的情况下增加CAR T细胞的杀伤能力。 治疗失败。为了克服这些局限性，我们开始开发监测体内动力学的方法， 基于免疫细胞放射性半抗原捕获概念的CAR T细胞。我们第一次证明了 T细胞可以成功地用DOTA抗体报告基因DAbR 1转导，使其能够在体内追踪 通过PET和SPECT。在目前的建议中，我们在这项工作的基础上，优化我们的翻译方法， 从动物到患者的免疫细胞放射性半抗原捕获，基于1） 具有皮摩尔结合亲和力的放射性半抗原捕获报告物scFv C825，和2）优化的放射性半抗原，下一个- Proteus-DOTA（Pr）系列适用于成像和靶向α治疗（达特）。主 本研究的目的是开发一种临床上适用的B细胞中CAR T细胞运输的PET成像策略。 进一步研究CD 40 L在抗同基因恶性肿瘤免疫抑制中的作用。 B细胞恶性肿瘤的模型。第二个目标是提供达特以增强T细胞的杀伤能力 在即将出现治疗失败的情况下，并提高CAR T细胞疗法的效力。 我们计划实现 我们的目标是通过产生第二代和装甲CD 19 CAR T细胞，表达细胞表面锚定的 scFv C825。CD 19 + B细胞恶性肿瘤的同源和免疫缺陷异种移植鼠模型，包括 将使用抗原丢失变体。 在成功转导后，我们将评估在体外的功能， CAR和报告基因，以及辐射毒性，然后是体内功能性，成像灵敏度， 生物分布，并开发一种基于装甲CAR T细胞的治疗诊断方法， [86Y]YPr/[225Ac]AcPr。最后，作为临床翻译这个新平台的前提，我们将进行研究， 在临床相关的异种移植模型中使用人类第二代和装甲CAR T细胞。的可用性 这样一个单一的平台将为更安全，更有效的临床试验提供关键信息。因此， 与我们目前的临床CD 19 CAR T研究和其他计划的CAR T研究具有直接的翻译相关性 细胞治疗试验

Project Summary/Abstract CD19-targeted chimeric antigen receptor (CAR) T cell therapy has shown remarkable treatment effects in B-cell malignancies, but many patients suffer from limited response and CD19-negative tumor relapse. Recently, we demonstrated that next-generation “armored” CAR T cells constitutively expressing the immune-stimulatory molecule CD40 ligand (CD40L) exhibited superior antitumor efficacy in preclinical studies but have not yet been fully explored in the context of CAR antigen-negative tumor relapse. There remains an urgent need for the non-invasive in vivo tracking of transfused T cells to determine their biodistribution, expansion, and functionality and to increase the CAR T cells’ killing capacity in case of imminent treatment failure. To overcome these limitations, we began developing methods for monitoring the in vivo kinetics of CAR T cells based on the concept of immune cell radiohapten capture. We demonstrated for the first time that T cells can be successfully transduced with a DOTA-antibody reporter, the DAbR1, enabling their in vivo tracking via PET and SPECT. In the current proposal, we build on this work and optimize our approach for translation of immune cell radiohapten capture from animals to patients, based on greatly improved components of 1) radiohapten capture reporter scFv C825 with picomolar binding affinity, and 2) optimized radiohaptens, the next- generation Proteus-DOTA (Pr) series suitable for imaging and targeted alpha therapy (TAT). The primary objectives of this study are to develop a clinically applicable PET imaging strategy of CAR T cell trafficking in B- cell malignancies and further study the effect of CD40L on counteracting the immune inhibition in syngeneic models of B-cell malignancies. The secondary objectives are to deliver TAT to enhance T cells’ killing capacity in cases of imminent treatment failure and improve the potency of CAR T cell therapies. We project to achieve our aims by generating second-generation and armored CD 19 CAR T cells expressing cell-surface anchored scFv C825. Syngeneic and immunodeficient xenograft murine models of CD19+ B cell malignancies including antigen-loss variants will be employed. After successful transduction, we will assess in vitro functionality of the CAR and the reporter, as well as radiation toxicity, followed by in vivo functionality, imaging sensitivity, and biodistribution, and develop an armored CAR T cell-based theranostic approach with the novel pair [86Y]YPr/[225Ac]AcPr. Finally, as a prerequisite to clinical translation of this novel platform, we will conduct studies using human second-generation and armored CAR T cells in clinically relevant xenograft models. The availability of such a single platform would provide crucial information for safer, more effective clinical trials. The aims thus have immediate translational relevance for our current clinical CD19 CAR T studies and other planned CAR T cell therapy trials.