Alteration of electrostatic surface potential enhances affinity and tumor killing properties of anti-ganglioside GD2 monoclonal antibody hu3F8 [Glycobiology and Extracellular Matrices]

April 7th, 2015 by Zhao, Q., Ahmed, M., Guo, H.-f., Cheung, I. Y., Cheung, N.-K. V.

Ganglioside GD2 is highly expressed on neuroectodermal tumors and an attractive therapeutic target for antibodies that have already shown some clinical efficacy. To further improve the current antibodies, which have modest affinity, we sought to improve affinity by using a combined method of random mutagenesis and in silico assisted design to affinity-mature the anti-GD2 monoclonal antibody hu3F8. Using yeast display, mutants in the Fv with enhanced binding over the parental clone were FACS sorted and cloned. In silico modeling identified the minimal key interacting residues involved in the important charged interactions with the sialic acid groups of GD2. Two mutations, D32H (L-CDR2) and E1K (L-FR1) altered the electrostatic surface potential of the antigen binding site, allowing for an increase in positive charge to enhance the interaction with the negatively charged GD2-pentasaccharide head group. Purified scFv and IgG mutant forms were then tested for antigen specificity by ELISA, tissue specificity by immunohistochemistry, affinity by BIACORE, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement mediated cytotoxicity (CMC) in vitro, and anti-tumor efficacy in xenografted humanized mice. The near 7-fold improvement in affinity of hu3F8 with single D32H (L-CDR2) mutation translated into a ~12-fold improvement in NK92MI-transfected CD16-mediated ADCC, 6-fold in CD32-mediated ADCC, and 2.5-fold in CMC, while maintaining restricted normal tissue cross-reactivity, and achieving substantial improvement in tumor ablation in vivo. Despite increasing GD2 affinity, the double mutation D32H (L-CDR2) and E1K (L-FR1) did not further improve anti-tumor efficacy.
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