Engineering Gp2 as a small ligand scaffold

将 Gp2 工程化为小型配体支架

• 批准号: 9219734
• 负责人: Benjamin Hackel
• 金额: $ 31.8万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9219734
• 关键词: Address; Advanced Development; Affinity; Amino Acids; Antibodies; Antibody Binding Sites; Area; Binding; Biodistribution; Bioinformatics; Biological; Biological Markers; Biology; Biophysics; Charge; Chemicals; Clinical; Consensus; Development; Engineering; Equilibrium; Evaluation; Evolution; Extravasation; Feedback; Frequencies; GP2 gene; Generations; Human; Image; Immunoglobulin Fragments; Lead; Libraries; Ligand Binding; Ligands; Modeling; Molecular; Molecular Target; Mus; Outcome; PDCD1LG1 gene; Penetration; Performance; Phylogenetic Analysis; Physiological; Positron-Emission Tomography; Production; Proteins; Research; Sensitivity and Specificity; Site; Solubility; Solvents; Surface; Testing; Time; Tissues; Variant; X-Ray Computed Tomography; combinatorial; comparative; cross reactivity; deep sequencing; design; fitness; hydrophilicity; imaging agent; immune checkpoint; improved; in vivo; innovation; molecular diagnostics; molecular imaging; molecular recognition; mutant; preclinical development; scaffold; screening; targeted agent; targeted treatment; tumor

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.

分子识别配体对于分子诊断、靶向治疗和生物学研究至关重要。 针对众多重要靶标的稳定、选择性亲和配体的稳健、有效发现将 加速这些领域的进步。尽管支架数量众多——从抗体到替代品 拓扑——是为了满足这些需求而开发的，但都有局限性。重要的是，能够高效地 将结合功能发展到超小支架上，同时保留生物物理完整性，将是一个 强大的推进。小尺寸有助于外渗、组织渗透和未结合背景的清除 配体可改善生理性能，特别是分子成像。而且，小单 结构域有利于生产、位点特异性缀合和设计师多功能融合。为此，我们 通过生物信息学方法发现了 45 个氨基酸的 Gp2 结构域，并且我们已经验证了其 作为配体的功效，能够强、特异性结合，同时保持稳定性。在此，我们建议推进 该脚手架的开发。 本研究的目的是设计 45 个氨基酸的框架和多样化互补位 Gp2 结构域可提高其作为分子靶向支架的实用性，并通过开发来举例说明实用性 PD-L1 的正电子发射断层扫描显像剂。该研究计划包括三个目标。 (1) 先进的组合文库设计——通过高通量配体识别位点多样性梯度 进化和深度测序反馈——能够直接选择 Gp2 中强、特异性的结合物 脚手架。数以千计的不同 Gp2 配体将从一个简单的组合库中进化而来。深的 测序将揭示位点氨基酸频率，从而指导第二代文库设计。 这些设计将针对进化适应性进行比较评估。进化的 Gp2 配体将在功能上 和生物物理特征。 (2) 设计 Gp2 框架以增强蛋白水解和热稳定性， 溶解度和生理被动性。两种创新的稳定性工程策略将与更多的策略进行比较 为进化提供信息并确定改进的 Gp2 框架的传统方法。调制 亲水性和电荷将进一步改善Gp2框架。 (3) 进行临床前开发 PD-L1 分子 PET 显像剂能够进行特异性、灵敏的早期时间点（~1 小时）成像。这 先进的互补位进化和 Gp2 框架将用于开发 5 kDa 结构域，选择性地 体内靶向 PD-L1。这些将与异种移植物中 PET 成像的抗体和片段进行比较 小鼠肿瘤模型。