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 a ∼12-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 anti-tumor efficacy.

• Authors: Qi Zhao, Mahiuddin Ahmed, Hong-fen Guo, Irene Y Cheung, Nai-Kong V Cheung
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 290 Issue 21 Pg. 13017-27 (May 22 2015) ISSN: 1083-351X [Electronic] United States
• PMID: 25851904 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Chemical References

• Antibodies, Monoclonal
• Gangliosides
• Immunoglobulin G
• ganglioside, GD2

Topics

• Animals
• Antibodies, Monoclonal (therapeutic use)
• Antibody Specificity
• Antibody-Dependent Cell Cytotoxicity (immunology)
• Cell Proliferation
• Computer Simulation
• Flow Cytometry
• Gangliosides (immunology)
• Humans
• Immunoenzyme Techniques
• Immunoglobulin G (immunology)
• Mice
• Mice, Inbred BALB C
• Models, Molecular
• Mutagenesis, Site-Directed
• Mutation (genetics)
• Neuroblastoma (immunology, pathology, prevention & control)
• Protein Conformation
• Static Electricity
• Surface Properties
• Tumor Cells, Cultured
• Two-Hybrid System Techniques
• Xenograft Model Antitumor Assays