Development of a Surgical Drug Delivery System for Enhancement of CAR T Cell Activity

项目概要和摘要 嵌合抗原受体（CAR）T细胞是基因工程化的T淋巴细胞，其被设计为感测 抗原并产生免疫反应。尽管CAR T细胞已经获得FDA批准用于治疗， 在几种血液恶性肿瘤中，实体瘤的成功受到缺乏特异性抗原的限制， 免疫抑制性肿瘤微环境和治疗限制性不良反应，如靶向、肿瘤外 毒性和细胞因子释放综合征。尽管研究人员报告了减轻这些限制的策略， 例如用于重塑肿瘤微环境的生物材料，以及逻辑门控的CAR T细胞，以防止非肿瘤细胞。 尽管存在特定的毒性，但没有提出的策略可以克服这些障碍中的每一个。为了克服这些限制，我 提出使用一种新的外科网片植入肿瘤切除腔内。该网将用于 与称为zipCAR的分裂CAR T细胞结合，其使用分离的衔接蛋白（“zipFv”）， 正义抗原该网由聚合物纳米纤维组成，其中沉积有壳聚糖基质。 毛孔网状物提供zipFv衔接蛋白、细胞因子（IL-15）和T细胞刺激性抗体（α- CD3/28）。我假设使用这种外科补片将克服CAR-T细胞治疗的障碍 在实体瘤中通过：（1）对抗T细胞无反应性并促进切除腔中的增殖，（2） 通过包封靶向多种抗原的zipFv防止抗原逃逸，和（3）赋予 在CAR T细胞活性的时空控制。该提案的目标1表明， 通过监测HER 2+乳腺癌鼠模型中的CAR T细胞增殖，证实了补片的优势。目的2 该提案的一部分证明了补片在手术模型中的有效性和安全性优势 卵巢癌的治疗为了证明预防抗原逃逸，ROR 1和HER 2缺陷型OVCAR 3 将使用CRISPR-Cas9敲除来创建细胞系。在抗原逃逸的鼠模型中，这些细胞将 用于在用zipCAR T细胞和负载有zipFv的网状物处理的小鼠中证明上级功效 针对两种抗原。为了证明上级安全性，将在相同的卵巢模型中使用补片 用经照射以上调骨中非造血干细胞中ROR 1表达的小鼠进行癌症研究 骨髓和脾脏，允许观察中靶、肿瘤外毒性。 该提案围绕关键研究和临床技能的四个关键组成部分构建，以支持我的 发展成为一个独立的医生科学家：（1）一个跨学科的研究项目，重点是 用于增强CAR T细胞活性的新型外科生物材料;（2）来自Dr. Grinstaff（生物材料），Wong（免疫疗法）;和Colson（临床医学，动物模型和 免疫学），（3）在研究行为和沟通技巧方面的学术医生科学家培训，（4） 承诺制定个人发展计划（IDP），以指导我的培训目标。

Project Summary and Abstract Chimeric antigen receptor (CAR) T cells are genetically engineered T lymphocytes designed to sense antigens and mount an immune response. Though CAR T cells have received FDA approval for the treatment of several hematologic malignancies, success in solid tumors is limited by a lack of specific antigens, the immunosuppressive tumor microenvironment, and treatment-limiting adverse effects such as on-target, off-tumor toxicity and cytokine release syndrome. Though investigators report strategies for mitigating these limitations such as biomaterials for reshaping the tumor microenvironment, and logic-gated CAR T cells to prevent non- specific toxicity, no proposed strategy has overcome each of these barriers. To surmount these limitations, I propose the use of a novel surgical mesh for implantation into the tumor resection cavity. This mesh will be used in conjunction with a split CAR T cell called a zipCAR, which uses a detached adaptor protein (a “zipFv”) to sense antigens. The mesh is composed of polymeric nanofibers with a matrix of chitosan deposited within the pores. The mesh supplies the zipFv adaptor protein, cytokines (IL-15), and T cell stimulatory antibodies (α- CD3/28). I hypothesize that the use of this surgical mesh will overcome the barriers to CAR T cell therapy in solid tumors by: (1) opposing T cell anergy and promoting proliferation in the resection cavity, (2) preventing antigen escape via encapsulation of zipFvs targeting multiple antigens, and (3) imparting spatiotemporal control over CAR T cell activity. Aim 1 of this proposal demonstrates the proliferation advantage of the mesh by monitoring CAR T cell proliferation in a murine model of HER2+ breast cancer. Aim 2 of this proposal demonstrates the efficacy and safety advantages of the meshes in a model of operative debulking of ovarian cancer. To demonstrate prevention of antigen escape, ROR1- and HER2-deficient OVCAR3 cell lines will be created using CRISPR-Cas9 knockouts. In a murine model of antigen escape, these cells will be used to demonstrate superior efficacy in mice treated with zipCAR T cells and meshes loaded with zipFvs against both antigens. To demonstrate superior safety, meshes will be utilized in the same model of ovarian cancer with mice that are irradiated to upregulate ROR1 expression in non-hematopoietic stem cells in the bone marrow and spleen, allowing observation of on-target, off-tumor toxicity. This proposal builds around four key components of critical research and clinical skills to support my development into an independent physician scientist: (1) an interdisciplinary research project focusing on novel surgical biomaterials for enhancement of CAR T cell activity; (2) multi-disciplinary mentoring from Drs. Grinstaff (biomaterials), Wong (immunotherapy); and, Colson (clinical medicine, animal models, and immunology), (3) academic physician scientist training in research conduct and communication skills, (4) commitment to an individual development plan (IDP) to guide my training goals.