Dock and Lock: novel protein engineering

项目简介：本研究的目的是开发新一代融合蛋白，通过预先靶向的方法，将抗体定位与诊断或治疗放射性核素的递送分离开来，用于改善癌症和其他疾病的成像和治疗。本申请描述了一种从两种不同的融合蛋白中轻松定量组装三价、双特异性结合结构的新技术，并提出了由三个Fab片段组成的原型分子，这些片段稳定地连接并保留了亲本抗体的结合亲和力。这些新型结构被纯化到均匀性，并被证明可用于将放射性标记的示踪剂预先靶向递送到裸鼠的人类结肠肿瘤异种移植物中，实现了特殊的肿瘤与非肿瘤比率。例如，99mtc标记的示踪剂注射后1h，肿瘤与血比高达66+5，肿瘤摄取超过每克注射剂量的30%。本申请的主要目的是进一步在体外和体内表征这种结构，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结构可以比以前从该组产生的重组双特异性抗体更有效和更高的生产力。在达到这些里程碑之后，我们应该能够提交II期申请，针对该药物的进一步开发进行初步临床试验，作为cea阳性癌症（如结肠癌）的预靶向放射免疫治疗。项目描述：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、成像和治疗各种具有抗原靶点的人类疾病，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.