Chemoenzymatic Glycan Remodeling of IVIG for Improved Anti-inflammatory Activity

项目摘要/摘要 拟议研究的目标是继续并扩大GlycoT在I期SBIR研究中的研究， 建立用于生产静脉内注射的高唾液酸化糖型的稳健且可扩展的方法， 免疫球蛋白（IVIG）具有显著增强的抗炎活性。我们将通过开发 一种创新的化学酶促糖基化重塑技术，用于生产糖工程IVIG， 在动物模型中测试其功效。IVIG是一种抗体的混合物， 健康的捐赠者高剂量IVIG（1- 2g/kg）已被广泛应用于治疗自身免疫性疾病， 慢性炎症，如类风湿性关节炎（RA），全球年销售额达数十亿美元。尽管 广泛的治疗应用，目前的IVIG制剂具有明显的局限性，包括长时间输注（4- 6小时），可变的疗效，供应短缺，和副作用。因此，IVIG的替代品具有更有效的 非常需要治疗效果（因此低得多的剂量）和最小的副作用。虽然精确的 IVIG抗炎活性的分子机制仍然没有很好的理解，最近的研究表明， 动物模型表明，IVIG的唾液酸化Fc糖型是一种次要组分， 为约10%的IVIG，可能是赋予抗炎活性的主要活性形式。已经 证明Fc唾液酸化糖型的富集可以在几种免疫抑制剂中实现IVIG的完全活性。 用小于10%的市售IVIG剂量的自身免疫性疾病小鼠模型。因此，我们认为， 用完全唾液酸化修饰IVIG指出了一种非常有前途的提高治疗效果的方法。 已经尝试通过IVIG的连续半乳糖基化和唾液酸化来增加IVIG的Fc唾液酸化， 但由于IVIG原始糖型的巨大异质性， 相对低的酶促唾液酸化效率和副反应。在这个应用中，我们应用一个 化学酶平台技术最初由马里兰州大学开发，以实现完全Fc唾液酸化 IVIG的糖型。在I期研究中，GlcyoT成功建立了一个高效、可扩展的 用于生产克级IVIG的高唾液酸化糖型的化学酶促方法。的Fc聚糖 工程IVIG是超过95%的四唾液酸化，并表现出10倍以上的抗炎活性， 在类风湿性关节炎的小鼠模型中的商业IVIG。拟议的第二阶段研究将进一步扩大规模 采用IVIG糖链改造技术，建立严格的质量控制体系， 有效性，毒性和药效学研究，为进一步的临床前研究和未来的人类 临床试验

Project Abstract/Summary The goal of the proposed studies is to continue and expand GlycoT’s research in a Phase I SBIR study to establish a robust and scalable process for production of hypersialylated glycoforms of intravenous immunoglobulin (IVIG) with markedly enhanced anti-inflammatory activity. We will achieve this goal by exploiting an innovative chemoenzymatic glycosylation remodeling technology to produce the glycoengineered IVIG and to test their efficacy in animal models. IVIG is a mixture of antibodies prepared from the pooled sera of thousands of healthy donors. High dosage of IVIG (1-2 g/kg) has been widely applied to treat autoimmune disease and chronic inflammation, such as rheumatoid arthritis (RA), with multi-billion-dollar annual sales globally. Despite its wide applications for treatment, current IVIG preparation has distinct limitations, including long time infusion (4- 6 h), variable efficacy, shortage of supplies, and side effects. As a result, alternatives for IVIG with more potent therapeutic efficacy (thus much lower doses) and minimal side effects are highly desired. While the precise molecular mechanisms for the anti-inflammatory activities of IVIG is still not well-understood, recent studies in animal models have suggested that the sialylated Fc glycoform of IVIG, which is a minor component consisting of ca. 10% of IVIG, could be the major active form that confers the anti-inflammatory activities. It has been demonstrated that the enrichment of the Fc sialylation glycoforms could achieve full activity of IVIG in several mouse models of autoimmune disease with less than 10% of the doses of commercial IVIG. Therefore, modification of IVIG with full sialylation points to a very promising approach to improving the therapeutic efficacy. Attempt to increase Fc sialylaton of IVIG by sequential galactosylation and sialylation of IVIG has been pursued, but met with only partial success due to the tremendous heterogeneity of the original glycoforms of IVIG, the relatively low efficiency of enzymatic sialylation, and side reactions. In this application, we apply a chemoenzymatic platform technology originally developed at University of Maryland to achieve fully Fc sialylated glycoform of IVIG. In the phase I study, GlcyoT has successfully established an efficient, scalable chemoenzymatic process for producing gram-scale of hypersialylated glycoforms of IVIG. The Fc glycan of engineered IVIG is more than 95% tetra-sialylated and exhibited 10-fold higher anti-inflammatory activity than the commercial IVIG in a mouse model of rheumatoid arthritis. The proposed Phase II study will further scale up the IVIG glycan-remodeling technology, establish a rigorous quality control system, and carry out intensive efficacy, toxicity, and pharmacodynamics study, paving the way for further preclinical studies and future human clinical trials.