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.

分子识别配体对于分子诊断，靶向治疗和生物学研究至关重要。强大的，有效地发现稳定的选择性亲和力配体对许多重要目标加速这些领域的进步。虽然有许多脚手架 - 从抗体到替代品拓扑 - 已经开发出来满足这些需求，都有局限性。重要的是，有效的能力在保留生物物理完整性的同时，进化的结合功能在超小脚手架上，将是一个强大的进步。小尺寸有助于渗出，组织穿透和无绑背的清除率配体可改善身体性能，尤其是用于分子成像的配体。而且，小单曲域最喜欢的生产，特定地点的共轭和设计师多功能融合。为此，我们已经通过生物信息学方法发现了45个氨基酸GP2域，我们已经验证了它作为能够具有强，特异性结合的配体的功效，同时保持稳定性。在此，我们建议进步这种脚手架的发展。这项研究的目的是设计45个氨基酸的框架和潜水副群 GP2域以推动其作为分子靶向支架的效用，并通过发展来体现效用 PD-L1的正电子发射断层扫描剂。研究计划包括三个目标。（1）高级组合图书馆设计 - 具有通过高通量配体鉴定的站点梯度的多样性梯度进化和深度测序反馈 - 以便在GP2中直接选择强，特定的粘合剂脚手架。成千上万的潜水员GP2配体将从幼稚的组合文库中进化。深的测序将揭示将指导第二代库设计的现场氨基酸频率。这些设计将对进化适应性进行比较评估。进化的GP2配体在功能上将是并具有生物物理特征。（2）设计GP2框架以增强蛋白水解和热稳定性，溶解度和物理消极。将两种创新的稳定工程策略与更多传统方法为进化提供信息并确定改进的GP2框架。调制亲水性和电荷将进一步改善GP2框架。（3）执行临床前发展 PD-L1的分子PET成像剂，能够具有特定敏感的早期时间点（〜1 h）成像。这 GP2的高级副副膜的演变和框架将用于开发5 kDa域，有选择地靶PD-L1体内。这些将与异种移植中的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。