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

项目总结 过继T细胞免疫治疗，特别是嵌合抗原受体T细胞(CART)产生 高度难治性CD19+B细胞恶性肿瘤患者的前所未有的反应。然而，只有有限的 接受抗CD19 CART治疗的患者数量将经历更长时间的缓解，而大多数患者要么 不要回应或最终故态复萌。此外，在实体癌的背景下，CART细胞通常是 令人失望。增加抗癌CART效应器的功能将代表着垂直方向的改善 过继T细胞免疫治疗领域。在这项建议中，我们的目标是增加CAR T的抗肿瘤效果 通过减少对CAR激活的抑制来抑制细胞。目前提高CART功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， 关于加强早期CAR和T细胞受体(TCR)信号传递的策略，这对有效的肿瘤杀伤至关重要， 尤其是在免疫抑制肿瘤微环境的背景下。为此，我们研究了 清道夫受体CD5。CD5与TCR复合体结合并通过几种途径抑制其激活 调解人，包括SHP-1、CBL、CBL-B和Grb2。这项研究的中心假设是CD5的缺失 通过增强TCR信号增强工程T细胞的抗肿瘤效果。在我们的预赛中 工作中，我们发现在CART细胞中敲除的CRISPR-Cas9 CD5增强了它们在 B细胞白血病(CART19)和T细胞淋巴瘤(CART5)的异种移植模型此外，我们还展示了， 在刺激下，CD5基因敲除的CART细胞表现出更高的关键CAR信号介质的磷酸化，如 与野生型相比。在目标1中，我们将测试T细胞中CD5的删除广泛增强的假设 过继T细胞免疫疗法抗肿瘤效果的研究--以CAR T细胞为背景 实体瘤的细胞。在目标2中，我们将检验CD5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抑制CAR信号。我们还将测试其安全性 这种方法的一部分。这一假设驱动的提议具有很高的创新性，因为它提出了对一部小说的研究 免疫调节靶标-CD5-并使用尖端技术，如数字空间图谱和单细胞 RNA测序以研究体内CD5缺失的CAR T细胞在人和小鼠模型中的作用。一位成功的 拟议的调查结果预计将对癌症免疫治疗领域产生重大影响， 提供一种合理和可推广的方法来改进T细胞免疫疗法，并最终推动 CD5基因敲除手推车治疗难治性癌症的首例人类临床试验。

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.