Oncofetal TNC CAR T Cell Therapy for Pediatric Sarcoma

项目摘要 该项目的长期目标是开发一种过继免疫疗法，用表达嵌合抗原的T细胞， 抗原受体（CAR）的复发性和难治性小儿肉瘤，其预后仍然很差。car T 细胞疗法已在临床研究中证明了对血液恶性肿瘤的成功;然而，CAR T细胞 在临床上对实体瘤的疗效不佳。虽然有限的疗效是多因素的，但 实体瘤CAR T细胞疗法的障碍是缺乏可靶向抗原。一种突出的ECM蛋白是 生腱蛋白C（TNC）和癌细胞，包括肉瘤细胞，表达癌胚生腱蛋白C（TNC）香料 变体，其可以包含额外的C结构域（C. TNC）。由于C.TNC表达与细胞凋亡相关， 恶性表型的癌细胞，我们认为它是一个理想的CAR靶标。为支持这项申请，我们 已经产生了具有CD28共刺激结构域的第二代C. TNC特异性CAR（C. TNC-CAR）。我现在 假设C.TNC-CAR T细胞识别并杀死C.TNC阳性肿瘤细胞， 优化以在临床前儿科肉瘤模型中具有有效的抗肿瘤作用。我们已经证明， C.TNC-CAR T细胞在体外识别和杀死C.TNC+肉瘤细胞系的初步研究。在这个项目中 现在，我们建议从两个相互关联的研究目标来探讨这一假设。目标1侧重于 理解我们的ECM靶向C. TNC-CAR T细胞的机制，然后Aim 2比较了 C.TNC-CAR T细胞在体内肉瘤异种移植模型中的效应子功能， 持久性和抗肿瘤活性。所采用的技术包括流式细胞术、ELISA、共培养 和细胞毒性测定以及CRISPR-Cas9基因组编辑。 这个项目的目的是训练我的技术，以产生和表征转基因T细胞 表达CAR，并将我引入不断发展的癌细胞治疗领域。这一建议和培训 将在圣裘德儿童研究医院进行，这是一个NCI指定的综合癌症中心。如果 如果成功的话，这个建议的结果可以立即应用于儿科肉瘤。此外还 应该对广泛的其他儿童和成人实体瘤产生重大影响， C.TNC.

Project Abstract The long-term goal of this project is to develop an adoptive immunotherapy with T cells expressing chimeric antigen receptors (CARs) for recurrent and refractory pediatric sarcoma, which prognosis remains poor. CAR T cell therapy has demonstrated success for hematological malignancies in clinical studies; however, CAR T cells have been less effective for solid tumors in the clinic. While limited efficacy is multifactorial, a significant roadblock for solid tumor CAR T cell therapy is the lack of targetable antigens. One prominent ECM protein is tenascin C (TNC), and cancer cells, including sarcoma cells, express oncofetal tenascin C (TNC) spice variants, which can contain an additional C domain (C.TNC). Since C.TNC expression correlates with the malignant phenotype of cancer cells, we posit that it is an ideal CAR target. In support of this application, we have generated a 2nd generation C.TNC-specific CAR with a CD28 costimulatory domain (C.TNC-CAR). I now hypothesize that C.TNC-CAR T cells recognize and kill C.TNC-positive tumor cells and can be optimized to have potent antitumor in preclinical pediatric sarcoma models. We have demonstrated in preliminary studies that C.TNC-CAR T cells recognize and kill C.TNC+ sarcoma cell lines in vitro. In this project now, we propose to investigate this hypothesis in two interrelated research aims. Aim 1 focuses on understanding the mechanism of our ECM-targeting C.TNC-CAR T cells, and Aim 2 then compares the effector function of C.TNC-CAR T cells in in vivo sarcoma xenograft models evaluating expansion, persistence, and anti-tumor activity. Techniques to be employed include flow cytometry, ELISAs, co-cultures and cytotoxicity assays, and CRISPR-Cas9 genome editing. This project was designed to train me in techniques to generate and characterize genetically modified T cells expressing CARs as well as introduce me into the evolving field of cancer cell therapy. This proposal and training will be performed at St. Jude Children’s Research Hospital, an NCI-designated comprehensive cancer center. If successful, the results of this proposal could have immediate applications for pediatric sarcomas. In addition, it should have a significant impact on a broad range of other pediatric and adult solid tumors that also express C.TNC.