Enzyme-delivery scaffold technology for targeted cancer killing.

用于靶向杀死癌症的酶递送支架技术。

• 批准号: 8034009
• 负责人: BRIAN KENNETH KAY
• 金额: $ 23.11万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8034009
• 关键词: Address; Affect; Affinity; Ankyrin Repeat; Antigens; Bacteria; Basic Science; Binding; Biodistribution; Blood; Breast; Carrier Proteins; Cell Death; Cell Surface Proteins; Cell surface; Cells; Cellular Structures; Clinical; Clinical Research; Code; Coupling; Cytokine Activation; Cytoplasm; Deoxycytidine Kinase; Disease; Down-Regulation; ERBB2 gene; Elements; Endocytic Vesicle; Engineering; Enzymes; Epitopes; Escherichia coli; Fc Receptor; Half-Life; Human; Lysosomes; Malignant Neoplasms; Malignant neoplasm of ovary; Methods; Molecular; Monoclonal Antibodies; Normal Cell; Normal tissue morphology; Penetration; Phage Display; Prodrugs; Production; Property; Proteins; Radioisotopes; Research; Roche brand of trastuzumab; Scaffolding Protein; Solid Neoplasm; Specificity; Surface; Surface Antigens; System; Techniques; Technology; Testing; Therapeutic; Thymidine; Thymidine Kinase; Time; Tissues; Toxin; Variant; Vesicle; Work; analog; base; cancer cell; cell killing; clinical application; complex biological systems; crosslink; design; killings; malignant breast neoplasm; new technology; novel; overexpression; receptor; scaffold; stem; success; tumor; uptake

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.

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大量应用。我们建议开发完成这项任务的细胞靶向系统，该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紧密且特异性的结合，然后有效内化并逃入细胞质区室。递送支架的这些特性将使其能够充当蛋白质进入细胞的转运蛋白。为了测试我们的技术，我们将提供人类脱氧胞苷激酶 (dCKEN) 的工程版本，它是一种新型酶变体，被赋予了胸苷激酶活性。我们将测试递送支架将 dCKEN 转运至 HER2 阳性细胞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该酶的细胞。通过这种方式，我们就可以开发出一种系统，可以用来根除 HER2 阳性细胞，同时不影响其他细胞。递送技术面临的挑战是发现可以在大肠杆菌中高产量获得的支架，该支架以低纳摩尔亲和力与细胞表面标记物结合，并且可以进行有效的内化并从内吞囊泡逃逸到细胞质中。这项工作的新颖性源于所使用的递送支架的类型，该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，以及在大肠杆菌中生产的能力。此外，递送支架的各个方面，从通过内化倾向的结合亲和力，到从囊泡逃逸到细胞质中，都将通过将噬菌体展示与适当的选择方法结合起来进行优化。 公共健康相关性：癌症治疗面临的一个主要技术障碍是如何利用癌细胞标记物来实现靶向治疗。该应用通过开发一种酶递送技术来满足这一需求，该技术将一种独特的酶运输到癌细胞的细胞内区室中。随后施用一种无毒的前药，通过独特的酶将其转化为有毒形式，从而消除目标癌细胞。重要的是，这种方法将保护健康组织。