Dock and Lock: novel protein engineering

描述(由申请人提供)：项目摘要：这项研究的目标是开发新一代融合蛋白，这种融合蛋白可用于通过预靶向方法改进癌症和其他疾病的成像和治疗，这种方法将抗体定位与诊断或治疗性放射性核素的输送分开。本申请描述了一种从两种不同的融合蛋白中简便、定量地组装三价、双特异性结合结构的新技术，并建议推进由三个稳定连接的Fab片段组成的原型分子，并保持亲本抗体的结合亲和力。这些新的构建物被提纯到均一，并被证明用于将放射性标记示踪剂预靶向人结肠癌裸鼠移植瘤，获得了特殊的肿瘤与非肿瘤比率。例如，在注射99mTc标记的示踪剂1h后，肿瘤与血液的比率高达66+5，肿瘤摄取超过每克注射剂量的30%。这项应用的主要目的是在体外和体内进一步鉴定这种结构，以便确定其未来用于预靶向治疗的临床开发的可行性。另一个关键目标是证明这种新的构建可以比以前从这个组产生的重组双特异性抗体更有效和更高的生产率。在达到这些里程碑后，我们应该能够提交第二阶段的申请，旨在进一步开发这种药物，用于初步临床测试，作为CEA阳性癌症(如结肠癌)的预靶向放射免疫疗法。项目简介：这项创新研究将产生多价、多功能、一般结构，特别是基于Fab的三价双特异性结合蛋白，可用于检测、成像和治疗各种以抗原为靶点的人类疾病，包括癌症、心血管疾病、自身免疫性疾病和传染病。

DESCRIPTION (provided by applicant): Project Summary: The objective of this research is to develop a new generation of fusion proteins that can be used for improved imaging and therapy of cancer and other diseases by the method of pretargeting, which separates antibody localization from the delivery of diagnostic or therapeutic radionuclides. This application describes a novel technology for the facile and quantitative assembly of trivalent, bispecific, binding structures from 2 distinct fusion proteins, and proposes to advance prototype molecules composed of three Fab fragments that are stably linked and retain the binding affinity of the parental antibodies. These novel constructs were purified to homogeneity and shown to be useful for pretargeted delivery of radio-labeled tracers to human colonic tumor xenografts in nude mice, achieving exceptional tumor-to-nontumor ratios. For example, at 1h post-injection of a 99mTc-labeled tracer, the tumor to blood ratio was as high as 66+5, with tumor uptake exceeding 30% injected dose per gram. The primary aim of this application is to further characterize 1 such construct both in vitro and in vivo, so that the feasibility of its future clinical development for pretargeted therapy can be defined. Another key aim is to show that this new construct can be prepared more efficiently and with higher productivity than previously-generated recombinant bispecific antibodies from this group. Upon meeting these milestones, we should be in a position to submit a Phase II application directed toward additional development of this agent for initial clinical testing as pretargeted radioimmunotherapy of CEA-positive cancers, such as colonic cancer. Project Narrative: This innovative research will produce multivalent, multifunctional, structures in general, and trivalent bispecific Fab-based binding proteins in particular, that can be useful for the detection, imaging, and therapy of various human diseases which have antigen targets, including cancers, cardiovascular diseases, autoimmune diseases, and infectious diseases.