Affinity maturation via in vitro evolution at the lab bench-top

通过实验室台式的体外进化实现亲和力成熟

• 批准号: 8625979
• 负责人: GEOFFREY A CHANG
• 金额: $ 23.36万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8625979
• 关键词: Affinity; maturation; via; vitro; evolution

We propose to develop a fully automated bench-top system that can generate tight-binding protein scaffolds called nanobodies against any target enzyme. The novelty of our approach lies in the ability to rapidly "evolve" these nanobodies using completely in vitro methods. We envision that the user injects a purified protein into this bench-top system and tight-binding Nbs will rapidly "evolve" within days. Our overall strategy is to mimic the genetic optimization themes found in nature and produce highly specific nanobody probes in an innovative animal-free system. The approach will include combining some newer, more risky technologies such as mRNA display and PCR-based DNA recombination methods as well as utilizing more established techniques like cell free protein expression and PCR amplification. The products of the system will be tight-binding polyclonal nanobodies against the target enzyme and the corresponding DNA, which can be used to generate larger quantities of monoclonal nanobodies. The Nbs can be directly used in everyday laboratory applications currently accomplished with traditional polyclonal antibodies. We envision that the rapid discovery, optimization, and production of nanobodies at the bench will have the same revolutionary impact that polymerase chain reaction (PCR) machines had on molecular biology decades ago. This innovative method will allow the broad biomedical research community to quickly generate highly specific biological probes, drugs, and other molecular tools for basic scientific research while circumventing the economic and time costs currently associated with the use of animals to generate antibodies. We propose to develop a fully in vitro bench-top system to create nanobodies de novo, allowing the scientific community to rapidly generate medicines, biological probes, and molecular tools for research in their own labs. The proposed idea would not require the use of animals and can be accomplished in at least one order of magnitude faster than today's in vivo methods. PUBLIC HEALTH RELEVANCE: We propose to develop a fully in vitro bench-top system to create nanobodies de novo, allowing the scientific community to rapidly generate medicines, biological probes, and molecular tools for research in their own labs. The proposed idea would not require the use of animals and can be accomplished in at least one order of magnitude faster than today's in vivo methods.

我们建议开发一种全自动台式系统，该系统可以生成针对任何目标酶的紧密结合的蛋白质支架，称为纳米抗体。我们方法的新颖性在于能够使用完全体外方法快速“进化”这些纳米抗体。我们设想用户将纯化的蛋白质注入这个台式系统中，紧密结合的 Nbs 将在几天内快速“进化”。我们的总体策略是模仿自然界中发现的遗传优化主题，并在创新的无动物系统中生产高度特异性的纳米抗体探针。该方法将包括结合一些更新、风险更大的技术，例如 mRNA 展示和基于 PCR 的 DNA 重组方法，以及利用更成熟的技术，例如无细胞蛋白表达和 PCR 扩增。该系统的产物将是针对目标酶和相应DNA的紧密结合的多克隆纳米抗体，可用于产生更大量的单克隆纳米抗体。 Nbs 可直接用于目前使用传统多克隆抗体完成的日常实验室应用。我们预计，纳米抗体的快速发现、优化和生产将产生与几十年前聚合酶链式反应 (PCR) 机器对分子生物学同样的革命性影响。这种创新方法将使广泛的生物医学研究界能够快速生成用于基础科学研究的高度特异性的生物探针、药物和其他分子工具，同时规避目前与使用动物生成抗体相关的经济和时间成本。我们建议开发一个完全体外的台式系统来从头创建纳米抗体，使科学界能够快速生成药物、生物探针和分子工具，用于在自己的实验室中进行研究。所提出的想法不需要使用动物，并且可以比当今的体内方法快至少一个数量级来完成。 公共健康相关性：我们建议开发一种完全体外的台式系统，以从头创建纳米抗体，使科学界能够快速生成药物、生物探针和分子工具，用于在自己的实验室进行研究。所提出的想法不需要使用动物，并且可以比当今的体内方法快至少一个数量级来完成。