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

项目摘要和摘要 嵌合抗原受体(CAR)T细胞是基因工程T淋巴细胞，旨在 抗原并启动免疫反应。尽管CAR T细胞已经获得FDA的批准用于治疗 在几种血液系统恶性肿瘤中，实体瘤的治疗成功受到缺乏特异性抗原的限制。 免疫抑制的肿瘤微环境，以及限制治疗的不良反应，如靶点上、肿瘤外 毒性和细胞因子释放综合征。尽管调查人员报告了缓解这些限制的策略 例如用于重塑肿瘤微环境的生物材料，以及用于防止非 对于特定的毒性，没有任何拟议的战略能够克服所有这些障碍。为了克服这些限制，我 提出一种用于肿瘤切除腔内植入的新型外科网片。将使用此网格 与被称为Zipcar的分裂的CAR T细胞结合在一起，Zipcar使用分离的适配器蛋白(ZipFv)来 感觉抗原。该网状物是由聚合物纳米纤维和壳聚糖基质沉积在 毛孔。Mesh提供ZipFv接头蛋白、细胞因子(IL-15)和T细胞刺激性抗体(α- CD3/28)。我推测，这种外科网状物的使用将克服CAR T细胞治疗的障碍 在实体瘤中：(1)在切除腔内对抗T细胞无能和促进增殖，(2) 通过包裹针对多个抗原的zipFv来防止抗原逃逸，以及(3)传递 对CAR T细胞活性的时空控制。该提案的目标1显示了核扩散 在HER2+乳腺癌小鼠模型中监测CAR T细胞增殖的网状细胞的优势。目标2 在手术模型中验证了网状结构的有效性和安全性。 摘除卵巢癌。为了证明防止抗原逃逸，缺乏ROR1和HER2的OVCAR3 将使用CRISPR-Cas9基因敲除技术创建细胞系。在抗原逃逸的小鼠模型中，这些细胞将 在Zipcar T细胞和zipFv网状物治疗的小鼠中显示出优越的疗效 抗这两种抗原。为了证明更好的安全性，网状物将被用于相同的卵巢模型 辐射上调骨中非造血干细胞中ROR1表达的小鼠患癌 骨髓和脾，允许观察靶点上，肿瘤外的毒性。 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 发展成为独立的内科科学家：(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)多学科指导。 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)学术内科科学家研究指导和沟通技能培训，(4) 致力于个人发展计划(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.