Third-party “off the shelf” mature or precursor CAR T cells to prevent or treat malignant relapse after allo HCT

摘要 复发仍然是异基因造血细胞移植后死亡的最重要原因 移植(allo-HCT)，在过去的几十年里进展甚微。嵌合体 针对CD19的抗原受体(CD19-CARS)重定向T细胞效应器功能以消除 表达CD19的白血病和淋巴瘤细胞。我们在临床前研究中发现同种异体 供者来源的CD19-CAR T细胞可以用最少的移植具有显著的抗淋巴瘤活性- 抗宿主病(GVHD)，这一观察结果在临床试验中得到了其他人的证实。我们 研究发现，这是由于CAR-T细胞耗尽和缺失所致 通过异种反应TCR和汽车发出信号。此外，我们还演示了 TCR基因缺失及CD19-CAR在T细胞中表达的可行性研究 CRISPR技术，这可能进一步降低这些细胞的潜在同种异体反应性。我们有 还开发了一种体外培养系统，以产生通用的第三方T细胞前体(Pret) 这可以被改造成在胸腺后表达CD19-CAR，以避免负选择。vt.在.的基础上 在allo-HCT接受者中过继转移，这些工程化的Pret细胞可以在宿主的 胸腺和发挥抗肿瘤活性，而不需要移植物抗宿主病。除了抗癌的潜力之外， 这些经过改造的Pret细胞还可以增强免疫重建。基于这些发现 以及过继转移的第三方抗病毒T细胞在allo-HCT受者中的临床疗效， 我们假设，第三方“现成”成熟细胞或表达CARS的Pret细胞的输注 靶向肿瘤相关抗原的TCR将促进抗肿瘤活性和 促进GVHD最少或无GVHD的allo-HCT受者的免疫重建。我们建议预先- 应用第三方成熟细胞和Pret细胞预防或治疗术后复发的临床研究 阿洛-HCT。在目标1中，我们将使用临床前allo-hct模型来评估其抗肿瘤作用。 内源性TCR已被激活的第三方T细胞的活性、GVHD潜能和持久性 使用CRISPR和表示(1.1)CD19-CAR、(1.2)三重抗原特异性CARS的删除，或 (1.3)CD19-CAR/WT-1特异性TCR。在目标2中，我们将研究第三方Pret细胞表达(2.1) CD19-CAR，(2.2)CD19-CAR和细胞因子，或(2.3)CD19-CAR/WT1特异性TCR。我们有 已经开发了两项使用供者来源的CD19-CAR T细胞或第三方的allo-HCT临床试验 Pret细胞，再加上我们在CAR T细胞方面的丰富临床经验，将促进 翻译拟议的临床前研究以减少allo-HCT患者的复发 使用第三方CAR T细胞治疗恶性血液病。

Abstract Relapse remains the most important cause of mortality after allogeneic hematopoietic cell transplantation (allo-HCT) and little progress has been made in the past decades. Chimeric antigen receptors targeting CD19 (CD19-CARs) redirect T cell effector functions to eliminate CD19-expressing leukemia and lymphoma cells. We found in pre-clinical studies that allogeneic donor-derived CD19-CAR T cells can have significant anti-lymphoma activity with minimal graft- versus-host disease (GVHD), an observation that was confirmed by others in clinical trials. We found that this was due to exhaustion and deletion of CAR-T cells attributable to cumulative signaling through both the alloreactive TCR and the CAR. In addition, we have demonstrated the feasibility of deletion of the endogenous TCR and expression of a CD19-CAR in T cells using CRISPR technology, which could further reduce potential alloreactivity of these cells. We have also developed an in vitro culture system to generate universal third-party T cell precursors (preT) that can be engineered to express CD19-CAR post-thymically to avoid negative selection. Upon adoptive transfer in allo-HCT recipients, these engineered preT cells can mature in the host’s thymus and exert anti-malignancy activity without GVHD. In addition to anti-malignancy potential, these engineered preT cells can also enhance immune reconstitution. Based on these findings and the clinical efficacy of adoptively transferred third party anti-viral T cells in allo-HCT recipients, we hypothesize that infusion of third-party “off-the-shelf” mature or preT cells expressing CARs and TCRs targeted against tumor-associated antigens will promote anti-malignancy activity and enhance immune reconstitution in allo-HCT recipients with minimal or no GVHD. We propose pre- clinical studies with engineered third-party mature and preT cells to prevent or treat relapse after allo-HCT. In Aim 1, we will use pre-clinical allo-HCT models to evaluate the anti-malignancy activity, GVHD potential and persistence of third-party T cells whose endogenous TCR has been deleted using CRISPR and that express (1.1) CD19-CAR, (1.2) triple-antigen-specific CARs, or (1.3) CD19-CAR/WT-1 specific TCR. In Aim 2, we will study third-party preT cells expressing (2.1) a CD19-CAR, (2.2) a CD19-CAR and cytokines, or (2.3) CD19-CAR/WT1 specific TCR. We have already developed two allo-HCT clinical trials with donor-derived CD19-CAR T cells or third-party preT cells, which together with our extensive clinical experience with CAR T cells will facilitate the translation of the proposed preclinical studies to decrease relapse in allo-HCT patients with hematologic malignancies using third party CAR T cells.