Pept?deos ligantes de c?lulas tumorais e de imunoglobulinas g espec?ficos do c?ncer de mama.

At? o momento, n?o existem biomoleculares que sejam efetivamente utilizados no diagn?stico do c?ncer de mama. Uma alternativa para auxiliar a busca por marcadores moleculares ? baseada na sele??o de ligantes de alta afinidade por meio de Phage Display (PD). O principal objetivo desta tese foi o desenvolvimento de novos biomarcadores envolvidos no c?ncer de mama selecionados contra uma linhagem celular tumoral altamente agressiva e contra IgG purificada espec?fica de tecido mam?rio de pacientes. Ap?s a sele??o PD em biblioteca aleat?ria de pept?deos, todos os clones imunorreativos foram caracterizados por sequenciamento, deduzidos in vitro e submetidos a an?lises in silico. Valida??es posteriores foram conduzidas com ELISA e an?lise histopatol?gica. Apesar de terem sido relatadas diversas varia??es da metodologia, a maior parte dos processos de sele??o usou ligantes obtidos depois de tr?s ciclos de sele??o. Os marcadores relatados na literatura foram pouco explorados devido ? complexidade dos processos de valida??o. Este trabalho demonstrou que as sele??es do primeiro e segundo ciclos s?o vi?veis e podem ser utilizadas para aumentar o n?mero de potenciais marcadores a serem testados. Os resultados com linhagem de c?lulas tumorais identificaram tr?s potenciais marcadores no terceiro ciclo de sele??o, dos quais foi selecionado aquele com melhor rela??o de absorb?ncia tumor/controle no ELISA para s?ntese qu?mica. O pept?deo sint?tico foi associado com uma marca??o menos intensa em tumores triplo negativos (Receptor de Estr?geno, Receptor de Progesterona e ErbB2). Outros pept?deos do segundo ciclo tamb?m demonstraram especificidade a tecidos tumorais. Para a estrat?gia de mapeamento de ant?genos contra IgG purificada espec?fica de tecido mam?rio, um pept?deo (F4) pode reconhecer tanto tecidos tumorais de mama quanto IgG circulante. Ambos clone F4 e pept?deo sint?tico SF4 alcan?aram diagn?stico de alta acur?cia, mas surpreendentemente, o clone F4 apresentou a melhor sensibilidade e especificidade (77,8% e 85,7%, respectivamente), sugerindo que pode ser utilizado no diagn?stico para triagem precoce de c?ncer de mama, anterior ?s an?lises por imagem e patol?gicas. A identifica??o e caracteriza??o do biomarcador putativo est? em investiga??o por meio de sele??o combinatorial de anticorpos scFv contra o fago F4 e o pept?deo SF4, os quais ser?o utilizados para captura imunol?gica do ant?geno verdadeiro e para an?lise por espectrometria de massas. Um sensor eletroqu?mico de baixo custo utilizando o pept?deo SF4 tamb?m foi desenvolvido.Currently there are no biomarkers that have effectively been used in breast cancer (BC) diagnosis, which has been accomplished mainly based on mammography and pathological analysis of biopsy specimens. An alternative technology for the discovery of markers is through selection of high affinity ligands by Phage Display (PD). The main objective of this investigation was the development of new biomarkers involved in breast cancer that were selected against a highly aggressive tumor cell lineage and against purified tissue-specific IgG from BC patients. After PD selections of random peptide libraries, all immunoreactive ligands were further characterized by sequencing, in vitro translated and submitted to bioinformatic analyses. Further validations were performed by ELISA and immunohistochemistry. Although many variations of the methodology have been reported, most of the selection processes have used ligands obtained after three or more cycles of selection, but successful markers have been marginally explored due to complex validation processes. We have demonstrated that selections in the first and second cycles are feasible and can be used to increase the number of potential markers to be tested. Furthermore, results from the tumor cell lineage also identified three potential peptide markers in the third cycle that presented the best relation was chosen for peptide synthesis. The synthetic peptide was associated to less intense labeling of triple negative tumors (Estrogen Receptor, Progesterone receptor, and ErbB2). Other peptides from the second cycle also seemed very specific to tumor tissues. For the antigen mapping strategy against purified tissue-specific IgG from patients? tumor biopsies, one peptide (F4) could recognize both breast tumor tissues as well as circulating IgG from patients. Both F4 clone and its synthetic peptide reached a very high accurate diagnosis, but surprisingly, the selected F4 clone presented the highest sensitivity and specificity, (77.8% and 85.7%, respectively), suggesting that it can be used as a diagnostic reagent for early BC screening prior to imaging and pathological analyses. Identification and characterization of the putative biomarker is under investigation through Fab combinatorial antibody selection against both F4 clone and peptide, which will be used for immunological capture of the true antigen and for mass spectrometry analysis. A low cost electrochemical sensor using the SF4 peptide was also developed