Background: Oncoantigens are oncogenic and high immunogenicity proteins specifically expressed in cancer cells, and are promising targets for immunotherapy. Methods: We have established a strategy as follows to identify new oncoantigens; (1) screening of highly expressed genes in the majority of 120 llung cancers using cDNA microarray representing 27,648 genes, (2) verification of no expression of each gene in normal tissues, (3) validation of the clinicopathological significance of its high level of expression with tissue microarray covering 400 lung cancers, (4) characterization of a critical role of each gene in the growth or invasiveness of cancer cells by RNAi and cell growth/invasion assays, (5) screening of the epitope peptides recognized by HLA-A*0201- or A*2402-restricted cytotoxic T lymphocyte (CTL) for clinical trials. Results: We identified 40 oncoantigens and identified dozens of 10-amino-acid peptides, each of which was a candidate to be presented on the surface of HLA-A*0201 or HLA-A*2402 that induced in vitro CTL response. We conducted a phase I study for HLA-A*2402-positive, advanced non-small cell lung cancer patients who failed to standard therapy, using the combination of 1, 2 or 3 mg/body of each peptides from LY6K, CDCA1, and KIF20A mixed with adjuvant once a week. 18 evaluable patients have been enrolled, and this cancer vaccine therapy demonstrated tolerability and had very high immunogenicity of even 1 mg/body dose to induce antigen-specific CTLs in cancer patients. The clinical response of the vaccination was evaluated according to RECIST; one complete response case was observed, 8 patients showed stable disease, and 9 showed progressive disease, indicating that disease control rate was 50%. We are also screening predictive and monitoring biomarkers for peptide vaccine therapy through immunogenomics approach by analyzing pattern of CTL response, genomic/proteomic profiles of CTLs and cancer tissues, and genetic variation of patients. Conclusions: The cancer vaccine therapy using the cocktail of three peptides demonstrated good tolerability as well as the promising disease control rate, and therefore warrants further clinical studies with screening of their companion diagnostics. Clinical trial information: NCT01069575.