Prevalence and characteristics of anti-PEG antibodies in humans

摘要 聚乙二醇(PEG)由于具有抵抗蛋白质吸附和降低Res清除率的能力而受到广泛关注。 广泛用于延长蛋白质和纳米粒的循环次数和提高疗效 治疗学。然而，由于包括洗涤剂在内的日常产品中不断接触到聚乙二醇酯 (肥皂、洗发水、牙膏等)、食品和化肥，相当大比例的人口 可能产生了某种程度的抗聚乙二醇化免疫。最近的动物研究表明，抗体 特定识别的聚乙二醇可在体内生成，导致聚乙二醇化的血液加速清除 治疗药物和纳米颗粒，从而降低了疗效。尽管先前存在的反聚乙二醇免疫很可能 损害了许多聚乙二醇化治疗的有效性，对抗聚乙二醇免疫知之甚少 在人类身上。在这项建议中，我们寻求将免疫学和分子生物学的最新进展应用于 确定人类体内抗聚乙二醇单抗的特征。在目标1中，我们将测量患病率和浓度 在北卡罗来纳大学医院血库收到的血液样本中检测到抗聚乙二醇IgG和免疫球蛋白M。我们还将 评估少数患者在治疗前和治疗后不同时间点的血清抗-聚乙二醇抗体水平 使用聚乙二醇化的治疗药物。在目标2中，我们将(A)分离并永生化人类B细胞，产生 抗聚乙二醇单抗，(B)鉴定其与聚乙二醇(Kon，Koff，Kd)的结合亲和力，(C)对高表达的DNA进行测序 亲和结合的单抗(MAb)，和(D)获得晶体结构，以揭示氨基酸是如何 Fab结合臂的取向特别识别聚乙二醇的环氧乙烷主链。在《目标3》中， 使用从Aim 2克隆到小鼠免疫球蛋白骨架上的人mAb，我们将评估(A) 抗-聚乙二醇免疫球蛋白，在反映在人类中发现的范围内的浓度(来自目标1)，影响生物- 聚乙二醇化微粒在小鼠体内的分布和循环动力学。(二)我们亦会决定会否预先- 在注射聚乙二醇化颗粒之前，以不同的剂量和时间注射游离的聚乙二醇酯，可以恢复天然的 饱和血清抗-PEGIg的分布和循环特征。这些研究将有助于 未来该领域研究的宝贵资源(完全的人抗聚乙二醇单抗和相应的多重链 分析)，提供了重要的结构洞察抗体如何特异性识别聚乙二醇胺和 潜在的其他聚合物，并促进下一代类聚乙二醇类聚合物的开发，这种聚合物可以避免 由抗聚乙二醇单抗结合。

Abstract Poly(ethylene glycol) (PEG), due to its ability to resist protein adsorption and reduce RES clearance, has been widely used to extend the circulation times and improve efficacy of protein- and nanoparticle- based therapeutics. However, because of constant exposure to PEG in everyday products including detergents (soap, shampoo, toothpaste, etc.), food products and fertilizers, a substantial proportion of the population has likely developed some degree of anti-PEG immunity. Recent animal studies show that antibodies that specifically recognize PEG can be generated in vivo, leading to accelerated blood clearance of PEGylated therapeutics and nanoparticles and thereby reduced efficacy. Although pre-existing anti-PEG immunity likely compromises the effectiveness of many PEGylated therapeutics, little is understood about anti-PEG immunity in humans. In this proposal, we seek to apply the latest advances in immunology and molecular biology to characterize anti-PEG antibodies in humans. In Aim 1, we will measure the prevalence and concentrations of anti-PEG IgG and IgM among blood samples received at the blood bank at the UNC Hospital. We will also evaluate serum anti-PEG antibody levels in a small number of patients before and at various time points post dosing with PEGylated therapeutics. In Aim 2, we will (A) isolate and immortalize human B cells producing anti-PEG antibodies, (B) characterize their binding affinity to PEG (kon, koff, KD), (C) sequence the DNA of high affinity binding monoclonal antibodies (mAb), and (D) obtain crystal structures to reveal how the amino acid orientation of the Fab binding arm specifically recognizes the ethylene oxide backbone of PEG. In Aim 3, using human mAb from Aim 2 cloned to a mouse IgG backbone, we will evaluate (A) how precise levels of anti-PEG IgG, at concentrations reflecting the range found in humans (from Aim 1), influence the bio- distribution and circulation kinetics of PEGylated particles in mice. (B) We will also determine whether pre- injection of free PEG, at various doses and times prior to injection of PEGylated particles, may restore native distribution and circulation characteristics by saturating serum anti-PEG IgG. These studies will contribute valuable resources for future studies in the field (fully human anti-PEG mAb and corresponding multiplex assays), provide important structural insights into how antibodies can specifically recognize PEG and potentially other polymers, and facilitate the development of next generation PEG-like polymers that can avoid binding by anti-PEG antibodies.