Engineering Cell-Cell Interactions by Chemically Self-Assembled CARS

 描述（由申请人提供）：对细胞表面进行工程化和重新编程的能力具有扩展和实现基于细胞的疗法用于组织再生和癌症的治疗用途的潜力。为了实现这一目标，已经设计了许多细胞表面工程方法。在临床上，细胞的基因工程是迄今为止最常用的修饰细胞表面的方法。然而，工程细胞的制备是耗时的，需要几个复杂的步骤，并可能导致长期的免疫毒性。我们的团队利用化学诱导蛋白质二聚化的能力开发了一种生产化学自组装纳米环（CSAN）的方法。我们证明了两个E.与单链抗体（scFv）或靶向肽融合的大肠杆菌二氢叶酸还原酶分子（DHFR 2）可以被工程化，以在加入化学二聚化剂双甲氨蝶呤（Bis-MTX）后自发地自组装成高度稳定的二价或八价合成抗体CSAN。最近，使用Bis-MTX磷脂缀合物，我们已经制备了快速（分钟）和稳定（天）插入细胞膜的CSAN。此外，当靶向EpCAM（癌和癌症干细胞标志物）的scFv与DHFR 2结构单元融合时，形成了我们的第一代抗EpCam化学自组装嵌合抗原受体（抗EpCAM-CS-CAR），并发现其指导EpCAM阳性乳腺癌细胞的选择性T细胞杀伤。我们的方法的独特特征是用无毒药物从T细胞中去除抗EpCAM-CS-CAR的能力。因此，我们提出制备结构不同的抗EpCAM-CS-CAR的小文库，并确定它们在体外和体内稳定功能化T细胞膜并诱导对EpCam阳性肿瘤细胞的细胞毒性的能力。这些初步数据将使我们能够建立化学自组装嵌合抗原受体（CS-CAR）的潜力，作为一种替代和补充方法，用于工程化稳定但不可逆的治疗性细胞-细胞相互作用。

 DESCRIPTION (provided by applicant): The ability to engineer and re-program the surfaces of cells has the potential to expand and enable the therapeutic use of cell based therapies for both tissue regeneration and cancer. To achieve this goal a number of cell surface engineering approaches have been devised. Clinically, genetic engineering of cells is by far the most commonly used approach for the modification of cell surfaces. Nevertheless, the preparation of the engineered cells is time consuming, requires several sophisticated steps and can lead to long term immune toxicities. Our group has employed the power of chemically induced protein dimerization to develop a method to produce chemically self-assembled nanorings (CSANs). We have shown that two E. coli dihydrofolate reductase molecules (DHFR2) fused to a single chain antibody (scFv) or targeting peptide can be engineered to spontaneously self-assemble, upon the addition of the chemical dimerizer, bis-methotrexate (Bis-MTX), into either highly stable bivalent or octavalent synthetic antibody CSANs. Recently, using a Bis-MTX phospholipid conjugate, we have prepared CSANs that rapidly (min) and stably (days) insert into cell membranes. In addition, when a scFv targeting EpCAM, a carcinoma and cancer stem cell marker, was fused to the DHFR2 building block, our first generation anti-EpCam chemically self-assembled chimeric antigen receptors (anti-EpCAM-CS-CARs) were formed and found to direct selective Tcell cell killing of EpCAM positive breast cancer cells. A unique feature of our approach is the ability to remove the anti-EpCAM-CS-CARs from the T-cells pharmacologically with a non-toxic drug. Therefore, we propose to prepare a small library of structurally different anti-EpCAM-CS-CARs and determine their ability to stably functionalize T-cell membranes and induce cytotoxicity toward EpCam positive tumor cells both in vitro and in vivo. This preliminary data will allow us to establish the potential for chemically self-assembled chimeric antigen receptors (CS-CARs) to serve as an alternative and complementary approach for the engineering of stable, yet pharmacologically reversible, therapeutic cell-cell interactions.