Harnessing the thymus for long-term tumor control with hematopoietic stem cell-derived naive CAR T cells

项目总结 嵌合受体抗原(CAR)T细胞通过提供肿瘤特异性、 分子靶向疗法。然而，尽管目前基于CAR T细胞的癌症的临床应用 免疫疗法如Kymriah或Yescarta在大多数情况下会导致缓解，长期的疾病控制，即 在患有高危恶性肿瘤的儿科和年轻成人癌症患者中尤其需要，仍然是主要的 临床挑战。事实上，恶性复发仍然是CAR T细胞治疗后死亡的主要原因。 体内CAR T细胞持久性不足是降低复发风险和改善的主要障碍 生死存亡。我们开发了一种新型的基于连续的肿瘤免疫监控平台 体内产生幼稚的CAR T细胞。这一建议是由假设驱动的，基于我们发布的和 未发表的数据显示，在完成初始疗程的强化化疗后，持续时间长的T细胞 通过使用造血干细胞和祖细胞(HSPC)可以建立对癌症抗原的免疫力 被设计成表达肿瘤细胞靶向CAR，并被输送到患者的胸腺。图像制导 胸腺内注射是一种微创的程序，它利用癌症患者的胸腺作为体内的 生物反应器，从而提供了一种创新的且相对简单和低毒的临床方法 从基因操纵的HSPC中生产高度有效的天真设计型T细胞。直接胸腺 HSPC的植入(绕过骨髓)消除了对骨髓清除性调节的需要，同时 保持预期的结果，即长期产生幼稚的抗原特异性T细胞。胸腺植入 通过胸腺照射结合胸腺内注射将细胞输送到胸腺或 通过过量表达胸腺来增强静脉注射HSPC的胸腺归巢能力 特定的归巢分子。我们将重点以CD19汽车为模型系统，建立我们的概念证明 因为CD19汽车已经成为评估新汽车技术的黄金标准。我们的 实验方法包括设计允许成功的胸腺阴性选择CD19的策略 车载HSPC。随着时间的推移，该项目预计将扩大到包括各种汽车细节。 来自HSPC的CAR T细胞的发展将在体外和体内进行分析，包括评估 胸腺上皮内HSPC的胸腺造血干细胞维持和T细胞分化 微环境。我们将在体内展示MOST的有效性(B细胞耗尽和抗肿瘤活性) 在同基因小鼠模型中有前景的CAR表达系统。人源化小鼠的翻译研究， 包括由患者衍生的儿童急性淋巴细胞白血病模型，将在最后一年进行 项目的一部分。总之，这项研究将测试体内CAR T细胞发育的新范式，有望 广泛使用CAR T细胞进行肿瘤免疫监测，作为高危患者的巩固后治疗 儿童和青壮年患者群体中的恶性肿瘤。

PROJECT SUMMARY Chimeric receptor antigen (CAR) T cells are transforming cancer treatment by providing tumor-specific, molecularly targeted therapies. However, even though current clinical applications of CAR T cell-based cancer immunotherapies such as Kymriah or Yescarta induce remission in most cases, long-term disease control, which is especially needed in pediatric and young adult cancer patients with high-risk malignancies, remains a major clinical challenge. In fact, malignant relapse continues to be the leading cause of death post CAR T cell therapy. Insufficient CAR T cell persistence in vivo is a major obstacle to reducing the risk of relapse and improving survival. We have developed a novel platform for long-lasting tumor immunosurveillance based on continuous in vivo generation of naïve CAR T cells. This proposal is driven by the hypothesis, based on our published and unpublished data, that after the completion of the initial course of intensive chemotherapy long-lasting T cell immunity to cancer antigens can be established by using hematopoietic stem and progenitor cells (HSPCs) engineered to express a tumor cell-targeting CAR and delivered into the patient’s thymus. Image-guided intrathymic injection is a minimally invasive procedure that harnesses the thymus of cancer patients as an in vivo bioreactor, thus offering an innovative and also relatively simple and low-toxic clinical method for sustainable production of highly potent naïve designer T cells from genetically manipulated HSPCs. Direct thymic engraftment of HSPCs (bypassing the bone marrow) eliminates the need for myelo-ablative conditioning while preserving the desired outcome, i.e., long-term generation of naïve antigen-specific T cells. Thymic engraftment will be facilitated by thymic irradiation combined with either cell delivery to the thymus by intrathymic injection or by enhancing the thymic homing capacity of intravenously administered HSPCs by overexpression of thymus- specific homing molecules. We will focus on CD19 CARs as a model system to establish proof of concept of our approach because CD19 CARs have become the gold standard for evaluating novel CAR technologies. Our experimental approaches include strategies designed to allow successful thymic negative selection of CD19 CAR-transduced HSPCs. Over time the project is expected to expand to include a variety of CAR specificities. CAR T cell development from HSPCs will be analyzed both in vitro and in vivo, including assays assessing thymic hematopoietic stem cell maintenance and T cell differentiation from HSPCs within the thymic epithelial microenvironment. We will demonstrate in vivo efficacy (B cell depletion and anti-tumor activity) of the most promising CAR expression system in syngeneic mouse models. Translational studies in humanized mice, including a patient-derived pediatric acute lymphoblastic leukemia model, will be performed during the final year of the project. In sum, this research will test the novel paradigm of CAR T cell development in vivo, promising to make tumor immunosurveillance by CAR T cells broadly available as post-consolidation therapy of high-risk malignancies in pediatric and young adult patient populations.