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

基本信息

批准号：
9751255
负责人：
Clifford Michael Csizmar
金额：
$ 2.58万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-04 至 2020-05-05
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9751255
关键词：
Adenocarcinoma 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 Cellular immunotherapy Chemical Structure Chemicals Chemistry Chimeric Proteins Clinical Consumption Data Development Dihydrofolate Reductase Elements Engineering Escherichia coli Evaluation FDA approved Fatty Acids Fibronectins Fostering Future Genetic Genetic Engineering Goals Hematologic Neoplasms 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 Structure Surface T-Cell Receptor T-Lymphocyte TACSTD1 gene Techniques Testing Therapeutic Time Treatment Efficacy Trimethoprim Variant Work Xenograft Model Yeasts anticancer treatment arm base cancer stem cell cancer therapy cell type chemical stability chimeric antigen receptor clinically relevant design dimer fluorophore genetic approach improved in vivo in vivo evaluation neoplastic cell non-genetic novel scaffold side effect small molecule libraries tissue regeneration tumor

项目摘要

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.

项目概要/摘要用嵌合抗原受体 (CAR) 修饰 T 细胞是一种经过临床验证的治疗以下疾病的方法一些 B 细胞恶性肿瘤。尽管有效，但这些细胞的基因修饰与显着的缺点。例如，基因工程技术繁琐、低效、不可行。适用于所有细胞类型。此外，这些修改是永久性的，会导致持久的、严重的和对患者产生不可逆转的副作用。由于这些缺点，使用替代的可逆支架来直接的治疗性细胞间相互作用将非常有益。之前已经证明，一个由两个大肠杆菌二氢叶酸还原酶 (DHFR2) 单位组成的融合蛋白将自发组装当与化学二聚剂双-结合时，形成化学自组装纳米环（CSAN）甲氨蝶呤（bisMTX）。最近，抗 EpCAM scFv 与 DHFR2 蛋白融合，并且磷脂与 bisMTX 物质缀合。这些物种的组装形成化学自组装嵌合体嵌入 T 细胞膜并驱动选择性识别的抗原受体 (CS-CAR) 并杀死 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一起孵育来表面，提供药理学机制停用 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 的结构对于细胞间相互作用的启动和维持是最佳的。因此，该项目提出的目标旨在进一步检验 CS-CAR 可以用作快速、为治疗目的而修饰细胞表面的可逆方法。具体而言，研究结果将进一步推动了解这些 CS-CAR 的结构组件，提供优化其结构的合理途径治疗功效。目标 1 将侧重于对 CS-CAR 的每个组成部分进行系统评估，生成一个小型 CS-CAR 结构库。这包括脂质种类、PEG/肽接头的变化长度和靶向元件身份（scFv 与通过酵母表面展示设计的新型纤连蛋白）。这 CS-CAR 在体外启动和维持特定的、可逆的细胞间相互作用的能力也将已确立的。目标 2 将评估用不同抗 EpCAM CS-CAR 修饰的 T 细胞的体外功效选择性识别并杀死 EpCAM 阳性 MCF7 乳腺癌细胞。这些结果将负担得起优化的 CS-CAR 适合进一步体内评估并增强该领域对设计的理解可逆的、治疗性的细胞间相互作用。因此，该提案不仅对基于细胞的恶性肿瘤治疗，也可用于利用定向细胞间相互作用的其他应用。此外，该应用程序提供了严格且明确的科学和指导框架，以促进申请人成为一名成功的学术医师科学家的目标。