%0 Journal Article %J International Journal of Molecular Medicine %D 2011 %T Analysis and optimization of interactions between peptides mimicking the GD2 ganglioside and the monoclonal antibody 14G2a %A Irena Horwacik %A Mateusz Kurcinski %A Malgorzata Bzowska %A Aleksandra K. Kowalczyk %A Dominik Czaplicki %A Andrzej Koliński %A Hanna Rokita %K Amino Acid Sequence %K Antibodies %K Binding Sites %K Cell Line %K Gangliosides %K Gangliosides: immunology %K Humans %K Models %K Molecular %K Molecular Mimicry %K Molecular Sequence Data %K Monoclonal %K Monoclonal: chemistry %K Monoclonal: immunology %K Neuroblastoma %K Neuroblastoma: genetics %K Neuroblastoma: immunology %K Peptide Library %K Peptides %K Peptides: chemistry %K Peptides: immunology %K Structure-Activity Relationship %K Tumor %X

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.

%B International Journal of Molecular Medicine %V 28 %P 47–57 %8 jul %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/21455557 %R 10.3892/ijmm.2011.655 %0 Journal Article %J Cancer Research %D 2005 %T DNA vaccine expressing the mimotope of GD2 ganglioside induces protective GD2 cross-reactive antibody responses %A Elizabeth Bolesta %A Aleksandra Kowalczyk %A Andrzej Wierzbicki %A Piotr Rotkiewicz %A Barbara Bambach %A Chun-Yen Tsao %A Irena Horwacik %A Andrzej Koliński %A Hanna Rokita %A Martin Brecher %A Xinhui Wang %A Soldano Ferrone %A Danuta Kozbor %K Active %K Active: methods %K Amino Acid Sequence %K Animals %K Antibodies %K Antibody %K Binding Sites %K Cancer Vaccines %K Cancer Vaccines: genetics %K Cancer Vaccines: immunology %K Cancer Vaccines: pharmacology %K Cross Reactions %K DNA %K DNA: genetics %K DNA: immunology %K DNA: pharmacology %K Female %K Gangliosides %K Gangliosides: genetics %K Gangliosides: immunology %K Humans %K Immunoglobulin G %K Immunoglobulin G: biosynthesis %K Immunoglobulin G: immunology %K Immunotherapy %K Inbred BALB C %K Melanoma %K Melanoma: immunology %K Melanoma: therapy %K Mice %K Molecular Sequence Data %K Monoclonal %K Monoclonal: genetics %K Monoclonal: immunology %K Neuroblastoma %K Neuroblastoma: immunology %K Neuroblastoma: therapy %K Peptide Library %K Peptides %K Peptides: genetics %K Peptides: immunology %K SCID %K Vaccines %K Xenograft Model Antitumor Assays %X The GD2 ganglioside expressed on neuroectodermally derived tumors, including neuroblastoma and melanoma, is weakly immunogenic in tumor-bearing patients and induces predominantly immunoglobulin (Ig)-M antibody responses in the immunized host. Here, we investigated whether interconversion of GD2 into a peptide mimetic form would induce GD2 cross-reactive IgG antibody responses in mice. Screening of the X(15) phage display peptide library with the anti-GD2 monoclonal antibody (mAb) 14G2a led to isolation of mimetic peptide 47, which inhibited the binding of 14G2a antibody to GD2-positive tumor cells. The peptide was also recognized by GD2-specific serum antibodies from a patient with neuroblastoma, suggesting that it bears an internal image of GD2 ganglioside expressed on the tumor cells. The molecular basis for antigenicity of the GD2 mimetic peptide, established by molecular modeling and mutagenesis studies, led to the generation of a 47-LDA mutant with an increased mimicry to GD2. Immunization of mice with peptide 47-LDA-encoded plasmid DNA elicited GD2 cross-reactive IgG antibody responses, which were increased on subsequent boost with GD2 ganglioside. The vaccine-induced antibodies recognized GD2-positive tumor cells, mediated complement-dependent cytotoxicity, and exhibited protection against s.c. human GD2-positive melanoma growth in the severe combined immunodeficient mouse xenograft model. The results from our studies provide insights into approaches for boosting GD2 cross-reactive IgG antibody responses by minigene vaccination with a protective epitope of GD2 ganglioside. %B Cancer Research %V 65 %P 3410–8 %8 apr %@ 7168458906 %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/15833876 %R 10.1158/0008-5472.CAN-04-2164