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）：目前，生物治疗剂构成了700亿美元的市场，但是它们的临床疗效通常会因蛋白水解降解，网细胞的吸收，网状内皮系统，肾脏去除和免疫力量的形成而受到限制。这可能导致在血液中达到和维持有效的治疗浓度方面遇到困难。延长蛋白质治疗的活性寿命的最流行的方法是与聚乙烯醇（Pegylation）结合。但是，没有通过体内的正常解毒机制消除PEG，而叶状蛋白的给药甚至可以产生抗PEG抗体。新兴的卵替代方法是多磷酸化，涉及将多氨酸聚合物（PSA）附着在蛋白质上。 PSA正在开发用于临床用途，胰岛素和红细胞生成素的多酰化版本表现出改善的耐受性和药代动力学。 PSA在体内在神经细胞粘附分子上合成，与PEG不同，被唾液酸酶代谢为天然糖分子。不幸的是，与Pegylation一样，PSA共轭过程在技术上是复杂且昂贵的。通过标准的PSA化学共轭导致具有随机依恋模式和不良异质性的产品，PSA共轭的体外体外过程更加复杂。 Glycobia专门从事糖化细菌，用作治疗性糖蛋白立体特异性生物合成的表达平台。当前提出的研究背后的特定假设是，糖化的大肠杆菌可用于在单个发酵中产生PSA偶联的蛋白，而无需体外化学修饰。 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).这种表达平台将代表生产PSA偶联的生物治疗剂的立体，定向，快速和具有成本效益的过程，该过程将带来PSA偶联蛋白的生产过程，并伴随其巨大的治疗潜力。 公共卫生相关性：蛋白质药物的疗效通常会因血液过早消除而损害，这导致不可接受的短暂治疗窗口和对医疗保健消费者的刺激性。多氨酸在治疗蛋白上的化学附着会提高耐受性和药代动力学，但是多氨酸偶联的过程在技术上具有挑战性且昂贵。这些提出的研究着重于在大肠杆菌发酵中产生多氨基酸偶联的蛋白，而无需体外化学修饰。