A novel pre-defined CDR library for selection of affinity reagents

 说明(申请人提供)：最常用的产生抗体的方法是对动物进行免疫。然而，这种方法通常是低通量、昂贵、耗时的，并且产生的抗体并不总是可再生的。重组抗体(RAb)与单链可变区(ScFv)一样，与杂交瘤产生的多克隆抗血清和单抗相比，具有许多吸引人的特性。它们可以通过在适当的异源宿主中过度表达而再生，它们很容易以DNA的形式存储和转移，它们可以作为各种酶、荧光蛋白和表位标签的融合基因工程。虽然生产重组亲和试剂的方法有很多，但当前技术的成本和产量是开发全面和广泛可用的可再生亲和试剂资源的重要障碍。我们相信，基因合成技术的改进和高通量DNA测序的可负担性的提高可以被用来创建基于合成生物学的抗体发现管道，与动物免疫系统相竞争。在这份提案中，我们将在短短21天内建成一条高通量的重组抗体开发管道。建议的平台利用预先设计的多样性、下一代序列分析和先进的分子生物学技术来实现特定抗体的快速鉴定。虽然我们的筛选平台是用单链可变区抗体(ScFv)开发的，但该技术既适用于Fab，也适用于酵母展示文库。单链抗体高通量转化为完整的免疫球蛋白G(IgG)将被整合到管道中，这样抗体就可以直接以所需的最终形式进行验证。在基因组学中，人们认为1000美元的基因组将是全基因组测序变得司空见惯的拐点。但即使每个基因组10,000美元，研究人员的吸收也是惊人的，使用下一代测序仪的新方法，如芯片序列、RNA-序列等都被发明了。以类似的方式，我们相信，在5000美元和4周的时间里，研究人员将开始开发新的应用程序，其中生产抗体的成本不再是一个相关因素，速度成为一切。低成本、高质量亲和试剂的现成将潜在地加速基础科学研究的方方面面，为 疾病生物标志物，并作为治疗概念的证明。就像1000美元的基因组一样，我们认为抗体鉴定和生产最终可以达到100美元以下，不到2周。在这一点上，对许多不同生物体和疾病状态的完整蛋白质组分析将成为可能。而新的方法将会出现。

 DESCRIPTION (provided by applicant): The most commonly used method for generating antibodies is through immunization of animals. However, this method is generally low-throughput, expensive, time-consuming, and the antibodies generated are not always renewable. Recombinant antibodies (rAb), like single chain variable fragments (scFv), have many attractive attributes compared to polyclonal antisera and monoclonal antibodies derived from hybridomas. They are renewable through overexpression in the appropriate heterologous host, they are easily stored and transferred as DNA, and they can be genetically engineered as fusions to various enzymes, fluorescent proteins, and epitope tags. While a number of approaches for generating recombinant affinity reagents exist, the cost and throughput of current technologies represent significant roadblocks to the development of a comprehensive and broadly available resource of renewable affinity reagents. We believe that improvements to both gene synthesis technologies and the increased affordability of high-throughput DNA sequencing can be leveraged to create antibody discovery pipelines based on synthetic biology that rival animal immune systems. In this proposal, we will build a high-throughput pipeline for recombinant antibody development in as few as 21 days. The proposed platform takes advantage of pre-designed diversity, next-generation sequence analysis, and advanced molecular biology techniques to enable the rapid identification of specific antibodies. Although our screening platform is being developed with single-chain variable fragment antibodies (scFvs), the technology is applicable to both Fab and yeast display libraries. High-throughput conversion of the scFvs to full immunoglobulin G (IgG) will be integrated within the pipeline so that the antibodies can be directly validated in the desired final format. In genomics, it was thought that the $1000 genome would be the inflection point at which whole genome sequencing would become commonplace. But even at $10,000 per genome, researcher uptake was phenomenal and new ways of using the NextGen sequencers, like ChIP Seq, RNA-seq, etc. were invented. In a similar fashion, we believe that at $5000 and 4 weeks, researchers will begin to develop new applications where the cost of producing antibodies is no longer a relevant factor and speed becomes everything. The ready availability of low cost, high quality affinity reagents will potentially accelerate all aspects of basic science research, provide diagnostics for disease biomarkers, and serve as a proof of concept for therapy. Like the $1000 genome, we think antibody identification and production can eventually go to under $100 and less than 2 weeks. At that point, whole proteome analyses for many different organisms and disease states will be possible. And new methods will arise.