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

• 批准号: 10324865
• 负责人: Qiang Yang
• 金额: $ 77.49万
• 依托单位: GLYCOT THERAPEUTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10324865
• 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; Structure; System; Testing; Therapeutic; Therapeutic antibodies; Universities; base; chemical synthesis; cost; enzyme activity; glycoprotein structure; 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

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/概要 本提案的目的是开发高效和简化的聚糖酶系统 通过酶的固定化，抗体和其他重要糖蛋白的重塑，可扩展的聚糖 生产和试剂盒开发。这种固定化酶，当与活化聚糖文库组合时， 试剂盒，可以帮助一般的学术和工业用户，特别是非专业人士， 用于结构和功能研究的糖蛋白。简化方法也可用于扩大规模 具有治疗潜力的同质糖蛋白的制备。蛋白质糖基化是最重要的 普遍存在的翻译后修饰。它深刻地影响了蛋白质的性质，如折叠，在体内 稳定性、免疫原性和药代动力学，并且还直接参与许多重要生物学 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。 糖蛋白研究中的一个主要问题是结构的异质性和分离的困难 用于详细的结构和功能研究。虽然取得了重大进展， 用于生产聚糖定义的糖蛋白，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工程化，可以实现的纯糖型仍然相当有限。 为了解决这一挑战，一种新的化学酶法来生产同质糖蛋白和 糖肽是最近开发出来的。这种聚合方法包括两个关键步骤： 然后将所需的活化的N-聚糖连接到糖蛋白上， 蛋白质-GlcNAc受体通过一种新的糖合酶。SBIR II应用程序是建立在 本研究（1 R43 GM 134816 -01）成功地完成了内切糖苷酶S2的固定化 （EndoS 2）从S.化脓性链球菌及其相应的糖合酶突变体EndoS 2-D184 M。EndoS2-D184M 通过位点定向的方法固定化糖合酶，几乎完全保留了酶的活性。我们 已经证明，固定化酶对于治疗剂的聚糖重塑是高效的。 抗体的在第二阶段的研究中，我们提出了以下三个具体目标。目标1：扩大 该固定策略与其它糖苷酶/糖苷酶结合以建立用于聚糖的酶的工具箱， 重塑目标2是优化和扩大我们的生产方案，为不同的恶唑啉和显着 降低生产成本。目的3是开发糖链重构试剂盒，建立可扩展的糖链重构模型 协议.