Enzymatic Modification of Antibody for Targeted Drug Delivery

Objective: The goal of this study is to chemically modify an antibody, a glycoprotein, specifically through its oligosaccharides using enzymatic oxidation for further application in targeted drug delivery. Background: In targeted drug constructs, it is critical to control the bioactivity of the targeting moiety, which is often an antibody. The preparation of such constructs often involves conjugation reactions directed to functional groups on amino acid side chains of the protein, which result in random conjugation, which may result in compromise in target binding. Thus, more specific method of modification is desired. If the drug is a glycoprotein, enzymatic oxidation may offer an alternative approach for therapeutic glycoproteins. Methods: Immunoglobulin G (IgG), a model antibody, was treated with galactose oxidase (GO) and horseradish peroxidase, followed by thiolation reaction using 2-pyridyldithiopropionic hydrazide (PDPH) at varying feed ratios. IgG was also randomly thiolated by using varying feed ratios of 2-pyridyldithiopropionic acid N-hydroxysuccimidyl ester (SPDP). The thiolated IgGs were isolated by Fast-Protein Liquid Chromatography (FPLC) on two Sephadex G-25 columns in series. The conjugates were stored at 4 degree C. The conjugates were characterized for the degree of modification by the treatment with 50 mM dithiotheitol to release pyridyl-2-thione, which can be quantified spectrophotometically at 343 nm. Results: The degree of thiolation varied from 0.5 – 2.5 per antibody molecule was achievable through either method of thiolation. However, the thiolated IgG under enzymatic oxidation exhibited superior stability compared to the conjugate prepared via random thiolation. Conclusion: IgG, a model antibody, was successfully modified via enzymatic oxidation using galactose oxidase. This method can be applied to a wide variety of antibodies with different targets presented for a disease. Grant: NSU HPD Grant, PFRDG