Administrative Supplement: Activity-based Discovery and Optimization

项目摘要 激动剂抗体能够通过在细胞表面的相互作用引发细胞信号传导事件， 作为疾病治疗的新范例出现，在临床试验中有几个有前途的候选人，包括T 细胞激活抗体。然而，具有特定生物学功能的罕见抗体的发现， 仍然是生物技术的主要瓶颈。为了解决这个问题，我试图采用直接基于功能的 利用哺乳动物细胞内报告基因的新兴生物医学技术抗体筛选方法 最近已经显示了用于发现激动剂抗体的概念验证的系统。我相信 这项创新技术如果能够推广，将具有革命性的潜力。因此， 本研究旨在建立新的基础方法，以广泛应用于基于功能的抗体 筛查，这可能最终为推进疾病治疗奠定基础。我最近 开发了新的模型系统，能够通过NF-κ B途径稳健地检测细胞内信号传导 从T细胞受体Ox40和CD137的细胞外活化开始。这些受体被选择用于 它们的明确定义的对照激动剂抗体的可用性对于系统验证是无价的。我建议 使用这些模型系统，以获得有关的基本机制和原则的新认识 在基于功能的抗体筛选，并利用这一知识对新的生物医学的发展， 技术和新疗法通过三个不同的目标。首先，我建议批判性地评估 在生物活性、抗体展示水平、受体展示水平和抗体分子形式之间， 确定最终提高激动剂抗体发现效率的筛选方法。二我 建议开发新的Ox40和CD137报告细胞系统，用于简化和改进基于功能的 通过基因工程的自催化激动剂抗体表达的抗体发现 受体特异性细胞内信号传导，以便通过简化信号传导和增强激动剂抗体发现。 检测和放大真正的阳性信号。第三，寻找新的Ox40和CD137激动剂， 通过直接基于功能的筛选获得具有改进活性的抗体。为了实现这一目标，我将 计算方法来告知文库设计，以增加文库变体 有效地接合活性位点附近的T细胞受体。我会用下一代测序技术 领先的候选人，然后将通过经典的T细胞活化试验进行严格的评估。最后要 严格分析与激动剂活性相关的前导序列，以发现潜在的分子 激动剂活性的决定因素和结构-功能关系。总的来说，拟议的研究具有 考虑到细胞信号传导不足涉及广泛的治疗影响， 病理学包括伤口愈合不足、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.