Platform for the High Throughput Generation and Validation of Affinity Reagents

摘要 我们提供了一个完整的重组单抗快速生成和验证的平台。这个 本方案的创新之处包括：(1)噬菌体通宵转化的帕西纳载体系统 将ScFv或Fab克隆展示为免疫球蛋白分子，(2)Epivolve法分离位点特异性 ABS，(3)将酵母展示物结合到PATENA中以允许结合的生物物理测量 不需要分离蛋白质的亲和力，以及(4)我们用于验证抗体的奶昔技术 用于IHC、WESTERN和EL ISA应用程序。这项提议是--而且在很大程度上是一种集中的 集成由委托人开发(并降低风险)的几项独立技术 调查员(PI)。我们现在希望整合这些技术并将它们添加到一个单一的Abbratech中 站台。本质上，该提案是一组非常强大的分子生物学模块，用于：(I)文库 构建，(Ii)使用噬菌体和酵母展示进行文库筛选，(Iii)生物物理和动力学分析， (Iv)定向进化；(V)亲和成熟(“AffMat”)；(Vi)蛋白质工程；(Vii)产生- 定向抗体，(Viii)整合到单一平台的蛋白质表达，以及(Viii)抗体流动验证 流式细胞术、免疫组化、酶联免疫吸附试验及西方应用。拟议工作流程的所有部分都可以- 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。 建议的平台代表了一种创新的方式来提供重组单抗，理想情况下是零售总量 成本低于数千美元(成本在商业化计划中讨论)。这 准确性、专一性、预计的时间表和成本目前是任何其他商业产品都无法实现的 可用的单抗发现平台。这项建议满足了一种尚未得到明显改善的需求 自从40年前出现了单抗和噬菌体展示，几乎是在同一时间。而且它可以 最后，将噬菌体展示作为多样性的主要引擎，取代了低效和耗时的 即使是噬菌体展示倡导者也依赖的免疫时间表。意义很高，团队 是合格的，创新的，战术的和战略的，是富有想象力的，即使这个范围 第二阶段令人望而生畏，球队已经做好了准备。

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.