Site-Specific PEGylation of Recombinant Immunotoxin DAB389IL-2: Structural and Functional Assessment

Background: DAB389IL-2 is considered a fusion immunotoxin and used for the treatment of Cutaneous T Cell Lymphoma (CTCL). It is composed of two distinct portions; the catalytic domain of diphtheria toxin and IL-2. Because of DAB389IL-2 free cysteine residue (Cys 513 in IL-2 part), it is prone to unwanted intramolecular and intermolecular disulfide bonds formation and aggregation problems. Aggregation is considered as the most common physical instability. PEGylation is a practical approach to increase the stability and half-life of therapeutic proteins. Materials and Methods: In this study, the PEGylation of recombinant DAB389IL-2 was performed by mPEG-vinylsulfone, through partial denaturation condition at 4° C for 24 h. To confirm the PEGylation reaction, SDS-PAGE and Dynamic Light Scattering (DLS) was used. The structure of DAB389IL-2 and its PEGylated immunotoxin form were analyzed using the Circular Dichroism (CD) and fluorescence methods. Also, the K562 cells line were treated with various concentrations of DAB389IL-2 and conjugated form. In the following, the nuclease activities of DAB389IL-2 and PEGylated form were determined. Results: The SDS-PAGE result confirmed the site-specific PEGylation of DAB389IL-2. Spectroscopy results exhibited that the PEGylation does not affect the protein original structure. Also, the cytotoxicity assay and nuclease activity test confirmed that PEGylated protein induces death in K562 cells line and DNA degradation, respectively. Conclusion: PEGylated immunotoxin DAB389IL-2 has a proper structure and function; thus, PEGylated immunotoxin requires more study because of its unique properties.