Identification of cyclin B1 as a new tumor antigen using a dendritic cell-based antigen discovery system

Successful immunotherapy of tumors relies in part on the identification of antigens capable of eliciting an effective immune response. To test a new method for tumor antigen discovery, we extracted peptides from immunoaffinity purified HLA class I molecules of an HLA-A2+ epithelial tumor cell line. The peptides were fractionated by RP-HPLC into 65 fractions and loaded onto in vitro generated dendritic cells to prime naive CD8+ T cells from a healthy donor. Twelve fractions that supported growth of tumor-specific CTLs were sequenced by electrospray ionization tandem mass spectrometry. One fraction yielded six peptides homologous to the human cyclin B1 sequence in the database. Four peptides were 9-mers (P1–P4) that differed only in the C terminal residue, and two were present as 10-mers (P5–P6). Tumor-derived peptides and the 9-mer (CB9) and 10-mer (CB10) from the database were synthesized and tested for binding to HLA-A2.1 and stimulation of CD8+ T cells. All peptides bound to HLA-A2.1. We were able to prime CD8+ T cell responses against P4 in a healthy donor and detect them by IFN-γ ELISPOT. Furthermore, four out of six breast and four out of five head & neck cancer patients exhibited strong secondary CD8+ T cell responses to cyclin B1 peptides. Immunohistochemical staining of the original tumor cell line used for peptide purification and a lung adenocarcinoma cell line showed marked overexpression of cyclin B1 compared to normal cells, with aberrant localization in the cytoplasm rather than the nucleus. This same pattern of overexpression and aberrant localization was also found in tumors of head and neck cancer patients who exhibited HLA class I-restricted T cell responses against the cyclin B1 peptides. Analysis of the genomic DNA and cDNA region encoding our peptide failed to detect any mutations in the DNA or transcription errors in the mRNA, suggesting that this may be the result of mistranslated defective ribosomal products, or DRiPs. Collectively, these results illustrate (a) a novel approach to tumor antigen discovery based on the in vitro priming of naive T cells with dendritic cells, and (b) the identification of cyclin B1 as a new epithelial shared tumor-associated antigen.