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

按需创建 Spiegel ERAbodies 的技术：生物稳定的通用抗体替代品

基本信息

批准号：
10510985
负责人：
Elisa Biondi
金额：
$ 23.18万
依托单位：
FOUNDATION FOR APPLIED MOLECULAR EVOLUTN
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-20 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10510985
关键词：
Affinity Amino Acids Anions Antibodies Area Attention Behavior Benchmarking Binding Binding Proteins Biological Biological Process Biological Products Biomedical Research Cancer Center Categories Charge Chemistry Clinical Consumption DNA Data Diagnostic Digestion Discrimination Disease Evolution Extracellular Protein Foundations Future Genetic Goals Gold Head Hour Image Immune system In Vitro Label Laboratories Letters Ligands Medical Methods Modification Molecular Evolution Nobel Prize Nucleotides Organism Palladium Patients Pattern Peptides Performance Plague Polymerase Problem Solving Procedures Process Proteins RNA RNA analysis RNA-Directed DNA Polymerase Reagent Reproducibility Research Research Personnel Ribonucleases Role Signal Transduction Specificity Structure System Techniques Technology Testing Therapeutic Therapeutic Agents Time Work aptamer base biological systems cancer cell chemical synthesis clinical practice cost density design functional group high risk innovation interest meetings nanomolar new technology overexpression prevent programmed cell death ligand 1 receptor scaffold technology development tool

项目摘要

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).

按需创建 Spiegel ERAbodies 的技术：生物稳定的通用抗体替换品应用分子进化基金会伊丽莎·比昂迪抽象的生物医学、诊断和临床领域的研究人员希望按需创建（或购买）试剂与他们正在研究的生物过程中可能涉及的蛋白质和其他目标结合。抗体- 长期以来，它们一直扮演着这一角色。然而，作为生物制剂，抗体正处于“不可重复性危机”的中心在生物医学研究中，即使在合适的情况下，也需要数月和数千美元才能完成。这有推动创建抗体替代品的努力，包括蛋白质（例如 Darpins）和 RNA（例如适体）。第一个难以操纵，而第二个稳定性低且亲和力令人失望。我们假设一个具有“扩展 RNA 字母表”（ERA）和额外功能的非自然平台具有额外结合潜力的群体将满足这一长期未得到满足的需求。扩展DNA字母表的同时现在已经很先进了，第一个创新是 ERA 尚未成为任何初步研究的目标数据。我们假设纳摩尔结合将经常实现，因为 ERAbodies 将能够访问通过使用 RNA 支架和通过 (a) 更高的信息密度，将导致 (b) 更明确的折叠具有支持折叠的功能，(c) 更大的结构多样性，为 ERAbody 提供更多模式紧密结合，以及（d）更多折叠图案，使 ERAbody 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，包括作为起点的价值后续几轮演进、使用信令实体的可修改性、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，这将允许研究人员订购或直接在几周内为他们自己选择的目标创建活页夹。我们假设进一步的创新通过将尚未探索的 ERA 技术与镜像对称的经典概念相结合。创造在生物系统中稳定的ERAbody，我们将以镜像（“Spiegel”）形式制作它们。目标 1. 单个 R21 演示项目将表明 ERAbodies 可以用构建模块来制造由钯化学创建。尽管该技术与应用程序无关（它可以创建任何目标的结合剂），该演示将通过以超大的医疗蛋白质为目标来产生影响意义，PD-L1（程序性死亡配体 1）。我们将检验 L-ERA 试剂的假设，就像标准 L-RNA 对生命系统中的 RNase 稳定。此工作流程步骤将耗时 18 个月。我们将扩展 ERA 测序方法并对酶合成的保真度进行基准测试。在工作流程，这将在前 6 个月内完成，因为音译模式通过各种反向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 区分度）和体外血清稳定性（2 小时内 RNase 降解<0.1%）。