Enzyme-delivery scaffold technology for targeted cancer killing.

描述（由申请人提供）：将活性状态的蛋白质递送到靶细胞中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开辟许多应用。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）和锚体（~ 9 kDa）的分子支架。这些支架将使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区室中。这些性质的交付支架将允许它作为一个转运蛋白进入细胞。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。我们将测试递送支架将dCKEN运送到HER 2阳性细胞中的能力的效率和选择性。这种工程酶的独特催化活性将使我们能够将胸苷类似物的激活仅限于已内化酶的细胞。通过这种方式，我们将开发出一种可用于根除HER 2阳性细胞而不影响其他细胞的系统。该技术面临的挑战是发现能够在大肠杆菌中高产率获得的支架。大肠杆菌，其以低纳摩尔亲和力结合细胞表面标记物，并且其经历有效的内化并从内吞囊泡逃逸到细胞质中。这项工作的新奇源于所使用的递送支架的类型，其比传统的基于单克隆的靶向系统小得多。使用这些更小的支架的优点包括更深地渗透到实体瘤中，由于单克隆抗体中不存在Fc区而减少非特异性结合，以及在大肠杆菌中产生的能力。杆菌此外，通过将噬菌体展示与适当的选择方法结合，将优化递送支架的每个方面，从结合亲和力到内化倾向，从囊泡逃逸到细胞质中。 公共卫生相关性：肿瘤治疗面临的一个主要技术障碍是如何利用癌细胞标志物实现靶向治疗。本申请通过开发将独特的酶转运到癌细胞的细胞内区室中的酶递送技术来解决这一需求。随后施用通过独特酶转化为其毒性形式的原本无毒的前药将导致靶向癌细胞的消除。重要的是，这种方法将保留健康组织。

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. PUBLIC HEALTH RELEVANCE: A major technological hurdle confronting cancer therapeutics is how to take advantage of cancer-cell markers to achieve targeted therapy. This application addresses this need by developing an enzyme delivery technology that transports a unique enzyme into the intracellular compartment of cancer cells. Subsequent administration of an otherwise non-toxic prodrug that is converted to its toxic form by the unique enzyme will result in the elimination of the targeted cancer cells. Importantly, this approach will spare healthy tissue.