RESUMO
Based on immune reactivity in response to a whole-cell colon tumor vaccine and using serological identification of Ags by recombinant cDNA expression cloning, we here describe the molecular and functional identification of a novel human tumor Ag. By screening a cDNA expression library derived from the coloncarcinoma cell line HT-29 with pooled colorectal cancer patients' sera, 26 clones reactive with IgG Abs could be identified. Characterization of these cDNA clones by sequence analysis and alignment, and detailed serological analysis revealed cancer-related immunoreactivity for the ErbB-3-binding protein-1 (Ebp1). Immunohistochemical staining of colorectal tumors and neighboring normal colon tissue indicated the observed cancer-related immunogenicity of Ebp1 to be related to overexpression. Via reverse immunology, five potential HLA-A2-restricted T cell epitopes were identified, of which two (Ebp1(45-54) and Ebp1(59-67)) bound HLA-A2 with intermediate and high affinity, respectively. Analysis of their immunogenicity in vitro indicated that only the high-affinity Ebp1(59) epitope gave rise to CD8(+) T cells capable of recognizing both exogenously loaded Ebp1 peptide and endogenously expressed Ebp1 on target cells. In addition, in vivo CD8(+) T cell responsiveness against the Ebp1(59) epitope could be detected in two of nine and three of six cancer patients PBMC and tumor draining lymph nodes, respectively, but not in nine of nine healthy donors tested. These data confirm that Ebp1 is an immunogenic protein, capable of eliciting CD8-mediated responses in vivo and in vitro, providing a rationale for further exploration of Ebp1 as a possible target for anticancer immunotherapy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Antígenos de Neoplasias/química , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/terapia , Imunização Passiva , Proteínas de Ligação a RNA/química , Receptor ErbB-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Clonagem Molecular , Neoplasias Colorretais/patologia , Testes Imunológicos de Citotoxicidade , DNA Complementar/biossíntese , Sistemas de Liberação de Medicamentos , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Biblioteca Gênica , Antígeno HLA-A2/imunologia , Células HT29 , Humanos , Imunização Passiva/métodos , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Linfócitos T Citotóxicos/imunologiaAssuntos
Adenocarcinoma/genética , Análise Mutacional de DNA/métodos , DNA de Neoplasias/análise , Genes ras/genética , Neoplasias Pulmonares/genética , Mutação Puntual , Escarro/química , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Exonucleases/metabolismo , Humanos , Escarro/citologia , Células Tumorais CultivadasRESUMO
In the last few years it has been shown that the humoral immune response in cancer patients is a rich source of putative cancer vaccine candidates. To fully explore the complex information present within the Ab repertoire of cancer patients, we have applied a method, serological Ag selection, to molecularly define tumor Ags recognized by the humoral immune response in colorectal cancer (CRC). First, we built a cDNA display library by cloning a cDNA library from CRC cell line HT-29 for expression as a fusion protein with a filamentous phage minor coat protein, pVI. This cDNA display library was then enriched on pooled sera from CRC patients who had undergone active specific immunization with autologous tumor. We identified a panel of 19 clones reactive with the serum pool. Seventeen of 19 (89%) clones showed reactivity with one or more of the eight Ag-reactive sera, conversely six of eight (75%) sera were reactive with at least one of the 19 clones. Sequencing revealed that these 19 clones represented 13 different Ags. A detailed serological analysis of the 13 different Ags showed preferential reactivity to sera of cancer patients for six different Ags. Four of these Ags displayed increased serum reactivity after the active specific immunization procedure. Furthermore, one of the six Ags, a novel Ag homologous to HSPC218, showed restricted expression in normal testis, suggesting that it belongs to the cancer-testis Ag family. Some of the Ags we have identified may be candidates for tumor vaccination, for sero-diagnosis of cancer, as prognostic markers, or as probes for monitoring tumor cell-based vaccination trials.
