Identification of beta-tubulin isoforms as tumor antigens in neuroblastoma

There is currently substantial interest in the identification of human tumo r antigens for diagnosis and immunotherapy of cancer. We have implemented a proteomic approach for the identification of tumor proteins that elicit a humoral response in cancer patients, which we have applied to neuroblastoma , Proteins from neuroblastoma tumors and cell lines were separated by two-d imensional PAGE and transferred to poly(vinylidene difluoride) membranes. S era from 23 newly diagnosed patients with neuroblastoma, from 12 newly diag nosed children with other solid tumors, and from 13 normal individuals were screened for IgG and IgM autoantibodies against neuroblastoma proteins by means of Western blot analysis. Sera from 11 patients with neuroblastoma an d from 1 patient with a primitive neuroectodermal tumor, but none of the ot her controls exhibited IgG-based reactivity against a protein constellation with an estimated M-r 50,000. NH2-terminal sequence and mass spectrometric analysis identified the major constituents of this constellation as beta-t ubulin isoforms I and III. The IgG antibodies were additionally characteriz ed to be of the subclass IgG1, Neuroblastoma patient sera that contained an ti-beta-tubulin IgG antibodies also contained IgM antibodies specific again st the full-length beta-tubulin molecule and against COOH-terminal beta-tub ulin cleavage products. Neuroblastoma patient sera that reacted with beta-t ubulin I and LII isoforms in neuroblastoma tissues did not react with beta- tubulin I and III isoforms found in normal brain tissue. Our findings indic ate the occurrence of beta-tubulin peptides in neuroblastoma, which are imm unogenic. The occurrence of immunogenic peptides in neuroblastoma may have utility in diagnosis and in immunotherapy of this aggressive childhood tumo r.