Interrogating the in vivo pharmacokinetics of armored CARs with radiohapten capture

项目摘要/摘要 CD19靶向嵌合抗原受体(CAR)T细胞治疗在B细胞中显示出显著的治疗效果 恶性肿瘤，但许多患者的反应有限，肿瘤CD19阴性复发。最近，我们 证明下一代“装甲”CAR T细胞结构性地表达免疫刺激作用 CD40分子配体(CD40L)在临床前研究中显示出优越的抗肿瘤效果，但尚未得到临床应用 在CAR抗原阴性肿瘤复发的背景下进行了充分的探索。 仍然迫切需要对输注的T细胞进行非侵入性体内追踪，以确定其 生物分布、扩增和功能，并在迫在眉睫的情况下增加CAR T细胞的杀伤能力 治疗失败。为了克服这些限制，我们开始开发监测体内动力学的方法 基于免疫细胞放射半抗原捕获概念的CAR T细胞。我们第一次证明了 可以用DOTA抗体报告基因DAbR1成功地转导T细胞，使其能够在体内进行跟踪 通过正电子发射计算机断层扫描和SPECT。在当前的提案中，我们在这项工作的基础上，优化了我们的翻译方法 免疫细胞放射半抗原从动物到患者的捕获，基于大大改进的1) 具有皮摩尔结合亲和力的放射半抗原捕获报告ScFv C825，以及2)优化的放射半抗原，下一步- 适用于成像和靶向阿尔法治疗(TAT)的新一代Proteus-DOTA(PR)系列。初级阶段 本研究的目的是建立一种可用于临床的CAR-T细胞在B-T细胞转运的PET成像策略。 细胞恶性肿瘤与CD40L对抗同基因免疫抑制的进一步研究 B细胞恶性肿瘤模型。次要目标是传递TAT以增强T细胞的杀伤能力 在治疗即将失败的情况下，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的CD19CAR T细胞来表达细胞表面锚定 单链抗体C825。CD19+B细胞恶性肿瘤同基因和免疫缺陷小鼠移植模型包括 将使用抗原丢失变种。 在成功转导后，我们将评估该基因的体外功能 Car和记者，以及辐射毒性，其次是体内功能，成像灵敏度，以及 生物分布，并开发了一种基于装甲车T细胞的治疗方法 [86Y]YPR/[225Ac]ACPR。最后，作为这一新平台临床翻译的前提，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车T细胞。可得性 这种单一平台的研究将为更安全、更有效的临床试验提供关键信息。目标是这样的 与我们当前的临床CD19 CAR T研究和其他计划中的CAR T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.