Platform for the High Throughput Generation and Validation of Affinity Reagents

摘要 我们提出了一个完整的平台，用于重组单克隆抗体的快速生成和验证。的 该建议的创新包括：（1）用于噬菌体过夜转化的pATHENA载体系统 将scFv或Fab克隆展示到IgG分子中，（2）Epivolve方法用于分离位点特异性 Abs，（3）将酵母展示并入pATHENA中以允许结合的生物物理测量 亲和性，而无需蛋白质分离，和（4）我们的MILKSHAKE技术用于抗体的验证 用于IHC、Western和ELISA应用。这一提议是一个高潮，主要是一个重点 集成由委托人开发（并降低风险）的多项独立技术 研究者（PI）。我们现在希望将这些技术整合到一个Abbratech中 平台本质上，该提案是一套非常强大的分子生物学模块，用于： 构建，（ii）使用噬菌体和酵母展示的文库筛选，（iii）生物物理和动力学分析， (iv)定向进化，（v）亲和力成熟（“AffMat”），（vi）蛋白质工程，（vii）位点- 定向Ab，（viii）整合到单个平台中的蛋白表达，和（viii）用于流动的Ab验证 流式细胞术、ICC、ELISA和Western应用。建议的工作流程的所有部分都可以是- 或者已经通过现成的机器人解决方案实现了高通量（HT）生产的自动化。 拟议的平台代表了一种创新的方式来提供重组单克隆抗体，在理想的零售总额 成本低于几千美元（成本在商业化计划中讨论）。这 准确性、特异性、预计时间轴和成本目前无法通过任何其他商业方法实现 mAb发现平台。这项建议满足了一个尚未得到明显改进的需要 自从四十年前单克隆抗体和噬菌体展示的出现以来，大约在同一时间。并且它可以 最终将噬菌体展示作为多样性的主要引擎，取代低效和耗时的 甚至噬菌体展示的拥护者也会依赖的免疫时间表。研究小组认为， 是合格的，创新，战术和战略，是富有想象力的，即使这一范围 第二阶段是令人生畏的，团队有能力做到这一点。

ABSTRACT We present a complete platform for the rapid generation and validation of recombinant mAbs. The innovations of this proposal include: (1) the pATHENA vector system for overnight conversion of phage display scFv or Fab clones into IgG molecules, (2) the Epivolve method for the isolation of site-specific Abs, (3) the incorporation of yeast display into pATHENA to allow biophysical measurement of binding affinities without the need for protein isolation, and (4) our MILKSHAKE technology for the validation of Abs for IHC, Western, and ELISA applications. This proposal is a culmination of- and largely a focused integration of several independent technologies that have been developed (and de-risked) by the principal investigator (PI). We now want to incorporate these technologies and add them into a single Abbratech platform. In essence, the proposal is a very robust set of molecular biology modules for: (i) library construction, (ii) library screening using both phage- and yeast display, (iii) biophysical and kinetic analysis, (iv) directed evolution, (v) affinity maturation (“AffMat”), (vi) protein engineering, (vii) production of site- directed Abs, (viii) protein expression integrated into a single platform, and (viii) Ab validation for flow cytometry, ICC, ELISA and Western applications. All parts of the proposed workflow have either can be- or have already been automated for high-throughput (HT) production with off-the-shelf robotic solutions. The proposed platform represents an innovative way to deliver recombinant mAbs, at ideally a retail total cost of less than several thousand dollars (costs are discussed in the Commercialization plan). This accuracy, specificity, projected timeline, and cost cannot be currently achieved by any other commercially available mAb discovery platform. This proposal meets a need that has not been markedly improved upon since the advent of monoclonal Abs forty years ago and phage display, around the same time. And it can finally place phage display as a primary engine of diversity, replacing the inefficient and time-consuming immunization schedules that even phage display advocates fall back on. The significance is high, the team is well-qualified, the innovation, tactical and strategic, is imaginative, and even though the scope of this Phase II is daunting, the team is well-equipped to do it.