Abstract NIR dyes are becoming increasingly popular due to their good tissue penetration, low tissue autofluorescence and resistance to hemoglobin quenching. However, they are still limited by lower hydrophilicity and poor chemical and photo-stability in-vivo. We aimed to try to improve these properties by covalent modification of these dyes by addition of polyethylene glycol (PEG) chains. We studied 2 commercially-available NIR dyes, DyLight 650 and 750, to evaluate how PEGylation affects the pharmacokinetics, biodistribution and imaging. PEGylated and non-PEGylated DyLight 650 and 750 dyes were conjugated to a chimeric anti-CEA antibody and were injected intravenously into nude mice. Serum samples were collected at various time points to determine serum concentrations and clearance kinetics. Biodistribution was determined by tissue sonication and analyzing tissue concentration profiles over time. PEGylated dyes had significantly higher serum concentrations than non-PEGylated dyes with p=0.005 for the 650 dyes and p<0.001 for the 750 dyes. PEGylated dyes had significantly lower liver concentrations (p=0.03 for the 650 dyes; p=0.002 for the 750 dyes) and higher renal concentrations (p=0.003 for the 650 dyes and p<0.001 for the 750 dyes) compared to non-PEGylated dyes. Human pancreatic tumors subcutaneously implanted into nude mice were labeled with antibody-dye conjugates and serially imaged. Labeling with PEGylated dyes resulted in significantly brighter tumors compared to the non-PEGylated dyes (p<0.001 for the 650 dyes; p=0.01 for 750 dyes). In a liver metastasis model of colon cancer, PEGylated dyes showed bright labeling of metastatic tissue with good contrast between normal and malignant tissue. PEGylation of the NIR dyes DyLight 650 and 750 significantly changes their pharmacokinetics and biodistribution, allowing higher serum concentrations, longer half-life, brighter tissue labeling and decreased accumulation in lymphoid organs. This allows for high fidelity imaging of metastatic disease to any lymphoid or hemoglobin dense organ such as the liver. Citation Format: Ali A. Maawy, Yukihiko Hiroshima, George A. Luiken, Yong Zhang, Robert M. Hoffman, Michael Bouvet. Bioconjugation of near infrared dyes by PEGylation improves pharmacokinetics and allows for better labeling and imaging of human gastrointestinal cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4306. doi:10.1158/1538-7445.AM2014-4306