Enzyme-delivery scaffold technology for targeted cancer killing.

描述(申请人提供)：将处于活性状态的蛋白质送入靶细胞是一个主要的技术障碍。然而，这样做的能力将在临床和基础研究环境中打开许多应用程序。我们建议开发完成这一任务的细胞靶向系统，由两个组件组成：细胞靶向模块和蛋白质货物。靶向模块将基于单链可变片段(scFv；~25 kDa)、设计的锚蛋白重复蛋白(DARPins；~15 kDa)和亲和体(~9 kDa)的分子支架。这些支架将利用噬菌体展示技术进行优化，以获得所需的属性。这些包括与细胞表面抗原的紧密和特异性结合，随后是有效的内化和逃逸到细胞质隔间。输送支架的这些特性将使其成为蛋白质进入细胞的运输者。为了测试我们的技术，我们将提供一个工程版本的人脱氧胞苷激酶(DCKEN)，这是一种新的酶变体，已被赋予胸苷激酶活性。我们将测试输送支架的效率和选择性，以了解它们将dCKEN运送到HER2阳性细胞的能力。这种工程酶独特的催化活性将允许我们将胸苷类似物的激活限制在已内化该酶的细胞上。这样，我们就可以开发出一种系统，可以用来根除HER2阳性细胞，而不会影响其他细胞。给药技术面临的挑战是发现在大肠杆菌中可以高产率获得的支架，它以低纳摩尔亲和力结合到细胞表面标记上，并经历有效的内化和内吞囊泡逃逸到细胞质中。这项工作的新颖性来自于所使用的输送支架的类型，这种支架比传统的基于单克隆的靶向系统小得多。使用这些较小支架的优势包括更深入地渗透到实体瘤中，由于单抗中没有Fc区域而减少了非特异性结合，以及在大肠杆菌中生产的能力。此外，通过将噬菌体展示与适当的选择方法相结合，传递支架的各个方面，从结合亲和力到内化倾向，再到从囊泡逃逸到细胞质，都将得到优化。

DESCRIPTION (provided by applicant): Delivering a protein in its active state into a targeted cell is a major technological hurdle. However, the ability to do so would open up numerous applications in both clinical and basic research settings. We propose to develop cell-targeting systems that fulfill this task, composed of two components: the cell targeting module, and the protein cargo. The targeting modules will be based on the molecular scaffolds of single chain variable fragments (scFvs; ~25 kDa), designed ankyrin repeat proteins (DARPins; ~15 kDa), and affibodies (~ 9 kDa). Theses scaffolds will undergo optimization using phage display technology in order to acquire the needed attributes. These include tight and specific binding to a cell surface antigen, which is followed by efficient internalization and escape into the cytoplasmic compartment. These properties of the delivery scaffold will allow it to act as a transporter of proteins into cells. To test our technology, we will deliver an engineered version of human deoxycytidine kinase (dCKEN), which is a novel enzyme variant that has been endowed with thymidine kinase activity. We will test the efficiency and selectivity of the delivery scaffolds for their ability to ferry dCKEN into HER2 positive cells. The unique catalytic activity of this engineered enzyme will allow us to confine the activation of thymidine analogs only to cells that have internalized the enzyme. In this way, we would have developed a system that can be used to eradicate HER2 positive cells while not affecting other cells. The challenges to the delivery technology are to discover scaffolds that can be obtained at high yield in E. coli, that bind to the cell-surface marker with low nanomolar affinity, and that undergo efficient internalization and escape from endocytic vesicles into the cytoplasm. The novelty of this work stems from the type of delivery scaffolds used, which are much smaller than conventional monoclonal-based targeting systems. The advantages of using these smaller scaffolds include deeper penetration into solid tumors, reduced non-specific binding due to the absence of an Fc region present in monoclonal antibodies, and the ability for production in E. coli. Moreover, every aspect of the delivery scaffold, from binding affinity via internalization propensity, to escape from vesicles into the cytoplasm will be optimized by coupling phage display with the appropriate selection method.