Abstract 1341: PRINT: A protein bioconjugation method with exquisite N-terminal specificity

The use of proteins and peptides for therapeutic applications are often compromised by low biological stability, high renal clearance, and non-optimal biodistribution. Chemical attachment of poly-(ethylene glycol) (PEGylation) is often considered the most effective way to improve these pharmacologic properties by increasing circulation half-life as well as reduced renal clearance, immunogenicity and protease mediated degradation. However, random conjugation results in heterogeneous derivatives with undefined composition and can substantially lower the bioactivity of the modified protein, leading to unpredictable in vivo behavior. Site-specific modification of proteins is therefore an attractive approach to circumvent the non-specificity resulting from random conjugation. We have developed a novel technique named PRINT (PRotect, INcise Tag) for N-terminal specific bioconjugation of proteins and peptides. Conceptually, PRINT can be performed on any protein that has any N-terminal tag for purification and a protease cleavage site following the tag. The recombinant protein is first treated with an excess of citraconic anhydride to reversibly block all reactive primary amine sites. Proteolytic cleavage then exposes only a single amine (the primary amine at the N-terminus) for desired bioconjugation by amine-reactive NHS ester chemistry. Lowering of reaction pH results in removal of the citraconates, leaving N-terminal specific mono PEGylated protein molecules. We used Tumor Necrosis Factor-α (TNF-α) as a model protein as it suffers from inherent instability and short biological half-life, and exhibits toxic side effects at therapeutic concentrations in both small animals and human patients. We demonstrate that PRINT results in a single product with exquisite selectivity and specificity in contrast to conventional reaction using the same NHS reagent, which was further confirmed by mass spectrometric analyses. Subsequent de-blocking generated an N-terminal protected TNF-α molecule with enhanced serum stability, superior pharmacokinetic properties, and reduced systemic toxicity. Importantly, N-terminal protection by PRINT did not affect the bioactivity of TNF-α. Existing site-selective bioconjugation approaches are either specific to amino acid tags or involve substantial non-trivial chemical or biotechnological manipulations to synthesize a desired bioconjugate. In contrast, PRINT employs ubiquitously used recombinant DNA techniques and easily acquired commercial reagents to generate exquisite N-terminal selective protection. We show that PRINT is a robust, reproducible and mild strategy which is able to target the α -amine and provide N-terminal specific protection to proteins or peptides that suffer from similar issues. We believe that this approach is strongly orthogonal to current methods and will be applicable to many biotherapeutics and bioprobes that are currently being designed to treat cancer. Citation Format: Surojit Sur, Yuan Qiao, Anja C. Fries, Robert N. O’Meally, Robert N. Cole, Kenneth W. Kinzler, Bert Vogelstein, Shibin Zhou. PRINT: A protein bioconjugation method with exquisite N-terminal specificity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1341.