Peptide-based epitope mapping in FabC4 antibody characterization

Phage Display is a molecular technique by which exogenous proteins are expressed on the virus surface. It is an extremely powerful tool to select peptides with specific binding properties from a wide number of variants. Regarding tumors, Phage Display is a promising technology for the selection of targets with clinical relevance, being capable of recognize cancer molecular diversity. Here we describe the characterization of a new FabC4 antibody, made with Phage Display technique, clinically relevant for breast cancer (BC) diagnosis and prognostic purposes. In this study, a bioprospecting assay was done against FabC4 in order to map its epitopes, after using a subtractive selection against an irrelevant Fab, and elution in two steps: with non-malignant proteins, which were discarded, and against BC proteins, which were amplified. By using Phage-ELISA, four of selected clones were able to differentiate serum samples of patients with BC, from serum of patients with benign disease and healthy women. The peptides were chemically synthesized (pA5, pA7, pC4 e pD6) and bound to FabC4. The pC4 demonstrated higher specificity when compared to the others peptides (p<0.05). However, only pD6 peptide was able to differentiate neoplastic samples. Also, we showed for the first time, by immunofluorescence, the cytoplasmic co-localization of annexin A2 (ANXA2) and cytokeratin 10 (CK10) in MDA-MB-231 cell line, suggesting a new molecular behavior of these proteins on triple-negative BC (TNBC). Molecular docking analysis confirmed interactions between FabC4 and the peptides, with better coverage and identity for pC4. This peptide also mimicked ANXA2 and CK10 protein regions that bind to FabC4. Therefore, we described new molecules for the diagnosis of TNBC and our strategy resulted on a successful mapping of FabC4 epitopes, opening new perspectives to better understand and treat TNBC.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorDissertação (Mestrado)Phage Display é uma técnica molecular pela qual proteínas exógenas são expressas na superfície de partículas virais. Esta é uma ferramenta extremamente poderosa para selecionar peptídeos ligantes específicos dentre um vasto número de variantes. Considerando tumores, é uma tecnologia promissora na seleção de alvos com relevância clínica, capaz de reconhecer sua diversidade molecular. Nós caracterizamos, por Phage Display, um novo anticorpo FabC4 com relevância clínica no diagnóstico e prognóstico do Câncer de Mama (CM). Neste estudo, foi realizado um ensaio de bioprospecção contra o FabC4 para mapear seus epítopos, após seleção subtrativa contra um Fab irrelevante e eluição em duas etapas: contra proteínas não malignas, que foram descartadas, e contra proteínas de CM, as quais foram amplificadas. Quatro dos clones selecionados diferenciaram, por Phage-ELISA, amostras de soros de pacientes com CM, de doença benigna da mama (DBM) e de mulheres saudáveis. Os peptídeos correspondentes quimicamente sintetizados (pA5, pA7, pC4 e pD6) se ligaram ao FabC4. O que apresentou maior afinidade ao FabC4 foi o pC4 (p