Engineering Reversible Cell-Cell Interactions with Chemically Self-Assembled CARs

利用化学自组装 CAR 设计可逆的细胞间相互作用

• 批准号: 9314991
• 负责人: Clifford Michael Csizmar
• 金额: $ 3.72万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-04 至 2020-07-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9314991
• 关键词: Adenocarcinoma Cell; Adverse effects; Alpha Cell; Antibiotics; Antibodies; B lymphoid malignancy; Back; Biological Markers; Breast Adenocarcinoma; Breast Cancer Cell; CD3 Antigens; Cancer Patient; Carcinoma; Cell Communication; Cell Maintenance; Cell Therapy; Cell membrane; Cell surface; Cells; Chemical Structure; Chemicals; Chemistry; Chimeric Proteins; Clinical; Data; Development; Dihydrofolate Reductase; Dimerization; Elements; Engineering; Escherichia coli; Evaluation; FDA approved; Fatty Acids; Fibronectins; Fostering; Future; Genetic; Genetic Engineering; Goals; Hematologic Neoplasms; Immunotherapy; In Vitro; Kinetics; Length; Libraries; Lipids; MCF7 cell; Malignant Epithelial Cell; Malignant Neoplasms; Membrane; Mentors; Methods; Methotrexate; Modification; Mus; Oligonucleotides; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Pharmacology; Phospholipids; Physicians; Population; Proteins; Receptor Cell; Role; Safety; Scientist; Stem cells; Surface; T-Lymphocyte; TACSTD1 gene; Techniques; Testing; Therapeutic; Time; Treatment Efficacy; Trimethoprim; Variant; Work; Xenograft Model; Yeasts; arm; base; cancer stem cell; cancer therapy; cell type; chemical stability; chimeric antigen receptor; clinically relevant; design; dimer; fluorophore; genetic approach; improved; in vivo; killings; neoplastic cell; non-genetic; novel; scaffold; small molecule libraries; tissue regeneration; tumor

PROJECT SUMMARY/ABSTRACT Modifying T cells with chimeric antigen receptors (CARs) is a clinically-validated approach for the treatment of some B-cell malignancies. Though efficacious, the genetic modification of these cells is associated with significant drawbacks. For instance, the genetic engineering techniques are tedious, inefficient, and not applicable to all cell types. Furthermore, the modifications are permanent, leading to persistent, severe, and irreversible patient side effects. Because of these drawbacks, the use of an alternative, reversible scaffold to direct therapeutic cell-cell interactions would be highly beneficial. It has been previously demonstrated that a fusion protein comprised of two units of E. coli dihydrofolate reductase (DHFR2) will spontaneously assemble into a chemically self-assembled nanoring (CSAN) when combined with the chemical dimerizer bis- methotrexate (bisMTX). Recently, an anti-EpCAM scFv was fused to the DHFR2 protein, and a phospholipid was conjugated to the bisMTX species. Assembly of these species formed chemically self-assembled chimeric antigen receptors (CS-CARs) that were embedded in the membrane of T cells and drove selective recognition and killing of EpCAM-positive carcinoma cells. Importantly, the CS-CARs were readily removed from the T cell surface via incubation with the FDA-approved antibiotic trimethoprim, affording a pharmacological mechanism to deactivate the CS-CARs. Despite these positive in vitro results, it remains unclear whether the current structure of the CS-CAR is optimal for the initiation and maintenance of cell-cell interactions. Therefore, the aims proposed in this project are designed to further test the hypothesis that CS-CARs can be used as a rapid, reversible method to modify cell surfaces for therapeutic purposes. Specifically, the results will further the understanding of the structural components of these CS-CARs, providing a rational pathway to optimize their therapeutic efficacy. Aim 1 will focus on a systematic evaluation of each component of the CS-CAR, generating a small library of CS-CAR constructs. This includes variation of lipid species, PEG/peptide linker lengths, and targeting element identity (scFv vs. novel fibronectin engineered via yeast surface display). The ability of the CS-CARs to initiate and maintain specific, reversible cell-cell interactions in vitro will be also established. Aim 2 will evaluate the in vitro efficacy T cells modified with different anti-EpCAM CS-CARs to selectively recognize and kill EpCAM-positive MCF7 breast adenocarcinoma cells. These results will afford optimized CS-CARs suitable for further evaluation in vivo and enhance the field's understanding of designing reversible, therapeutic cell-cell interactions. Therefore, this proposal has broad implications not only for the cell-based treatment of malignancies, but also for other applications utilizing directed cell-cell interactions. Moreover, this application provides a rigorous, yet defined scientific and mentoring framework to foster the applicant's goals of becoming a successful academic physician-scientist.

项目总结/摘要 用嵌合抗原受体（汽车）修饰T细胞是一种临床上验证的用于治疗免疫缺陷综合征的方法。 B细胞恶性肿瘤尽管有效，但这些细胞的遗传修饰与 重大缺陷。例如，基因工程技术是繁琐的，效率低下的， 适用于所有细胞类型。此外，修改是永久性的，导致持续的，严重的， 不可逆的患者副作用。由于这些缺点，使用替代的可逆支架来 直接的治疗性细胞-细胞相互作用将是非常有益的。先前已经证明， 融合蛋白由两个E.大肠杆菌二氢叶酸还原酶（DHFR 2） 当与化学二聚剂双- 甲氨蝶呤（bisMTX）。最近，将抗EpCAM scFv与DHFR 2蛋白融合，并将磷脂 与bisMTX物质缀合。这些物种的组装形成化学自组装嵌合体 抗原受体（CS-CAR），嵌入T细胞膜并驱动选择性识别 和杀死EpCAM阳性癌细胞。重要的是，CS-CAR很容易从T细胞中去除 通过与FDA批准的抗生素甲氧苄啶孵育， 来解除CS-CAR尽管有这些积极的体外结果，但目前仍不清楚目前是否 CS-CAR的结构对于细胞-细胞相互作用的起始和维持是最佳的。因此 该项目中提出的目标旨在进一步检验CS-CAR可用作快速， 可逆的方法来修饰细胞表面用于治疗目的。具体而言，这些结果将进一步促进 了解这些CS-CAR的结构组分，提供合理的途径来优化其 疗效目标1将侧重于系统评价CS-CAR的每个组成部分， 产生CS-CAR构建体的小文库。这包括脂质种类、PEG/肽接头的变化 长度和靶向元件同一性（scFv与通过酵母表面展示工程化的新型纤连蛋白）。的 CS-CAR在体外启动和维持特异性的、可逆的细胞-细胞相互作用的能力也将是 确立了习目的2将评估用不同抗EpCAM CS-CAR修饰的T细胞对免疫应答的体外功效。 选择性识别并杀死EpCAM阳性MCF 7乳腺癌细胞。这些结果将提供 优化的CS-CAR适合于进一步的体内评价，并增强了该领域对设计的理解。 可逆的治疗性细胞间相互作用因此，这项建议不仅对 恶性肿瘤的基于细胞的治疗，但也用于利用定向细胞-细胞相互作用的其它应用。 此外，该应用程序提供了一个严格的，但明确的科学和指导框架，以促进 申请人的目标是成为一个成功的学术物理学家，科学家。