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CAREER: Generation of Highly Selective Inhibitory Antibodies by Novel Paratope Design, Function-Based Screening, and Deep Sequencing

职业：通过新型互补位设计、基于功能的筛选和深度测序生成高选择性抑制抗体

基本信息

批准号：
1453645
负责人：
Xin Ge
金额：
$ 50万
依托单位：
University of California-Riverside
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2021-06-30
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1453645&HistoricalAwards=false
关键词：
CAREER Generation Highly Selective Inhibitory

项目摘要

1453645Ge, Xin This research will significantly advance our understanding on the mechanisms of enzymatic inhibition and virus neutralization, and thus will lead to development of a panel of novel methods for the generation of highly potent inhibitory antibodies for a broad range of applications in pharmaceutical and biotechnological industries. Specifically, the work aims to develop antibodies that interfere and inhibit the binding of protein cleaving enzymes or proteases. The project will (i) increase US technological competitiveness; (ii) develop a globally competitive STEM workforce; (iii) increase participation of women and underrepresented minorities; and (iv) contribute to undergraduate and graduate STEM education. UC Riverside is the minority serving institution with the largest Hispanic student population among all UC campuses. More specifically, the PI plans to first prepare UCR STEM undergraduates for a global marketplace and dynamic scientific communities through collaboration with two Chinese universities; and second increase university/college enrollment of high school graduates in Riverside and San Bernardino Counties, especially those from minority and other disadvantaged socio-economic backgrounds, by inspiring their interests in science and engineering programs through lectures, workshops and interacting events. As extremely important signaling molecules, proteases precisely control a wide variety of physiological processes, and thus represent one of the largest families of potential pharmaceutical targets. Considering that ~2% of the human genome is estimated to encode proteases, specificity is highly desired for any protease inhibition therapy. However, proteases share high amino acid similarity among the same class of proteases and their active sites are extensively conserved. It has been a challenging task to develop small molecule inhibitors that can deliver required specificities. Therefore, antibodies are emerging as a very attractive alternative for highly selective inhibition. To date, at least three obstacles make the routine discovery of protease-inhibiting monoclonal antibodies (mAbs) considerably difficult: (i) low antigenicity of the proteolytic active sites, (ii) lack of a function-based selection method, and (iii) loss of beneficial clones during the selection. The long-term goal of this CAREER award is to develop therapeutic monoclonal antibodies (mAbs) or biologics that inhibit specific proteases for biomedical applications. The objective of this research is to overcome the technical hurdles and establish general methodologies that facilitate the identification of inhibitory antibodies. The hypothesis is that convex antigen-binding sites (paratopes) are inhibition-prone. This central hypothesis will be tested by the following three specific approaches: (1) Clearly verify inhibition mechanisms by design, construction and optimization of synthetic human antibody libraries enriched with convex paratopes; (2) Efficiently identify inhibitory antibodies by developing a function-based high-throughput screening method; (3) Systematically understand sequence-inhibition landscapes by deep sequencing and data mining.

1453645葛鑫这项研究将大大推进我们对酶抑制和病毒中和机制的理解，从而将导致开发一组新方法，用于产生在制药和生物技术行业广泛应用的高效抑制性抗体。具体来说，这项工作旨在开发干扰和抑制蛋白质切割酶或蛋白酶结合的抗体。该项目将（i）提高美国的技术竞争力;（ii）发展具有全球竞争力的STEM劳动力;（iii）增加妇女和代表性不足的少数民族的参与;（iv）促进本科和研究生STEM教育。加州大学滨江分校是少数族裔服务机构，拥有加州大学所有校区中最大的西班牙裔学生人口。更具体地说，PI计划首先通过与两所中国大学的合作，为UCR STEM本科生进入全球市场和充满活力的科学界做好准备;第二，通过讲座激发他们对科学和工程项目的兴趣，增加滨江县和圣贝纳迪诺县高中毕业生的大学/学院入学率，特别是那些来自少数民族和其他弱势社会经济背景的高中毕业生，研讨会和互动活动。蛋白酶作为极其重要的信号分子，精确地控制各种生理过程，因此代表了潜在药物靶标的最大家族之一。考虑到估计约2%的人类基因组编码蛋白酶，特异性对于任何蛋白酶抑制疗法都是高度期望的。然而，蛋白酶在同类蛋白酶中具有高度的氨基酸相似性，并且它们的活性位点是广泛保守的。开发能够提供所需特异性的小分子抑制剂一直是一项具有挑战性的任务。因此，抗体正在成为高度选择性抑制的非常有吸引力的替代方案。迄今为止，至少有三个障碍使得蛋白酶抑制性单克隆抗体（mAb）的常规发现相当困难：（i）蛋白水解活性位点的低抗原性，（ii）缺乏基于功能的选择方法，以及（iii）在选择期间损失有益克隆。该CAREER奖项的长期目标是开发治疗性单克隆抗体（mAb）或生物制剂，抑制生物医学应用中的特定蛋白酶。本研究的目的是克服技术障碍，建立通用的方法，促进抑制性抗体的鉴定。假设是凸抗原结合位点（互补位）易于抑制。这一中心假设将通过以下三种具体方法进行验证：（1）通过设计、构建和优化富含凸互补位的合成人抗体文库，明确验证抑制机制;（2）通过开发基于功能的高通量筛选方法，有效鉴定抑制性抗体;（3）通过深度测序和数据挖掘，系统地了解序列抑制景观。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Active-site MMP-selective antibody inhibitors discovered from convex paratope synthetic libraries

DOI：
10.1073/pnas.1609375114
发表时间：
2016-12-27
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
影响因子：
11.1
作者：
Dong Hyun Nam;Rodriguez, Carlos;Ge, Xin
通讯作者：
Ge, Xin

Direct production of functional matrix metalloproteinase-14 without refolding or activation and its application for in vitro inhibition assays: Periplasmic Production of MMP

无需重折叠或激活的功能性基质金属蛋白酶-14的直接生产及其在体外抑制测定中的应用：MMP的周质生产