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Engineering Gp2 as a small ligand scaffold

将 Gp2 工程化为小型配体支架

基本信息

批准号：
9895785
负责人：
Benjamin Hackel
金额：
$ 31.94万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-01 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9895785
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 Emission-Computed Tomography 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 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 Xenograft procedure 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 programmed cell death ligand 1 scaffold screening targeted agent targeted treatment tumor tumor xenograft

项目摘要

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.

分子识别配体在分子诊断、靶向治疗和生物学研究中起着重要作用。稳健、有效地发现稳定、选择性的亲和配体对许多重要的目标，加快这些领域的发展。尽管有许多支架-从抗体到替代药物，拓扑结构-已经被开发来满足这些需求，但都有局限性。重要的是，在保持生物物理完整性的同时，将结合功能发展到超小支架上，将是一种强大的进步。小尺寸有助于外渗、组织渗透和未结合背景的清除配体用于改善生理性能，特别是用于分子成像。此外，小单结构域促进生产、位点特异性缀合和设计的多功能融合。为此我们我们通过生物信息学方法发现了45个氨基酸的Gp 2结构域，并且我们已经验证了其作为配体的功效，能够强的特异性结合，同时保持稳定性。在此，我们建议这个支架的发展。本研究的目的是设计45-氨基酸的框架和多样的互补位， Gp 2结构域，以提高其作为分子靶向支架的效用，并通过开发 PD-L1的正电子发射断层扫描成像剂。研究计划包括三个目标。（一）先进的组合库设计-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Gp 2中的强特异性结合剂脚手架数千种不同的Gp 2配体将从一个幼稚的组合库中进化出来。深测序将揭示位点氨基酸频率，其将指导第二代文库设计。这些设计将进行比较评估的进化适应性。进化的Gp 2配体将在功能上并进行生物药理学表征。(2)工程化Gp 2框架以增强蛋白水解和热稳定性，溶解性和生理被动性。两个创新的稳定性工程战略将比较更多传统的方法来告知进化和识别改进的Gp 2框架。调制亲水性和电荷将进一步改善Gp 2框架。(3)进行临床前开发 PD-L1的分子PET成像剂，能够进行特异性、灵敏的早期时间点（~1 h）成像。的 Gp 2的高级互补位进化和框架将被应用于开发选择性地体内靶向PD-L1。这些将与用于异种移植中PET成像的抗体和片段进行比较。小鼠肿瘤模型。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Ligand Engineering via Yeast Surface Display and Adherent Cell Panning.

通过酵母表面展示和贴壁细胞淘选进行配体工程。

DOI：
10.1007/978-1-4939-9853-1_17
发表时间：
2020
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
0
作者：
Stern,LawrenceA;Lown,PatrickS;Hackel,BenjaminJ
通讯作者：
Hackel,BenjaminJ

Extended yeast surface display linkers enhance the enrichment of ligands in direct mammalian cell selections.

扩展的酵母表面展示接头增强了直接哺乳动物细胞选择中配体的富集。

DOI：
10.1093/protein/gzab004
发表时间：
2021
期刊：
Protein engineering, design & selection : PEDS
影响因子：
0
作者：
Lown,PatrickS;Cai,JessyJ;Ritter,SethC;Otolski,JacobJ;Wong,Ryan;Hackel,BenjaminJ
通讯作者：
Hackel,BenjaminJ

Engineered Charge Redistribution of Gp2 Proteins through Guided Diversity for Improved PET Imaging of Epidermal Growth Factor Receptor.

通过引导多样性设计 Gp2 蛋白的电荷重新分布，以改善表皮生长因子受体的 PET 成像。