Development of streamlined chemoenzymatic glycan remodeling systems for antibodies and other important glycoproteins

开发抗体和其他重要糖蛋白的简化化学酶聚糖重塑系统

基本信息

批准号：
10459621
负责人：
Qiang Yang
金额：
$ 71.37万
依托单位：
GLYCOT THERAPEUTICS, LLC
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10459621
关键词：
Address Affect Antibodies Award Binding Biological Process Carbohydrates Cell Adhesion Cellulose Chimeric Proteins Communities Complex Development Diagnosis Drug Kinetics Endoglycosidases Engineering Enzymes Escherichia coli Extravasation Glycopeptides Glycoproteins Glycoside Hydrolases Goals Heterogeneity Hybrids Immobilization Immobilized Enzymes Immune response Industrialization Legal patent Libraries Maryland Methods Modification N-terminal Oligosaccharides Pathway interactions Phase Phytolacca dodecandra Polysaccharides Post-Translational Protein Processing Preparation Price Production Property Protein Glycosylation Proteins Protocols documentation Research Research Proposals Site Small Business Innovation Research Grant Streptococcus pyogenes System Testing Therapeutic Therapeutic antibodies Universities base chemical synthesis cost enzyme activity glycosylation immunogenicity in vivo maltose-binding protein mutant novel pathogen phase 1 study phase 2 study scale up success sugar technology development tool tumor progression

项目摘要

Project abstract/summary The objectives of this proposal are to develop efficient and streamlined cheomoenzymatic systems for glycan remodeling of antibodies and other important glycoproteins, through enzyme immobilization, scalable glycan production, and kits development. Such immobilized enzymes, when combined with activated glycan library as kits, can help general academic and industrial users, particularly non-specialists, to prepare glycan-defined glycoproteins for structural and functional studies. The streamlined approach can also be applied in scale-up preparation of homogenous glycoproteins with therapeutic potential. Protein glycosylation is one of the most ubiquitous posttranslational modifications. It profoundly affects a protein’s properties such as folding, in vivo stability, immunogenicity, and pharmacokinetics, and also directly participate in many important biological processes, including cell adhesion, cancer progression, host-pathogen interactions, and immune responses. A major issue in glycoprotein studies is the structural heterogeneity and the difficulty in isolating homogeneous glycoforms for detailed structural and functional studies. Although significant progresses have been made for producing glycan-defined glycoprotein such as total chemical synthesis and biosynthetic pathway engineering in different host expression system, the pure glycoforms that can be achieved are still quite limited. To address this challenge, a novel chemoenzymatic method to produce homogeneous glycoprotein and glycopeptides has been developed recently. This convergent approach consists of two key steps: deglcosylation of glycoproteins with an endoglycosidase and subsequent attachment of a desired activated N-glycan to the protein-GlcNAc acceptor by a novel glycosynthase. This SBIR II application is built on the success of the Phase I study (1R43GM134816-01), where we have successfully finished the immobilization of endoglycosidase S2 (EndoS2) from S. pyogenes and its corresponding glycosynthase mutant EndoS2-D184M. The EndoS2-D184M glycosynthase was immobilized by a site-oriented approach which almost fully retains the activity of enzyme. We have demonstrated that the immobilized enzymes are highly efficient for glycan remodeling of therapeutic antibodies. In this Phase II research, we propose to pursue the following three specific aims. Aim 1 is to expand this immobilization strategy to other glycosidases/glycosynthases to establish a tool box of enzymes for glycan- remodeling. Aim 2 is optimize and scale up our production protocol for different oxazolines and significantly lower their production cost. Aim 3 is to develop glycan remodeling kits and establish scalable glycan remodeling protocols.

项目摘要/总结该提案的目标是开发高效且精简的聚糖化学酶系统通过酶固定化、可扩展的聚糖来重塑抗体和其他重要的糖蛋白此类固定化酶与活化聚糖文库结合时的生产和试剂盒开发。试剂盒，可以帮助一般学术和工业用户，特别是非专业人士，制备聚糖定义的糖蛋白的结构和功能研究也可以应用于放大。具有治疗潜力的同质糖蛋白的制备是最具治疗潜力的方法之一。普遍存在的翻译后修饰深刻影响蛋白质的体内特性，例如折叠。稳定性、免疫原性和药代动力学，也直接参与许多重要的生物学过程过程，包括细胞粘附、癌症进展、宿主-病原体相互作用和免疫反应。糖蛋白研究的一个主要问题是结构异质性和分离的困难尽管在详细的结构和功能研究方面取得了重大进展。用于生产聚糖定义的糖蛋白，例如全化学合成和生物合成途径在不同的宿主表达系统中进行改造，可以获得的纯糖型仍然相当有限。为了应对这一挑战，一种新的化学酶方法来生产均质糖蛋白和糖肽是最近开发出来的，这种聚合方法包括两个关键步骤：去糖基化。糖蛋白与糖苷内切酶的结合，随后将所需的活化 N-聚糖附着到该 SBIR II 应用是建立在该阶段成功的基础上的。我研究（1R43GM134816-01），我们已经成功完成了糖苷内切酶S2的固定化 (EndoS2) 来自化脓性链球菌及其相应的糖合酶突变体 EndoS2-D184M。糖合酶通过定点方法固定，几乎完全保留了酶的活性。已经证明固定化酶对于治疗性药物的聚糖重塑非常有效在这项二期研究中，我们建议追求以下三个具体目标：扩大规模。这种对其他糖苷酶/糖合酶的固定化策略，建立了聚糖酶的工具箱目标 2 是优化和扩大我们不同恶唑啉的生产方案，并显着提高。目标 3 是开发聚糖重塑试剂盒并建立可扩展的聚糖重塑。协议。