Rapid isolation of high-affinity human antibodies from large synthetic libraries

描述（由申请人提供）：目前有超过300种治疗性蛋白质处于临床试验的各个阶段，通往更健康未来的道路将需要生产更安全和更便宜的重组蛋白质的新方法。特别地，衍生自单克隆抗体（例如Fab、scAb、scFv、免疫毒素等）的下一代治疗剂在治疗包括细菌和病毒感染、癌症、炎性疾病和神经退行性疾病在内的一系列人类疾病中显示出巨大的临床前景。目前，细菌Sec蛋白输出途径是蛋白展示的主要机制（例如，噬菌体展示、细胞表面展示），并且还用于小的非糖基化抗体片段的表达和工程化。然而，由于与Sec输出相关的许多限制，用于临床前和临床试验的高产率的稳定、高亲和力抗体片段的细菌生产可能是抗体药物管道中的严重瓶颈。因此，该提案的总体目标是通过利用最近发现的细菌双精氨酸易位（达特）途径的显著性质来开发通用抗体表达和工程化平台。为了这个目标，本发明的提议包括以下具体目标：（1）创建单链Fv（scFv）抗体片段的合成文库;和（2）应用专有的基于Tat的遗传选择策略，用于有效分离针对疾病相关抗原的超稳定、超高亲和力的人scFv。预期所提出的研究将产生用于发现在细胞内或细胞外环境中折叠和起作用的scFv序列的快速单步选择平台。拟议研究的成功完成预计将产生一种强大的新技术，用于生产有效的基于抗体的生物制药、诊断和研究试剂。 公共卫生相关性：到2008年，工程抗体片段预计将占生物技术市场所有收入的30%以上，并将用于治疗各种人类疾病，包括细菌和病毒感染、癌症、炎症性疾病和神经退行性疾病。由于抗体疗法在开发中的药物中占越来越大的比例，随着药物开发成本的不断升级增加，对有效抗体片段的生产或发现的任何改进将对人类健康产生显著影响。因此，该提议寻求开发一种新的细菌选择策略，用于快速、低成本地分离针对几乎任何感兴趣的靶抗原的有效人抗体。

DESCRIPTION (provided by applicant): With over 300 therapeutic proteins currently in various stages of clinical trials, the road to a healthier future will require new methods for producing safer and less expensive recombinant proteins. In particular, next generation therapeutics derived from monoclonal antibodies (e.g. Fab, scAb, scFv, immunotoxins, etc) show great clinical promise in treating a range of human disorders including bacterial and viral infections, cancer, inflammatory diseases and neurodegenerative disorders. Currently, the bacterial Sec protein export pathway is the dominant mechanism for protein display (e.g., phage display, cell surface display) and also for the expression and engineering of small, non-glycosylated antibody fragments. However, due to a number of limitations associated with Sec export, bacterial production of stable, high-affinity antibody fragments in high yields for preclinical and clinical trials can be a serious bottleneck in the antibody drug pipeline. Therefore, the overall objective of the proposal is to develop a versatile antibody expression and engineering platform by capitalizing on the remarkable properties of the recently discovered bacterial twin-arginine translocation (Tat) pathway. Towards this objective, the current proposal encompasses the following specific aims: (1) creation of synthetic libraries of single-chain Fv (scFv) antibody fragments; and (2) application of a proprietary Tat-based genetic selection strategy for efficient isolation of super-stable, ultra- high affinity human scFvs against disease-relevant antigens. The proposed studies are expected to result in a rapid, single-step selection platform for the discovery of scFv sequences that fold and function in intra- or extra-cellular environments. Successful completion of the proposed studies is expected to result in a powerful new technology for generating potent antibody-based biopharmaceuticals, diagnostics and research reagents. PUBLIC HEALTH RELEVANCE: By 2008, engineered antibody fragments are predicted to account for >30% of all revenues in the biotechnology market and will be used to treat a wide array of human diseases including bacterial and viral infections, cancer, inflammatory diseases and neurodegenerative disorders. Since antibody therapies are an increasingly large fraction of the drugs in development, with ever escalating increases in the cost of drug development, any improvements to the production or discovery of efficacious antibody fragments will have a significant impact on human health. Thus, this proposal seeks to develop a novel bacterial selection strategy for rapid, low-cost isolation of potent human antibodies against virtually any target antigen of interest.