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: 9978120
• 负责人: Qiang Yang
• 金额: $ 15万
• 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 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978120
• 关键词: Address; Adopted; Affect; Antibodies; Biological Process; Cell Adhesion; Chitosan; Complex; Coupling; Development; Drug Kinetics; Endoglycosidases; Engineering; Enzymes; Fucosidase; Glycopeptides; Glycoproteins; Heterogeneity; Hybrids; Immobilization; Immobilized Enzymes; Immune response; Industrialization; International; Lactobacillus casei; Legal patent; Libraries; Magnetism; Maryland; Methods; Pathway interactions; Phase; Polysaccharides; Post-Translational Protein Processing; Preparation; Procedures; Process; Property; Protein Glycosylation; Proteins; Protocols documentation; Reaction; Research; Research Proposals; Sepharose; Site; Streptococcus pyogenes; Structure; System; Testing; Therapeutic; Transglutaminases; United States National Institutes of Health; Universities; chemical synthesis; crosslink; glycoprotein structure; glycosylation; immunogenicity; in vivo; mutant; new technology; novel; particle; pathogen; phase 1 study; prospective; protein purification; research study; scaffold; scale up; success; technology development; tumor progression

Project abstract/summary This proposal responds to the NIH PA-16-157 “New Technologies for the Glycosciences (R43/R44)”. We aim to develop efficient and streamlined cheomoenzymatic systems for glycan remodeling of antibodies and other important glycoproteins, through optimizing enzyme immobilization and simplifying the reaction procedures. 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 a number of 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 in multiple approaches to prepare glycan-defined glycoprotein (such as total chemical synthesis and glycosylation through glycosylation 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. In the proposed study, GlycoT Therapeutics attempts to streamline and simplify this enzymatic glycan remodeling method through immobilizing the enzymes and developing easy-to-use kits and protocols, which are particularly useful for non- specialists. Immobilization will offer multiple valuable benefits: 1) stabilizing the enzyme for sustained activity, repeated use, and easy storage; 2) removing the protein purification steps for protein-GlcNAc acceptor and final products, thus greatly simplifying the process; 3) eliminating the potential problem of wild type enzyme contamination from the deglycosylation step. To achieve these objectives, we propose to pursue the following two specific aims in Phase I study. Aim 1 is to immobilize key enzymes for glycan remodeling of antibodies, which include: the endoglycosidase S2 (Endo-S2) from Streptococcus pyogenes, the Endo-S2 glycosynthase mutant (D184M), and an α1,6-fucosidase for defucosylation. Different methods of immobilization will be investigated. Aim 2 is to develop reaction kits and simply protocols for streamlined application of the enzymatic glycan remodeling of antibodies. In prospective phase II research, the study will expand to other endoglycosidases, to cover other glycoproteins/glycopeptides, for both academic and industrial applications.

项目摘要/概要 该提案响应了NIH PA-16-157“糖科学新技术（R43/R44）”。我们 旨在开发用于抗体聚糖重塑的高效和简化的cheomoenzymatic系统， 通过优化酶的固定化和简化反应步骤， 这种固定化酶，当与活化聚糖库组合作为试剂盒时，可以帮助一般学术和 工业用户，特别是非专业人员，制备聚糖定义的糖蛋白，用于结构和功能 问题研究该简化方法也可用于大规模制备同质糖蛋白 具有治疗潜力蛋白质糖基化是最普遍的翻译后修饰之一。 它深刻地影响蛋白质的性质，如折叠、体内稳定性、免疫原性和免疫原性。 药物代谢动力学，并且还直接参与许多重要的生物过程，包括细胞 粘附、癌症进展、宿主-病原体相互作用和免疫应答。的一个主要问题 糖蛋白研究的主要问题是结构的异质性和分离同质糖型的困难， 详细的结构和功能研究。尽管在多个领域取得了重大进展， 制备聚糖限定的糖蛋白的方法（例如全化学合成和通过 不同宿主表达系统中的糖基化途径工程化），可以是 所取得的成就仍然相当有限。为了应对这一挑战，一种新的化学酶法来生产 最近已经开发了均一的糖蛋白和糖肽。这种趋同的办法包括 由两个关键步骤组成：用糖苷内切酶使糖蛋白去糖基化， 通过新型糖合酶将所需的活化N-聚糖转化为蛋白-GlcNAc受体。在拟议的研究中， GlycoT Therapeutics试图通过以下方式简化和简化这种酶促聚糖重塑方法： 固定酶和开发易于使用的试剂盒和方案，这是特别有用的非- 专家。固定化将提供多种有价值的益处：1）稳定酶以保持活性， 2）去除蛋白质-GlcNAc受体和最终的蛋白质纯化步骤， 产品，从而大大简化了过程; 3）消除了野生型酶的潜在问题 来自去糖基化步骤的污染。为了实现这些目标，我们建议实现以下目标 第一阶段研究的两个具体目标。目的1是固定化抗体聚糖重塑的关键酶， 其包括：来自化脓性链球菌的内切糖苷酶S2（Endo-S2）、Endo-S2糖合酶（Endo-S2 glycosynthase 突变体（D184 M）和用于去岩藻糖基化的α 1，6-岩藻糖苷酶。将采用不同的固定方法 研究了目的2是开发反应试剂盒和简单的方案，以简化酶的应用。 抗体的聚糖重塑。在未来的第二阶段研究中，该研究将扩展到其他 内切糖基化酶，以涵盖其他糖蛋白/糖肽，用于学术和工业应用。