Engineering Escherichia coli for sialylation of therapeutic proteins

工程大肠杆菌用于治疗性蛋白质的唾液酸化

• 批准号: 8647417
• 负责人: Judith H Merritt
• 金额: $ 60.22万
• 依托单位: GLYCOBIA, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-10 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8647417
• 关键词: Address; Anabolism; Animal Model; Animals; Aranest; Bacteria; Benchmarking; Biological Assay; Biomanufacturing; Bioreactors; Biotechnology; Blood; Capital; Carbohydrates; Cell Culture Techniques; Cells; Charge; Chemicals; Clinical; Collaborations; Data; Drug Kinetics; Drug Stability; Drug Targeting; Engineering; Enzymes; Erythrocytes; Erythropoietin; Escherichia coli; Eukaryotic Cell; Excision; Feedback; Fermentation; Generations; Genetic Engineering; Glycolipids; Glycopeptides; Glycoproteins; Goals; Half-Life; Human; Human body; Immune response; Immunoassay; In Vitro; Kidney; Lead; Link; Marketing; Modeling; Mus; Pathway interactions; Patients; Peptides; Performance; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Polysaccharides; Preclinical Testing; Process; Production; Property; Protein Glycosylation; Proteins; Rattus; Recombinant Proteins; Recombinants; Relative (related person); Renal clearance function; Reticuloendothelial System; Serum; Sialic Acids; Site; Solubility; Solutions; Somatropin; Structure; Technology; Therapeutic; Thompson-Friedenreich Antigen; Time; Validation; asparaginase; base; chemical stability; commercialization; cost effective; design; dolichyl-diphosphooligosaccharide - protein glycotransferase; drug candidate; glycosylation; immunogenicity; improved; link protein; neutralizing antibody; phase 1 study; pre-clinical; public health relevance; sialylation; stability testing; success; therapeutic protein; uptake

Project Summary Glycoengineering is a clinically-validated strategy to enhance the therapeutic properties of protein and peptide drugs. This strategy involves the attachment and manipulation of carbohydrates (i.e., glycans) to improve the stability, solubility, serum half-life, and activity of these drugs. A key factor in most glycoengineering is the inclusion of terminal sialic acid residues on glycans by a process known as sialylation. Sialic acid is large and carries a negative charge which serves to improve stability, decrease aggregation, slow clearance, and impede immune response. Nearly all examples of glycoengineering require eukaryotic cell culture and/or the in vitro conjugation of glycans. Unfortunately, eukaryotic cell culture can be expensive, time consuming, and can result in inconsistent and incomplete sialylation. Although in vitro glycosylation can result in similar effects, the process is expensive, difficult, and has not been scalable to a commercial level. Glycoengineering would be greatly improved if a simple host cell such as Escherichia coli was used for production of sialylated therapeutic proteins. Glycobia specializes in genetically engineering bacteria for the bottom up glycoengineering (BUG) of therapeutic glycoproteins. Since E. coli lacks native protein glycosylation pathways of any kind, BUG can produce tailored glycan structures that can be site-specifically conjugated to target proteins. The specific hypothesis behind this proposal is that glycoengineered E. coli can produce enhanced therapeutic proteins by sialylation in a short, single fermentation. In Phase I of this project we engineered E. coli to attach humanlike, sialyl-T glycans to recombinant proteins. The sialyl-T glycan is a sialylated Thomsen- Friedenreich antigen that can be found on erythrocytes in the human body. This type of glycosylation is simply not possible in any other known expression host. We also show that bacterial glycosylation improves the in vitro stability of therapeutic proteins expressed in E. coli. We anticipate that our BUG expression platform will be capable of producing sialylated proteins in a controlled, rapid, cost-effective manner. The objective of this proposal is to synthesize and advance our first drug targets from glycoengineered E. coli into preclinical testing by: (i) expressing, purifying, and characterizing glycosylated drug candidates from E. coli and (ii) testing stability, pharmacokinetics, and immunogenicity of these drug candidates in animal models. We will compare their performance to aglycosylated and asialylated versions of these same drugs to isolate the effects of sialylation. The benchmark of success for this project is the generation of positive preclinical validation data to further advance commercialization of this technology. This bacterial expression platform represents a transformative solution to the unanswered biomedical challenge of generating cost-effective glycoengineered protein drugs for both companies and patients.

项目概要 糖工程是一种经过临床验证的策略，可增强蛋白质和肽的治疗特性 药物。该策略涉及碳水化合物（即聚糖）的附着和操作，以改善 这些药物的稳定性、溶解度、血清半衰期和活性。大多数糖工程的一个关键因素是 通过称为唾液酸化的过程将末端唾液酸残基包含在聚糖上。唾液酸含量大， 带有负电荷，可提高稳定性、减少聚集、减缓清除并阻碍 免疫反应。几乎所有糖工程的例子都需要真核细胞培养和/或体外 聚糖的结合。不幸的是，真核细胞培养可能昂贵、耗时，并且可能导致 唾液酸化不一致和不完全。尽管体外糖基化可以产生类似的效果， 该过程昂贵、困难，并且尚未扩展到商业水平。糖工程将是 如果使用简单的宿主细胞（例如大肠杆菌）来生产唾液酸化的产物，则效果会大大改善 治疗性蛋白质。 Glycobia 专注于自下而上的基因工程细菌 治疗性糖蛋白的糖工程（BUG）。由于大肠杆菌缺乏天然蛋白质糖基化途径 任何种类的 BUG 都可以产生定制的聚糖结构，可以与目标位点特异性结合 蛋白质。该提案背后的具体假设是，糖工程大肠杆菌可以产生增强的 在短暂的单一发酵中通过唾液酸化产生治疗性蛋白质。在该项目的第一阶段，我们设计了 E. 大肠杆菌将类人唾液酸-T 聚糖附着到重组蛋白上。唾液酸-T 聚糖是唾液酸化的 Thomsen- Friedenreich 抗原可以在人体内的红细胞上找到。这种类型的糖基化是 在任何其他已知的表达宿主中根本不可能。我们还表明细菌糖基化 提高大肠杆菌中表达的治疗蛋白的体外稳定性。我们预计我们的BUG 表达平台将能够以受控、快速、经济高效的方式生产唾液酸化蛋白 方式。该提案的目的是合成并推进我们的第一个药物靶点 通过以下方式将糖基化大肠杆菌纳入临床前测试：(i) 表达、纯化和表征糖基化 来自大肠杆菌的候选药物以及 (ii) 测试这些药物的稳定性、药代动力学和免疫原性 动物模型的候选者。我们将把它们的性能与无糖基化和无唾液酸化版本进行比较 这些相同的药物可以隔离唾液酸化的影响。该项目成功的标准是 生成积极的临床前验证数据，以进一步推进该技术的商业化。这 细菌表达平台代表了尚未解决的生物医学挑战的变革性解决方案 为公司和患者生产具有成本效益的糖工程蛋白药物。