Engineering Gp2 as a small ligand scaffold

分子识别配体对分子诊断、靶向治疗和生物学研究具有重要意义。 稳健、高效地发现稳定的、选择性的、针对众多重要靶标的亲和配体将 加快推进这些领域的建设。尽管有许多支架-从抗体到替代 拓扑-已开发来满足这些需求，但都有局限性。重要的是，能够高效地 将绑定功能进化到超小型支架上，同时保持生物物理完整性，将是一种 强有力的进步。小尺寸有助于渗出、组织穿透和清除未结合的背景 改善生理性能的配体，特别是用于分子成像的。此外，小单曲 结构域有助于生产、特定部位的结合和设计者的多功能融合。为此，我们 已经通过生物信息学的方法发现了45个氨基酸的GP2结构域，并且我们已经验证了它 作为一种配基，在保持稳定性的同时，能够强烈地、特异地结合。在此，我们建议推进 这种脚手架的发展。 这项研究的目的是设计45个氨基酸的框架和不同的对位。 GP2结构域作为分子靶向支架的应用前景及发展前景 PD-L1正电子发射断层显像剂。该研究计划包括三个目标。(1) 高级组合文库设计--通过高通量配基识别多样性的位置梯度 进化和深度测序反馈-支持直接选择GP2中的强、特定结合剂 脚手架。数以千计不同的GP2配体将从一个天真的组合库进化而来。深沉 测序将揭示不同部位的氨基酸频率，这将指导第二代文库设计。 这些设计将进行进化适应性的比较评估。进化的GP2配体将在功能上 并具有生物物理学特征。(2)改造GP2框架以提高蛋白质分解和热稳定性， 溶解性和生理被动性。两种创新的稳定性工程策略将与更多 通知演进和确定改进的GP2框架的传统方法。调制方式 亲水性和带电性将进一步完善GP2框架。(3)进行临床前开发 用于PD-L1的分子PET显像剂能够特异、灵敏地进行早期时间点(~1h)的成像。这个 将应用先进的对位基因进化和GP2框架来开发具有选择性的5 kDa结构域 体内靶向PD-L1。这些将与用于异种移植的PET成像的抗体和片段进行比较 小鼠肿瘤模型。

Molecular recognition ligands are critical for molecular diagnostics, targeted therapy, and biological study. Robust, efficient discovery of stable, selective affinity ligands towards the multitude of important targets would accelerate advances in these fields. Though numerous scaffolds – ranging from antibodies to alternative topologies – have been developed to fill these needs, all have limitations. Importantly, the ability to efficiently evolve binding functionality onto an ultra-small scaffold, while retaining biophysical integrity, would be a powerful advance. Small size aids extravasation, tissue penetration, and clearance of unbound background ligand for improved physiological performance, particularly for molecular imaging. Moreover, small single domains facilitate production, site-specific conjugation, and designer multi-functional fusions. To this end, we have discovered the 45-amino acid Gp2 domain via a bioinformatics approach, and we have validated its efficacy as a ligand capable of strong, specific binding while retaining stability. Herein, we propose to advance development of this scaffold. The objective of this research is to engineer the framework and diverse paratope of the 45-amino acid Gp2 domain to advance its utility as a molecular targeting scaffold and exemplify utility by development of positron emission tomography imaging agents for PD-L1. The research plan consists of three aims. (1) Advance combinatorial library design – with a sitewise gradient of diversity identified via high-throughput ligand evolution and deep sequencing feedback – to enable direct selection of strong, specific binders in the Gp2 scaffold. Thousands of diverse Gp2 ligands will be evolved from a naïve combinatorial library. Deep sequencing will reveal sitewise amino acid frequencies that will guide second-generation library designs. These designs will be comparatively evaluated for evolutionary fitness. Evolved Gp2 ligands will be functionally and biophysically characterized. (2) Engineer the Gp2 framework to enhance proteolytic and thermal stability, solubility, and physiological passivity. Two innovative stability-engineering strategies will be compared to more conventional approaches to inform evolution and identify an improved Gp2 framework. Modulation of hydrophilicity and charge will further improve the Gp2 framework. (3) Perform preclinical development of molecular PET imaging agents for PD-L1 capable of specific, sensitive early time point (~1 h) imaging. The advanced paratope evolution and framework of Gp2 will be applied to develop 5 kDa domains that selectively target PD-L1 in vivo. These will be compared to antibodies and fragments for PET imaging in xenografted mouse tumor models.