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.