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）：将其活性状态的蛋白质传递到目标细胞中是一个主要的技术障碍。但是，这样做的能力将在临床和基础研究环境中开放众多应用。我们建议开发符合此任务的细胞靶向系统，这些系统由两个组成部分组成：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的分子支架（SCFVS; 〜25 kDa），设计了Ankyrin重复蛋白（DARPINS； 〜15 kDa）和Affibodies（〜9 kDa）。这些脚手架将使用噬菌体显示技术进行优化，以获取所需的属性。这些包括与细胞表面抗原的紧密结合，随后是有效的内在化并逃脱到细胞质室中。递送支架的这些特性将使其充当蛋白质的转运蛋白到细胞中。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，它是一种新型的酶变体，已赋予胸苷激酶活性。我们将测试输送支架的效率和选择性，以便它们将DCKEN渡轮到HER2阳性细胞中。该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。这样，我们将开发一种可以用来消除HER2阳性细胞的系统，同时不影响其他细胞。递送技术的挑战是发现可以在大肠杆菌中以高产量获得的支架，这些支架与低纳摩尔亲和力结合的细胞表面标记，并经历有效的内在化并从内吞囊泡进入细胞质。这项工作的新颖性源于所使用的输送支架的类型，这些脚手架比常规的基于单克隆的靶向系统小得多。使用这些较小的支架的优点包括更深的渗透到实体瘤，由于单克隆抗体中存在FC区域而导致的非特异性结合以及大肠杆菌中生产的能力。此外，从结合亲和力通过内在化倾向到从囊泡进入细胞质的各个方面，将通过与适当的选择方法耦合噬菌体显示来优化囊泡进入细胞质。 公共卫生相关性：面对癌症治疗剂的主要技术障碍是如何利用癌细胞标记来实现有针对性的治疗。该应用程序通过开发一种将独特的酶传输到癌细胞细胞内室的酶递送技术来满足这种需求。随后通过独特的酶转化为毒性形式的原本无毒前药将导致消除靶向癌细胞。重要的是，这种方法将避免健康的组织。