Administrative Supplement: Activity-based Discovery and Optimization

项目总结 激动剂抗体能够通过细胞表面的相互作用启动细胞信号事件，具有 成为疾病治疗的新范例，临床试验中有几个很有希望的候选者，包括T 细胞激活抗体。然而，具有特定生物学功能的稀有抗体的发现 仍然是生物技术的一个主要瓶颈。为了解决这个问题，我寻求使用直接基于函数的 抗体筛选方法--一种使用哺乳动物细胞内报告的新兴生物医学技术 最近发现激动剂抗体的概念验证系统。我相信 如果这项创新技术能够推广，它就有可能成为革命性的。因此，目标是 本研究旨在为基于功能的抗体的广泛应用建立新的基本方法 筛查，这最终可能为推进疾病治疗奠定基础。我最近有过 开发了新的模型系统，能够通过NF-κB途径强大地检测细胞内信号 起始于T细胞受体OX40和CD137的细胞外激活。这些受体是为 他们可获得定义明确的控制激动剂抗体，这对系统验证是无价的。我提议 使用这些模型系统对所涉及的基本机制和原理有新的理解 在基于功能的抗体筛选中，并将这一知识用于开发新的生物医学 技术和新疗法通过三个不同的目标。首先，我建议对两国关系进行批判性评估 生物学活性、抗体展示水平、受体展示水平和抗体分子形式之间的关系 确定最终提高激动剂抗体发现效率的筛选方法。第二，我 建议开发新的OX40和CD137报告细胞系统，以简化和改进基于功能的系统 通过基因工程发现抗体依赖于自身催化激动剂抗体的表达 受体特异的细胞内信号转导，以通过简化信号来改进激动剂抗体的发现 检测和放大真正信号。第三，我寻求寻找新的OX40和CD137激动剂 通过直接基于功能的筛选，获得活性提高的抗体。为了实现这个目标，我将聘请 为库设计提供信息的计算方法，以增加库变体 有效地激活活性部位附近的T细胞受体。我将使用下一代测序来识别 然后，将通过经典的T细胞激活分析对其进行严格评估。最后，我会 严格分析前导序列与激动剂活性的关系，以发现潜在的分子 激动剂活性和结构-功能关系的决定因素。总体而言，拟议的研究具有 潜在的广泛的治疗影响，因为细胞信号不充分涉及广泛的 病理包括伤口愈合不足、自身免疫性疾病和癌症。

PROJECT SUMMARY Agonist antibodies, capable of initiating cellular signaling events through interaction at the cell surface, have emerged as a new paradigm in disease treatment with several promising candidates in clinical trials including T cell activating antibodies. However, the discovery of rare antibodies possessing specific biological functions remains a major biotechnological bottleneck. To address this issue, I seek to employ direct function-based antibody screening methods, an emergent biomedical technology using mammalian intracellular reporter systems for which proof-of-concept has recently been shown for the discovery of agonist antibodies. I believe that this innovative technology has the potential to be revolutionary if it can be generalized. Thus, the goal of the proposed research is to establish new fundamental methods for broad use in function-based antibody screening, which could ultimately lay the foundation for advancing disease treatment. I have recently developed novel model systems that enable robust detection of intracellular signaling via the NF-κB pathway initiated from extracellular activation of the T-cell receptors Ox40 and CD137. These receptors were chosen for their availability of well-defined control agonist antibodies that are invaluable for system validation. I propose to use these model systems to gain new understanding of the fundamental mechanisms and principles involved in function-based antibody screening and employ this knowledge toward the development of new biomedical technology and novel therapeutics via three distinct aims. First, I propose to critically evaluate the relationship between biological activity, antibody display level, receptor display level, and antibody molecular format to identify screening methods that ultimately improve the efficiency of agonist antibody discovery. Second, I propose to develop new Ox40 and CD137 reporter cell systems for simplified and improved function-based antibody discovery via genetic engineering of autocatalytic agonist antibody expression that depends on receptor-specific intracellular signaling in order to improve agonist antibody discovery by both simplifying signal detection and amplifying true positive signals. Thirdly, I seek to identify new Ox40 and CD137 agonist antibodies with improved activity by direct function-based screening. Toward this goal, I will employ computational approaches to inform library design in order to increase the likelihood that library variants effectively engage T cell receptors near the active site. I will employ next-generation sequencing to identify lead candidates, which will then be rigorously evaluated via classic T cell activation assays. Finally, I will critically analyze lead sequences in relationship to agonist activity in order to uncover potential molecular determinants of agonist activity and structure-function relationships. Overall, the proposed research has the potential for broad therapeutic impact given that insufficient cellular signaling is involved in a wide range of pathologies including insufficient wound healing, autoimmune disease and cancer.