Homology modeling and in silico docking analysis of a murine IL-12 engineered to block its heparin binding activity and fused to a therapeutic monoclonal antibody.

FERRERO S.; DANIELS T. R.; HELGUERA G.; CANDELARIA P.; PENICHET M. L.; LURIA-PÉREZ R.; RODRÍGUEZ J. A.

Mar del Plata

Jornada; Reunión Conjunta de Sociedades de Biociencias; 2019

Sociedad Argentina de Investigación Clínica (SAIC)

The cytokine interleukin-12 (IL-12) is a heterodimeric immune-modulator with heparin binding activity. This property favors the binding of the cytokine to the cell surface on glycosaminoglycans (GAGs) at the extracellular matrix and has been implicated in modulating IL-12 bioactivity. We have constructed an antibody-cytokine fusion protein with murine single-chain IL-12 genetically fused to a human IgG3 specific for the human tumor-associated antigen HER2/neu. It maintains cytokine bioactivity, antigen binding, anti-tumor activity, and IL-12 heparin-binding activity. Previous studies indicate that the domain responsible of the heparin-binding activity is at the p40 subunit of human and murine IL-12, but the absence of X-ray crystallography studies of the complex heparin-p40 precludes the structural analysis of this interaction. Here we used an in silico docking analysis in order to identify the pocket responsible of this interaction. Using as a template the structure of the human p40 subunit (PDB ID 3DUH), we generated a homology model of mouse p40 subunit using SWISS-MODEL and performed the in silico docking to the heparin molecule using ClusPro. In both cases the analysis of predicted contacts of best models placed the heparin binding pocket in a conserved cluster of basic arginine (R) and lysine (K) residues (a.a. 254?260 RKKEKMK) of the murine p40, which is conserved in human p40. A homology model replacing the basic amino acids by the neutral non-polar amino acid alanine (A), predicted a significant reduction of heparin atom contacts in this pocket. Moreover, after generation of the corresponding mutant fusion protein, ELISA and flow cytometry studies showed that it lacks heparin-binding activity but retains antigen binding. Therefore, these studies suggest that in silico docking analysis tools could be useful to guide the rational design of antibody-cytokine fusion protein with tailored properties to improve their specificity, efficacy and safety profile.