Conjugation of polysialic acid to biologics in glycoengineered Escherichia coli

描述（由申请人提供）：生物治疗药物目前构成了700亿美元的市场，但其临床疗效经常受到蛋白质水解降解、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有效的治疗浓度。最流行的方法来延长有效寿命的蛋白质治疗已偶联到聚乙二醇（聚乙二醇化）。然而，PEG不能通过体内正常的解毒机制消除，PEG化蛋白甚至可以产生抗PEG抗体。聚唾液酰化是聚唾液酰化的一种新兴替代方法，它涉及将聚唾液酸（PSA）的聚合物附着到蛋白质上。PSA正在开发用于临床，胰岛素和促红细胞生成素的多唾液化版本已显示出改善的耐受性和药代动力学。PSA是在体内的神经细胞粘附分子上合成的，与PEG不同，它作为天然糖分子被唾液酸酶代谢。不幸的是，与聚乙二醇化一样，PSA偶联过程在技术上是复杂和昂贵的。PSA的标准化学偶联会导致产物具有随机的附着模式和不良的异质性，这使得体外多步骤的PSA偶联过程进一步复杂化。glybia专门研究糖工程细菌，作为立体特异性生物合成治疗糖蛋白的表达平台。目前提出的研究背后的具体假设是，糖工程大肠杆菌可以在一次发酵中产生psa结合蛋白，而不需要体外化学修饰。基于这些观察结果，本研究的目的是通过克隆和表达糖工程大肠杆菌中PSA合成的遗传机制（ai1），并在糖工程大肠杆菌的周质中将PSA与重组人胰岛素结合（Aim 2），在糖工程大肠杆菌中生成PSA结合重组蛋白。这种表达平台将代表一种立体特异性、定向、快速和经济高效的过程，用于生产psa偶联生物治疗药物，这将使psa偶联蛋白的生产过程与其巨大的治疗潜力相一致。

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.