Alteration of electrostatic surface potential enhances affinity and tumor killing properties of anti-ganglioside GD2 monoclonal antibody hu3F8 Journal Article
| Authors: | Zhao, Q.; Ahmed, M.; Guo, H. F. ; Cheung, I. Y.; Cheung, N. K. V. |
| Article Title: | Alteration of electrostatic surface potential enhances affinity and tumor killing properties of anti-ganglioside GD2 monoclonal antibody hu3F8 |
| Abstract: | 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-CDR1) 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 headgroup. Purified scFv and IgG mutant forms were then tested for antigen specificity by ELISA, for tissue specificity by immunohistochemistry, for affinity by BIACORE, for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity in vitro, and for anti-tumor efficacy in xenografted humanized mice. The nearly 7-fold improvement in affinity of hu3F8 with a single D32H (L-CDR1) mutation translated into ã12-fold improvement in NK92MI-transfected CD16-mediated ADCC, a 6-fold improvement in CD32-mediated ADCC, and a 2.5-fold improvement in complement-mediated cytotoxicity 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-CDR1) and E1K (L-FR1) did not further improve antitumor efficacy. |
| Keywords: | immunohistochemistry; controlled study; human tissue; unclassified drug; human cell; drug dose comparison; drug efficacy; nonhuman; animal cell; mouse; cell death; mus; animal experiment; animal model; in vivo study; antineoplastic activity; cytotoxicity; in vitro study; tumor xenograft; enzyme linked immunosorbent assay; monoclonal antibodies; monoclonal antibody; antigens; antigen specificity; neuroblastoma; tumors; antibodies; cross reaction; therapeutic targets; neuroectodermal tumors; ganglioside gd2 antibody; tissue; antibody affinity; antibody dependent cellular cytotoxicity; complement dependent cytotoxicity; tissue specificity; cloning; tumor ablation; drug products; ganglioside antibody; antibody combining site; electrostatics; sialic acid; anti-tumor efficacy; human; priority journal; article; surface potential; antibody-dependent cell-mediated cytotoxicities; antigen-binding sites; cytotoxicity in vitro; electrostatic surfaces; monoclonal antibody hu3f8; electrical potential parameters; electrostatic surface potential |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 290 |
| Issue: | 21 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2015-05-22 |
| Start Page: | 13017 |
| End Page: | 13027 |
| Language: | English |
| DOI: | 10.1074/jbc.M115.650903 |
| PROVIDER: | scopus |
| PUBMED: | 25851904 |
| PMCID: | PMC4505556 |
| DOI/URL: | |
| Notes: | Export Date: 2 July 2015 -- Source: Scopus |
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