Conjugation of polysialic acid to biologics in glycoengineered Escherichia coli

聚唾液酸与糖工程大肠杆菌中的生物制剂结合

• 批准号: 7911940
• 负责人: Adam Charles Fisher
• 金额: $ 19.9万
• 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 2011-09-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7911940
• 关键词: Address; Amination; Amines; Anabolism; Antibodies; Asparagine; Bacteria; Biological Response Modifier Therapy; Blood; C-terminal; Cells; Chemicals; Clinical; Cloning; Complex; Coupling; Drug Kinetics; Drug Metabolic Detoxication; ERBB2 gene; Engineering; Erythropoietin; Escherichia coli; Excision; Fermentation; Genes; Genetic; Glycoproteins; Granulocyte Colony-Stimulating Factor; Half-Life; Healthcare; Heterogeneity; Human; Hydration status; Immune system; In Vitro; Insulin; Interferons; Kidney; Laboratories; Lead; Life; Link; Lipids; Location; Marketing; Modification; N-Acetylneuraminic Acid; Neural Cell Adhesion Molecules; O Antigens; Pattern; Peptides; Pharmaceutical Preparations; Plasmids; Polymers; Polysaccharides; Polysialic Acid; Process; Production; Property; Proteins; Recombinant Proteins; Recombinants; Reticuloendothelial System; Sialic Acids; Site; Structure; Sulfhydryl Compounds; Technology; Therapeutic; Time; Tissues; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Western Blotting; asparaginase; base; capsule; chemical standard; clinical efficacy; cost; glycosylation; immunogenicity; improved; in vivo; nanoparticle; periplasm; premature; public health relevance; residence; stoichiometry; sugar; therapeutic protein; uptake

DESCRIPTION (provided by applicant): Biotherapeutics currently constitute a $70 billion market, but their clinical efficacy is often compromised by limitations arising from proteolytic degradation, uptake by cells of the reticuloendothelial system, renal removal, and immunocomplex formation. This can lead to difficulties in reaching and maintaining effective therapeutic concentrations in the blood. The most popular approach to lengthen the active life of a protein therapeutic has been conjugation to polyethyleneglycol (PEGylation). However, PEG is not eliminated via normal detoxification mechanisms in the body and the administration of PEGylated proteins can even generate anti-PEG antibodies. An emerging alternative to PEGylation is polysialylation which involves attachment of polymers of polysialic acid (PSA) to a protein. PSA is being developed for clinical use and polysialylated versions of insulin and erythropoietin have displayed improved tolerance and pharmacokinetics. PSA is synthesized in the body on neural cell adhesion molecule and, unlike PEG, is metabolized as a natural sugar molecule by sialidases. Unfortunately, as with PEGylation, the PSA conjugation process is technically complex and expensive. The multi-step, in vitro process of PSA conjugation is further complicated by the fact that standard chemical conjugation of PSA results in products with random attachment patterns and undesirable heterogeneity. Glycobia specializes in glycoengineering bacteria for use as an expression platform for the stereospecific biosynthesis of therapeutic glycoproteins. The specific hypothesis behind the current proposed studies is that glycoengineered E. coli can be used to produce PSA-conjugated proteins in a single fermentation without the need for in vitro chemical modification. Based on these observations, the objective of this proposal is to generate PSA-conjugated recombinant protein in glycoengineered E. coli by: cloning and expressing the genetic machinery for PSA synthesis in glycoengineered E. coli (Aim1) and conjugating PSA to recombinant human insulin in the periplasm of glycoengineered E. coli (Aim 2). Such an expression platform will represent a stereospecific, directed, rapid, and cost-effective process for the production of PSA-conjugated biotherapeutics that will bring the production process of PSA-conjugated proteins in concert with their tremendous therapeutic potential. PUBLIC HEALTH RELEVANCE: The efficacy of protein drugs is often compromised by premature elimination from the blood, which results in unacceptably short therapeutic windows and costs that are prohibitive to the healthcare consumer. The chemical attachment of polysialic acid to therapeutic proteins results in improved tolerance and pharmacokinetics, but the process of polysialic acid conjugation is technically challenging and expensive. These proposed studies focus on producing polysialic acid-conjugated proteins in Escherichia coli fermentation without the need for in vitro chemical modification.

描述（由申请人提供）：生物治疗目前构成了一个 700 亿美元的市场，但其临床疗效常常受到蛋白水解降解、网状内皮系统细胞摄取、肾脏去除和免疫复合物形成等限制的影响。这可能导致难以达到和维持血液中的有效治疗浓度。延长蛋白质治疗剂活性寿命的最流行方法是与聚乙二醇缀合（聚乙二醇化）。然而，PEG 不会通过体内正常的解毒机制消除，施用 PEG 化蛋白质甚至可以产生抗 PEG 抗体。聚唾液酸化是一种新兴的聚唾液酸化替代方案，涉及将聚唾液酸 (PSA) 聚合物附着到蛋白质上。 PSA 正在开发用于临床用途，胰岛素和促红细胞生成素的聚唾液酸化形式已显示出改善的耐受性和药代动力学。 PSA 在体内通过神经细胞粘附分子合成，与 PEG 不同，PSA 被唾液酸酶代谢为天然糖分子。不幸的是，与聚乙二醇化一样，PSA 缀合过程在技术上复杂且昂贵。 PSA 缀合的多步骤体外过程因 PSA 的标准化学缀合导致产物具有随机附着模式和不良异质性而进一步复杂化。 Glycobia 专门研究糖工程细菌，用作治疗性糖蛋白立体特异性生物合成的表达平台。目前提出的研究背后的具体假设是，糖工程化的大肠杆菌可用于在单次发酵中生产 PSA 结合蛋白，而无需体外化学修饰。基于这些观察，本提案的目标是通过以下方式在糖基工程大肠杆菌中生成 PSA 缀合的重组蛋白：在糖基工程大肠杆菌 (Aim1) 中克隆和表达用于 PSA 合成的遗传机制，并将 PSA 与重组人胰岛素缀合糖工程化大肠杆菌的周质（目标 2）。这样的表达平台将代表一种立体特异性、定向、快速且经济高效的 PSA 结合生物治疗药物的生产工艺，使 PSA 结合蛋白的生产过程与其巨大的治疗潜力相协调。 公众健康相关性：蛋白质药物的功效常常会因过早从血液中消除而受到损害，这会导致治疗窗口短得令人无法接受，并且成本高昂，令医疗保健消费者望而却步。聚唾液酸与治疗性蛋白质的化学连接可改善耐受性和药代动力学，但聚唾液酸缀合的过程在技术上具有挑战性且昂贵。这些拟议的研究重点是在大肠杆菌发酵中生产聚唾液酸缀合蛋白，而不需要体外化学修饰。