Henle, Andrea M.

Abstract HER2 is a prime target for cancer therapeutics due to its high overexpression in tumors. Advanced clinical trials, in breast and prostate cancer, investigating the use of the HLA-A2-binding HER2 peptide p369-377 (E75) as a vaccine are ongoing. In the present study, we investigated the proteasomal processing of p369-377 and other epitopes from HER2. We did not detect processing of p369-377 by either the proteasome or immunoproteasome in vitro, suggesting that tumor cells do not naturally process this epitope from HER2 for presentation on HLA-A2. However, other HER2 peptides were found to be consistently processed by the proteasome and immunoproteasome. One newly identified processed peptide, HER2 p373-382, was found to bind HLA-A2 stronger than p369-377, despite algorithms that predicted p369-377 as a stronger binder. Cytotoxic T lymphocytes (CTL) specific for p373-382 recognized target cells pulsed with either p373-382 or p369-377. CTL specific for p373-382 also lysed HER2+HLA-A2+ human breast cancer cells more efficiently than p369-377-specific CTL. These results suggest that cancer cells are naturally processing and presenting p373-382. Furthermore, our results suggest that T cells generated against p369-377 in previous studies may have been cross-reacting with p373-382. Thus, the newly identified p373-382 peptide may be a more appropriate candidate for vaccines with this new evidence that it is processed and presented by HLA-A2 for recognition by HER2-specific CTL.

Abstract Patients with HER-2/neu–expressing breast cancer remain at risk for relapse following standard therapy. Vaccines targeting HER-2/neu to prevent relapse are in various phases of clinical testing. Many vaccines incorporate the HER-2/neu HLA-A2–binding peptide p369–377 (KIFGSLAFL), because it has been shown that CTLs specific for this epitope can directly kill HER-2/neu–overexpressing breast cancer cells. Thus, understanding how tumors process this epitope may be important for identifying those patients who would benefit from immunization. Proteasome preparations were used to determine if p369–377 was processed from larger HER-2/neu–derived fragments. HPLC, mass spectrometry, cytotoxicity assays, IFN-γ ELISPOT, and human breast cancer cell lines were used to assess the proteolytic fragments. Processing of p369–377 was not detected by purified 20S proteasome and immunoproteasome, indicating that tumor cells may not be capable of processing this Ag from the HER-2/neu protein and presenting it in the context of HLA class I. Instead, we show that other extracellular domain HER-2/neu peptide sequences are consistently processed by the proteasomes. One of these sequences, p373–382 (SLAFLPESFD), bound HLA-A2 stronger than did p369–377. CTLs specific for p373–382 recognized both p373–382 and p369–377 complexed with HLA-A2. CTLs specific for p373–382 also killed human breast cancer cell lines at higher levels than did CTLs specific for p369–377. Conversely, CTLs specific for p369–377 recognized p373–382. Peptide p373–382 is a candidate epitope for breast cancer vaccines, as it is processed by proteasomes and binds HLA-A2.