Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein

• Autores: Luria Pérez, Rosendo; Candelaria, Pierre V.; Daniels Wells, Tracy R.; Rodríguez, José A.; Helguera, Gustavo Fernando; Penichet, Manuel L.
• Año de publicación: 2019
• Idioma: inglés
• Tipo de recurso: artículo
• Materia: MONOCLONAL; ANTIBODY; INTERLEUKIN 12; CYTOKINE
• Institución: Consejo Nacional de Investigaciones Científicas y Técnicas

An antibody-cytokine fusion protein, composed of the murine single-chain cytokine interleukin-12 (IL-12) genetically fused to a human IgG3 specific for the human tumor-associated antigen HER2/neu maintains antigen binding, cytokine bioactivity, and IL-12 heparin-binding activity. This latter property is responsible for the binding of the cytokine to glycosaminoglycans (GAGs) on the cell surface and the extracellular matrix and has been implicated in modulating IL-12 bioactivity. Previous studies indicate that the p40 subunit of human and murine IL-12 is responsible for the heparin-binding activity of this heterodimeric cytokine. In the present study we used bioinformatic analysis and site-directed mutagenesis to develop a version of the antibody-(IL-12) fusion protein without heparin-binding activity. This was accomplished by replacing the basic arginine (R) and lysine (K) residues in the cluster of amino acids 254–260 (RKKEKMK) of the murine IL-12 p40 subunit by the neutral non-polar amino acid alanine (A), generating an AAAEAMA mutant fusion protein. ELISA and flow cytometry demonstrated that the antibody fusion protein lacks heparin-binding activity but retains antigen binding. A Tcell proliferation assay showed IL-12 bioactivity in this construct. However, the IL-12 bioactivity is decreased compared to its non-mutated counterpart, which is consistent with an ancillary role of the heparin-binding site of IL-12 in modulating its activity. Thus, we have defined a cluster of amino acid residues with a crucial role in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein.
Fil: Luria Pérez, Rosendo. University of California at Los Angeles; Estados Unidos. Hospital de Niños de México "Federico Gómez"; México
Fil: Candelaria, Pierre V.. University of California at Los Angeles; Estados Unidos
Fil: Daniels Wells, Tracy R.. University of California at Los Angeles; Estados Unidos
Fil: Rodríguez, José A.. University of California at Los Angeles; Estados Unidos
Fil: Helguera, Gustavo Fernando. University of California at Los Angeles; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina
Fil: Penichet, Manuel L.. University of California at Los Angeles; Estados Unidos