Engineering CAR T cells to potentiate innate and adaptive immunity

项目总结/摘要 嵌合抗原受体（CAR）T细胞疗法重定向T细胞以激活并随后杀死抗原- 表达癌细胞。这是通过偶联癌抗原特异性细胞外单链 可变IgG片段（scFv）与胞质内内源性T细胞活化信号传导结构域的结合。CAR T细胞 治疗已显示出治疗造血系统恶性肿瘤的前景;然而，抗原阴性的造血系统恶性肿瘤的复发， 肿瘤仍然是这些患者失败的重要原因。此外，几乎没有成功， 用免疫抑制微环境治疗实体瘤。用CAR T细胞和 封锁检查站是克服这些障碍的一个可能办法。检查点阻断疗法 拮抗抑制免疫系统的信号通路。当前的检查站封锁策略 一直专注于改变T细胞与肿瘤的相互作用，但最近的研究也显示， 免疫检查点，特别是CD 47-SIRPα信号轴。这条路径被称为“不要吃我” 信号，阻止抗体介导的巨噬细胞吞噬作用和T细胞的主动交叉致敏， 树突状细胞，并因此参与抑制先天性和适应性免疫过程。癌细胞 选择了这条途径来逃避免疫攻击。然而，抗CD 47药物的早期临床试验 显示贫血和血小板减少的全身毒性。 我们的长期目标是设计一种更有效的CAR T细胞，可以克服抗原丢失复发和免疫缺陷。 免疫抑制肿瘤微环境。为了实现这一点，我们建议研究组合 CAR T细胞治疗与内在SIRPα蛋白分泌激活抗体治疗和抗原 介绍，因为这种组合应该有效地参与先天和适应性免疫，以导致更多的 完全抗肿瘤反应。我们已经改造了人类CD 19 CAR T细胞，使其分泌一种小的 分子，高亲和力，SIRPα模拟物，CV1.这些分泌CV1的CAR T细胞，称为OrexiCAR T细胞，保留了 它们的细胞毒性功能和细胞分泌的CV1可以增强mAb治疗。此外，我们还表明， 体外OrexiCART细胞的癌抗原刺激导致分泌的CV1大量增加。这里我们 我建议在完全免疫活性的同源环境中研究OrexiCAR T细胞，以确定 机制有助于其效力。我们相信，拟议的研究将有助于更好地了解 OrexiCAR T细胞功效及其临床适用性。目的：1）构建小鼠CD 19 将OrexiCAR载体转染入原代小鼠细胞，并在体外验证CAR和CV1的功能 和2）评估mOrexiCAR T细胞在免疫活性的同基因重组小鼠中的抗肿瘤作用。 小鼠模型，揭示和描述免疫机制

Project Summary/Abstract Chimeric antigen receptor (CAR) T cell therapy redirects T cells to activate and subsequently kill antigen- expressing cancer cells. This is achieved by coupling a cancer antigen-specific extracellular single-chain variable IgG fragment (scFv) to intracytoplasmic, endogenous T cell activation signaling domains. CAR T cell therapy has shown promise for treating hematopoietic malignancies; however, relapse of antigen-negative tumors remains a significant source of failure for these patients. Further, little success has been seen in treating solid tumors with immunosuppressive microenvironments. Combination therapy with CAR T cells and checkpoint blockade is a possible approach to overcome these obstacles. Checkpoint blockade therapy antagonizes the signaling pathways that suppress the immune system. Current checkpoint blockade strategies have focused on altering T cell-tumor interactions, but recent studies also show promise in abrogating innate immune checkpoints, specifically the CD47-SIRPα signaling axis. This pathway, known as the “do not eat me” signal, prevents both antibody mediated macrophage phagocytosis and active cross priming of T cells by dendritic cells, and is thus involved in suppressing both innate and adaptive immune processes. Cancer cells have co-opted this pathway to evade immune attack. However, early stage clinical trials of anti-CD47 agents show systemic toxicities of anemia and thrombocytopenia. Our long-term goal is to engineer a more potent CAR T cell that can overcome antigen loss relapse and the immunosuppressive tumor microenvironment. To accomplish this, we propose to investigate the combination of CAR T cell therapy with intrinsic SIRPα protein secretion to activate antibody therapy and antigen presentation, as this combination should potently engage both innate and adaptive immunity to lead to a more complete antitumor response. We have already engineered human CD19 CAR T cells to secrete a small molecule, high affinity, SIRPα mimic, CV1. These CV1-secreting CAR T cells, named OrexiCAR T cells, retain their cytotoxic function and the cell-secreted CV1 can potentiate mAb therapy. In addition, we have shown that cancer antigen stimulation of the OrexiCAR T cells in vitro leads to a large increase in secreted CV1. Here, we propose to study OrexiCAR T cells in a fully immunocompetent, syngeneic setting to determine which mechanisms contribute to their potency. We believe the proposed research will allow a better understanding of OrexiCAR T cell efficacy and its applicability to the clinic. The Aims are: 1) To construct mouse CD19 OrexiCAR vectors, transduce into primary mouse cells, and validate functions of CAR and CV1 in vitro and 2) To evaluate the anti-tumor effect of mOrexiCAR T cells in an immunocompetent, syngeneic mouse model and to discover and describe the immunologic mechanism