Selection of novel peptide mimics of the GD2 ganglioside from a constrained phage-displayed peptide library

Abstract

Abstract. Aberrant glycosylation is a universal feature of cancer cells. There are quantitative and qualitative changes in expression of gangliosides observed in tumors of a neuroectodermal origin such as neuroblastoma, melanoma and astrocytoma. The presence of large amounts of GD2 ganglioside on neuroblastoma cells, as compared to normal cells, opens the possibilities to use the tumor-associated carbohydrate antigen in diagnosis and immunotherapeutic approaches. In the quest for immunogens potentially capable of eliciting anti-GD2 ganglioside immune responses, we performed affinity purification of phage-displayed peptides from the LX-8 library (12-mer containing disulphide bridge). The library was screened with the biotinylated anti-GD2 ganglioside 14G2a mAb monoclonal antibody. Our goal was to isolate and characterize peptide mimics of GD2 ganglioside. Numerous individual phage clones that bound 14G2a mAb were identified with the application of immunoblotting technique in the phage pools yielded from the pannings. The phage-borne peptides were tested for their anti-GD2 ganglioside antibody binding ability using ELISA. Among these clones five different phage-displayed peptide sequences were identified. Moreover, we showed that the secondary structure of the peptides, stabilized by the disulfide bridging between cysteine residues at positions 2 and 11, was crucial for the binding of the peptides to 14G2a mAb. In a separate set of experiments, we observed a competition of the peptides, expressed on phages as well as in their synthetic form, with the nominal antigen GD2 ganglioside expressed on IMR-32 neuroblastoma cells for binding to 14G2a mAb. Based on the obtained results we concluded that all of these 5 peptides were mimics of the GD2 ganglioside.

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

“…Finally, we were able to confirm the particularly important role of the disulphide bridge, formed by the c residue at positions 2 and 11, in the observed binding of the free peptides. The finding was previously reported for all our phage-display peptides based on the observed abrogation of their mAb 14G2a binding in the presence of two agents, dithiothreitol and N-ethylmaleimide, reducing and preventing them from reforming a disulphide bond (20). Altogether, for both the free and the phage-displayed peptides, we can assume that cyclic peptides could adopt or maintain the antigenically reactive structure necessary for Gd2 mimicry.…”

“…Peptide sequences of Gd2 mimetics (#8, #65, #85, #94, #d) expressed on phages and #0-phage (a phage without any fusion peptide) were isolated during biopanning of the LX-8 library with mAb 14G2a [for a detailed description of the biopanning and the preparation of the phage-expressed peptides see Horwacik et al (20)]. The major coat protein p8 is used for peptide display in the LX-8 library (24).…”

“…Recently, we have described a series of 12-amino acid constrained peptides capable of binding the anti-Gd2 mAb, 14G2a, isolated from the LX-8 phage-displayed peptide library (20). The mAb, 14G2a, can effectively mediate in vitro and in vivo cytotoxicity against neuroblastoma cells (21) and has a well-established record of medical applications (22,23).…”

Application of Molecular Mimicry to Target GD2 Ganglioside

“…Immunoblotting and ELISA can be used to analyse phage clones isolated from peptide libraries (bottom). The examples from the screening of the LX-8 library with the 14.G2a were included (see Horwacik et al, 2007 for more details). Replicas of bacteria infected with phages on nitrocellulose membranes were incubated with the 14.G2a antibody and the secondary HRP-conjugated antibody, and then developed (bottom left).…”

“…The sera samples obtained from immunisations with the DNA construct alone, as well as in combination with the GD2 ganglioside boost, mediated complement dependent lysis of the GD2 positive IMR-32 neuroblastoma, and HT-144 melanoma cells (Bolesta et al, 2005). Yet another 5 sequences binding to the 14G2a antibodies were isolated, but this time from the constrained library LX-8 (Horwacik et al, 2007). The library displays 12-amino acid peptides.…”