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.

项目摘要/摘要用嵌合抗原受体（CAR）修饰T细胞是一种用于治疗的临床验证方法一些B细胞恶性肿瘤。尽管有效，但这些细胞的遗传修饰与重要的缺点。例如，基因工程技术乏味，效率低下，而不是适用于所有细胞类型。此外，修改是永久的，导致持久，严重和不可逆的患者副作用。由于这些缺点，使用替代，可逆的脚手架直接的治疗细胞 - 细胞相互作用将是非常有益的。以前已经证明融合蛋白由两个大肠杆菌二氢叶酸二酸酯还原酶（DHFR2）组成与化学二聚体BIS- 甲氨蝶呤（BISMTX）。最近，将抗EPAM SCFV融合到DHFR2蛋白上，磷脂与BISMTX物种共轭。这些物种的组装形成化学自组装的嵌合嵌入在T细胞膜中并驱动选择性识别的抗原受体（CS-CARS）并杀死EPCAM阳性癌细胞。重要的是，CS卡车很容易从T细胞中删除通过与FDA批准的抗生素甲氧苄啶孵育，提供药理机制停用CS卡车。尽管这些积极的体外结果，但仍不清楚电流是否 CS-CAR的结构对于细胞 - 细胞相互作用的启动和维护是最佳的。因此，该项目中提出的目的旨在进一步检验以下假设：CS-CAR可以用作快速，快速的，可逆方法用于治疗目的。具体而言，结果将进一步了解这些CS-CAR的结构成分，提供了一种合理的途径来优化其治疗功效。 AIM 1将专注于对CS卡的每个组件的系统评估，生成一个小型CS卡车构造的库。这包括脂质物种，PEG/肽接头的变化长度和靶向元素身份（SCFV与新型纤连蛋白通过酵母表面显示设计）。这 CS-CARS在体外启动和维持特定的可逆细胞 - 细胞相互作用的能力也将是已确立的。 AIM 2将评估用不同的抗EPAM CS-CARS修饰的体外功效T细胞有选择地识别并杀死EPCAM阳性MCF7乳腺癌细胞。这些结果将负担得起优化的CS-CAR适用于体内进一步评估，并增强该领域对设计的理解可逆的治疗细胞 - 细胞相互作用。因此，该提议不仅对基于细胞的恶性肿瘤治疗，但也用于使用定向细胞相互作用的其他应用。此外，该应用程序提供了一个严格但定义的科学和指导框架，以促进申请人成为成功的学术医师科学家的目标。