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输送到目标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，由两个部分组成：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（scFvs, ~25 kDa）、设计的锚蛋白重复蛋白（DARPins, ~15 kDa）和粘附体（~ 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，随后有效内化并逃逸到细胞质室。递送支架的这些特性将使其成为蛋白质进入细胞的转运体。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，这是一种具有胸苷激酶活性的新型酶变体。我们将测试递送支架将dCKEN运送到HER2阳性细胞的效率和选择性。这种工程酶的独特催化活性将使我们能够将胸腺嘧啶类似物的激活仅限于内化酶的细胞。通过这种方式，我们将开发出一种可以用来根除HER2阳性细胞而不影响其他细胞的系统。递送技术面临的挑战是发现在大肠杆菌中可以高产量获得的支架，以低纳摩尔亲和力与细胞表面标记物结合，并且能够有效地内化并从内吞囊泡逃逸到细胞质中。这项工作的新颖之处在于所使用的递送支架的类型，它比传统的单克隆靶向系统小得多。使用这些较小的支架的优点包括更深入地渗透到实体肿瘤中，由于单克隆抗体中不存在Fc区而减少了非特异性结合，并且能够在大肠杆菌中生产。此外，从结合亲和力到内化倾向，再到从囊泡逃逸到细胞质，递送支架的各个方面都将通过噬菌体展示和适当的选择方法进行优化。