Conjugation of polysialic acid to biologics in glycoengineered Escherichia coli

描述(申请人提供)：生物疗法目前构成了700亿美元的市场，但其临床疗效经常受到蛋白质降解、网状内皮系统细胞摄取、肾脏切除和免疫复合体形成等限制的影响。这可能导致难以达到和保持血液中有效的治疗浓度。延长蛋白质疗法有效寿命的最受欢迎的方法是与聚乙二醇偶联物(聚乙二醇化)。然而，聚乙二醇并不能通过体内正常的解毒机制被清除，服用聚乙二醇化的蛋白质甚至可以产生抗聚乙二醇抗体。聚乙二醇化的一种新的替代方法是聚唾液酸化，它涉及聚唾液酸聚合物(PSA)与蛋白质的结合。PSA正在开发用于临床，多唾液酸化版本的胰岛素和促红细胞生成素已经显示出更好的耐受性和药代动力学。PSA是在体内通过神经细胞黏附分子合成的，与聚乙二醇不同，它是通过唾液酸酶作为天然糖分子进行代谢的。不幸的是，与聚乙二醇化一样，PSA偶联过程在技术上复杂且昂贵。PSA的多步骤体外偶联过程进一步复杂化，因为PSA的标准化学偶联会导致产物具有随机的附着模式和不希望看到的异质性。Glycobia专门研究糖工程菌，作为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，糖工程大肠杆菌可以在一次发酵中生产PSA偶联蛋白，而不需要体外化学修饰。基于这些观察结果，本研究的目的是在糖工程大肠杆菌(Aim1)中克隆和表达PSA合成的遗传机制，并在糖工程大肠杆菌(Aim1)周质中将PSA与重组人胰岛素偶联(Aim 2)，从而在糖工程大肠杆菌中产生PSA偶联的重组蛋白。这样的表达平台将代表一种立体特异性的、定向的、快速的和低成本的生产PSA偶联生物疗法的过程，它将使PSA偶联蛋白质的生产过程与其巨大的治疗潜力相一致。 与公共卫生相关：蛋白质药物的疗效往往因过早从血液中消除而受到影响，这导致治疗窗口短到令人无法接受的程度，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结合可改善耐受性和药代动力学，但聚唾液酸偶联过程在技术上具有挑战性且成本高昂。这些拟议的研究集中于在不需要体外化学修饰的情况下在大肠杆菌发酵中生产聚唾液酸结合蛋白。

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.