Analysis and optimization of interactions between peptides mimicking the GD2 ganglioside and the monoclonal antibody 14G2a

Abstract

Abstract. Overexpression of the Gd2 ganglioside (Gd2) is a hallmark of neuroblastoma. The antigen is used in neuroblastoma diagnosis and to target newly developed therapies to cancer cells. Peptide mimetics are novel approaches in the design of antigens for vaccine development. We previously reported the isolation of five GD2-mimicking peptides from the LX-8 phage display library with the monoclonal antibody (mAb) 14G2a. The goal of our current study was to analyze and optimize the binding of the peptide mimetics to the mAb 14G2a. Therefore, we performed further experiments and supported them with molecular modeling to investigate structure-activity relationships that are the basis for the observed mimicry of Gd2 by our peptides. Here, we show that the peptides have overlapping binding sites on the mAb, 14G2a and restricted specificity, as they did not crossreact with other ganglioside-specific antibodies tested. In addition we demonstrate that the phage environment was involved in the process of selection of our peptides. The AAEGd sequence taken from the viral major coat protein, p8, and added to the c-termini of the peptides #65, #85 and #94 significantly improved their binding to the mAb, 14G2a. By application of analogs with amino acid substitutions and sequence truncations, we elucidated the structure-activity relationships necessary for the interactions between the 14G2a mAb and the peptide #94 (RCNPNMEPPRCF). We identified amino acids indispensable for the observed Gd2-mimicry by #94 and confirmed a pivotal role of the disulphide bridge between the cysteine residues of #94 for binding to the mAb 14G2a. More importantly, we report five new peptides demonstrating a significant improvement of mAb 14G2a binding. The experimental data were supported and expanded with molecular modeling tools. Taken together, the experimental results and the in silico data allowed us to probe in detail the mechanism of the molecular mimicry of Gd2 by the peptides. Additionally, we significantly optimized binding of the leading peptide sequence #94 to the mAb 14G2a. We can conclude that our findings add to the knowledge on factors governing selections of peptide mimetics from phage-display libraries.

Structural Basis of GD2 Ganglioside and Mimetic Peptide Recognition by 14G2a Antibody

“…Our study provides a detailed insight into how the tested peptides imitate GD2 in its binding to specific antibody. The provided here structural data perfectly explains the results of previous mutagenesis studies within the predecessor of peptide 1 (for mutagenesis data see (15); for a detailed discussion see supplemental Information and Table S2). Collectively, this information shall facilitate rational modification of peptides to improve their utility in active immunization.…”

“…Peptide 2 binding relies considerably less on water mediated interactions. The functional rather than structural nature of the mimicry is further corroborated by the fact that the tested peptide mimetics interact only with 14G2a, but not other tested antibodies specific for GD2 (15). Therefore the observed mimicry is receptor rather than antigen dependent, as required by the "functional" model.…”

“…Peptide 1 (RCNPNMEPPRC-WAAEGD) or peptide 2 (VCNPLTGALLCSAAEGD; both from Genscript) (15) were incubated overnight with the Fab fragment at 3:1 molar ratio at 4°C and used for crystallization. GD2 sugar (GalNAc␤4(Neu5Ac␣8Neu5Ac␣3)Gal␤4Glc; Elicityl, Crolles, France) was incubated with the Fab fragment in conditions similar to those used for both peptides.…”

“…The major conclusions are verified by directed mutagenesis and antibody variant with increased affinity toward GD2 is developed using structure guided approach. The binding modes of two largely divergent peptide mimics of GD2 (15) at the antigen-binding site of 14G2a antibody are reported and compared with that of the carbohydrate. Mouse 14G2a antibody was chosen for this study because it contains the same antigen binding region as the ch14.18 chimeric antibody recently approved by FDA (8).…”

Application of Molecular Mimicry to Target GD2 Ganglioside

“…Additionally, the peptides were shown to occupy overlapping binding sites on the 14.G2a. This finding was supported by in silico models of interactions of the peptides with the Fab fragment of the 14.G2a (Horwacik et al, 2011). Fig.…”