MODULATION OF CD5 SIGNALING TO ENHANCE ADOPTIVE T-CELL THERAPIES FOR CANCER

项目摘要 诱导性T细胞免疫疗法，特别是产生的嵌合抗原受体T细胞（CART）， 在高度难治性CD 19 + B细胞恶性肿瘤患者中获得前所未有的反应。然而，只有有限的 许多接受抗CD 19 CART治疗的患者将经历长期缓解，而大多数患者 没有反应或最终复发。此外，在实体癌的情况下，CART细胞通常是 失望增加CART效应子对抗癌症的功能将代表在癌症治疗中的纵向改善。 过继性T细胞免疫疗法领域。在这项提案中，我们的目标是增加CAR T的抗肿瘤功效。 通过减少CAR激活的抑制作用来抑制细胞。目前提高CART疗效的方法主要集中在 通过靶向PD-1/PD-L1轴减少长期T细胞耗竭。然而，缺乏调查 增强早期CAR和T细胞受体（TCR）信号传导的策略，这对有效杀死肿瘤至关重要， 特别是在免疫抑制肿瘤微环境的设置中。为此，我们研究了 清道夫受体CD 5。CD 5与TCR复合物结合，并通过几种途径抑制其活化。 包括SHP-1、CBL、CBL-B和GRB 2的介质。这项研究的中心假设是， 通过增强TCR信号传导增加工程化T细胞的抗肿瘤功效。在我们的初步调查中 我们已经发现，CRISPR-Cas9 CD 5在CART细胞中的敲除增强了它们的抗肿瘤活性， B细胞白血病（CART 19）和T细胞淋巴瘤（CART 5）的异种移植模型。此外，我们证明， 在刺激后，CD 5敲除的CART细胞显示出关键CAR信号传导介质的更高磷酸化， 与野生型相比。在目标#1中，我们将检验T细胞中CD 5的缺失广泛增强T细胞中CD 5的表达的假设。 过继性T细胞免疫疗法的抗肿瘤功效，通过研究CAR T背景下的CD 5敲除 实体瘤的细胞。在目标#2中，我们将检验CD 5缺失增强T细胞抗肿瘤活性的假设 通过进行体外和体内机制研究来解除CAR信号传导。我们还将测试 这种方法。这种假设驱动的建议是高度创新的，因为它提出了一个小说的研究 免疫调节靶点-CD 5-并使用尖端技术，如数字空间分析和单细胞 RNA测序以在人和鼠模型中研究体内CD 5缺失的CAR T细胞。一个成功 预期所提出的研究的结果将显著影响癌症免疫治疗领域， 提供了一种合理的和可推广的方法来改善T细胞免疫疗法，并最终推动 开发CD 5敲除CART用于难治性癌症的首次人体临床试验。

PROJECT SUMMARY Adoptive T-cell immunotherapies and, in particular, chimeric antigen receptor T cells (CART) generated unprecedented responses in patients with highly refractory CD19+ B cell malignancies. However, only a limited number of patients treated with anti-CD19 CART will experience prolonged remission while most patients either do not respond or eventually relapse. Moreover, in the setting of solid cancers, CART cells have generally been disappointing. Increasing CART effector function against cancer would represent a vertical improvement in the field of adoptive T-cell immunotherapy. In this proposal, we aim at increasing the anti-tumor efficacy of CAR T cells by reducing the inhibition of CAR activation. Current approaches to enhance CART efficacy focus on reducing long-term T cell exhaustion by targeting the PD-1/PD-L1 axis. However, there is a lack of investigation on strategies to enhance early CAR and T cell receptor (TCR) signaling, which is critical for effective tumor killing, especially in the setting of the immunosuppressive tumor microenvironment. To this goal, we studied the scavenger receptor CD5. CD5 associates with the TCR complex and inhibits its activation through several mediators, including SHP-1, CBL, CBL-B, and GRB2. The central hypothesis of this grant is that CD5 deletion increases the anti-tumor efficacy of engineered T cells through enhancement of TCR signaling. In our preliminary work, we have found that CRISPR-Cas9 CD5 knock out in CART cells enhances their anti-tumor activity in xenograft models of B-cell leukemia (CART19) and T-cell lymphoma (CART5). Moreover, we demonstrated that, upon stimulation, CD5 knocked-out CART cells show higher phosphorylation of key CAR signaling mediators as compared to wild-type. In Aim#1, we will test the hypothesis that the deletion of CD5 in T cells broadly enhances the anti-tumor efficacy of adoptive T-cell immunotherapies, by studying CD5 knockout in the setting of CAR T cells for solid tumors. In Aim#2, we will test the hypothesis that CD5 deletion augments T-cell anti-tumor activity by disinhibiting CAR signaling by performing in vitro and in vivo mechanistic studies. We will also test the safety of this approach. This hypothesis-driven proposal is highly innovative as it proposes the study of a novel immunomodulatory target - CD5 - and uses cutting edge technologies like digital spatial profiling and single-cell RNA sequencing to investigate CD5 deleted CAR T-cell in vivo in both human and murine models. A successful outcome of the proposed investigations is expected to impact the field of cancer immunotherapy significantly, providing a rational and generalizable approach to improving T-cell immunotherapies and ultimately drive the development of a first-in-human clinical trial of CD5 knocked-out CART for refractory cancers.