Technology to Create Spiegel ERAbodies on Demand: Biostable Universal Antibody Replacements

按需生产Spiegel ERAbodies的技术：生物稳定的通用抗体 替代品 应用分子进化基金会 Elisa Biondi 摘要 生物医学、诊断和临床领域的研究人员希望按需创建（或购买） 与蛋白质和其他可能参与他们正在研究的生物过程的目标结合。抗体 长期以来，它们一直扮演着这一角色。然而，作为生物制品，抗体正处于“不可再生性危机”的中心 在生物医学研究中，即使在合适的时候，也需要几个月和数千美元的时间来制造。这 推动了创建抗体替代品的努力，包括蛋白质（例如Darpins）和RNA（例如适体）。第一 难以操作，而第二种稳定性低，亲和力令人失望。 我们假设一个具有“扩展RNA字母表”（ERA）和额外功能的非天然平台， 具有额外结合潜力的群体将满足这一长期未满足的需求。当扩展的DNA字母表 虽然电子逆向拍卖目前已经取得了进展，但第一项创新是，电子逆向拍卖尚未成为任何初步审查的目标， 数据我们假设，纳摩尔结合将常规实现，因为ERA体将有机会进入 （a）更高的信息密度，这将导致（B）更好地定义折叠，通过使用RNA支架和 具有支持折叠的功能性，（c）更大的结构多样性，其给予ERAbody更多的模式， 紧密结合，和（d）更多的折叠基序，其允许ERA体具有更紧凑的结构。他们是 还假设具有经典适体的所有优点，包括作为 后续几轮进化、使用信号实体的可修改性、低成本、快速周转和直接 化学合成 这个R21项目将证明这个新技术平台的价值，它将允许研究人员订购或 直接创建在几周内，活页夹的目标，他们自己选择的。我们假设一个更进一步的创新 通过将尚未开发的ERA技术与镜像对称的经典概念相结合。创建 ERAbodies在生物系统中是稳定的，我们将使这些在镜像（“明镜”）的形式。 目标1.一个单一的R21示范项目将表明，ERAbodies可以与积木 由钯化学创造。尽管这项技术在应用程序方面是不可知的（它可以创建 任何目标的粘合剂），这一证明将通过靶向具有超大医疗价值的蛋白质产生影响。 PD-L1（程序性死亡配体1）。我们将测试假设，L-ERA试剂，很像 标准L-RNA对生命系统中的RNA酶是稳定的。此工作流程步骤将耗时18个月。 我们将扩展ERA测序方法，并对酶促合成的保真度进行基准测试。在 工作流程，这将是在第6个月内完成，作为各种反音译模式 转录酶将用于定义适用于各种6-和8-字母ERA系统的测序程序。 目标指标是结合亲和力（纳摩尔）、结合特异性（100：1区分）和体外结合特异性（100：1区分）。 血清稳定性（在2小时内<0.1%RNase降解）。

Technology to Create Spiegel ERAbodies on Demand: Biostable Universal Antibody Replacements Foundation for Applied Molecular Evolution Elisa Biondi ABSTRACT Researchers in biomedical, diagnostic, and clinical areas want to create (or buy), on demand, reagents that bind to proteins and other targets that may be involved in a biological process that they are studying. Antibod- ies have long served this role. However, as biologics, antibodies are at the center of an "irreproducibility crisis" in biomedical research, and, even when suitable, take months and thousands of dollars to make. This has driven efforts to create antibody replacements, both protein (e.g. Darpins) and RNA (e.g. aptamers). The first are difficult to manipulate, while the second have low stability and disappointing affinity. We hypothesize that an unnatural platform with an "Expanded RNA Alphabet" (ERA) and extra functional groups with extra binding potential will meet this long-standing unmet need. While expanded DNA alphabets are now advanced, a first innovation is that ERAs have not yet been the target of any preliminary data. We hypothesize that nanomolar binding will be routinely achieved because ERAbodies will have access to (a) higher information density that will lead to (b) better defined folds, both by using an RNA scaffold and by having functionality that supports folding, (c) greater structural diversity that gives ERAbodies more modes for tight binding, and (d) more folding motifs that allow ERAbodies to have more compact structures. They are also hypothesized to have all of the advantages of classical aptamers, including value as the starting points for subsequent rounds of evolution, modifiability using signaling entities, low cost, fast turnaround, and direct chemical synthesis. This R21 project will prove the value of this new technology platform, which will allow researchers to order or directly create in weeks, binders for targets that they themselves select. We hypothesize a further innovation by merging yet unexplored ERA technology with the classical concept of mirror symmetry. To create ERAbodies that are stable in biological systems, we will make these in mirror image ("Spiegel") form. Aim 1. A single R21 demonstration project will show that ERAbodies can be made with building blocks created by palladium chemistry. Although this technology is agnostic with respect to applications (it can create binders for any target), this demonstration will have impact by targeting a protein with outsized medical significance, PD-L1 (Programmed Death Ligand 1). We will test the hypothesis that L-ERA reagents, much like standard L-RNA, are stable against RNases in living systems. This workflow step will consume 18 months. We will expand methods to sequence ERA and benchmark the fidelity of enzymatic synthesis. In the workflow, this will be completed in the first 6 months, as patterns of transliteration by various reverse transcriptases will be used to define a sequencing procedure applicable to various 6- and 8-letter ERA systems. The target metrics are binding affinity (nanomolar), binding specificity (100:1 discrimination), and in vitro sera stability (<0.1% RNase degradation over 2 hours).