Abstract 1271: A novel method to identify HER2-derived HLA-A2 epitopes for use in combination immunotherapy with Trastuzumab.
Authors: Qiao Na , Chawla Akhil , Alatrash Gheath , Philips Anne , Peoples George , Ponniah Sathibalan , Mittendorf Elizabeth A. 
Published: August 2014

Introduction: Several MHC class I HER2-derived peptides have been identified that elicit a peptide-specific, cytotoxic T lymphocyte (CTL) response. We have postulated that Trastuzumab (Tz) binding may protect or modulate enzymatic cleavage sites on HER2 normally accessible by proteasomes thereby altering the cleavage pattern and resulting peptide profile. We have previously reported that Tz-treated breast cancer cells are recognized and killed more efficiently than untreated cells by T cells stimulated with the HER2-derived peptide E75 (aa:369-377). In the current study, we show that unique peptides may be located within the Tz binding sequence and that treatment with Tz may generate novel immunogenic peptides that could be used as vaccines in combination immunotherapy.

Methods: The binding site of Tz on the HER2 protein was analyzed for HLA-A2 binding peptides. Five nonameric peptides were identified, synthesized and tested. HLA-stabilization assays using T2 cells were performed to confirm HLA-A2 binding. HER577 (aa:577-585; FGPEADQCV) demonstrated the highest affinity and was further studied. PBMC from healthy donors were stimulated with HER577 and tested in standard cytotoxicity assays (CTX) against HER2-expressing tumor cell lines that were untreated or pre-treated with Tz. NK cell depletion was performed to ensure that the effects were not due to ADCC. The effect of Tz treatment on cell surface MHC class I protein expression was also determined.

Results: HER577 (aa:577-585; FGPEADQCV) showed binding affinity to HLA-A2 stronger than that of E75 (MFI = 532 for HER577 and 332 for E75). HER577 and E75 both had a n off-rate of t1/2 &gt; 8 hr. CTX assays using MCF7 (HLA-A2+, low HER2) showed specific cytotoxicity by HER577-CTL (45±3%) that was similar to E75-CTL (51±4%). CTX assays evaluating Her577-CTL versus MCF7 cells pretreated with Tz resulted in a 16% increase in specific cytotoxicity. In MCF7 cells transfected to overexpress HER2 (HER18) the specific lysis was 30±5% which increased to 51±5% (p=0.05) with Tz treatment. Similarly, with SKBr3-A2 cells (SKBr3 cells transfected to express HLA-A2), the specific lysis was 44±5% which increased to 62±3% (p=0.02) with Tz treatment. Tz treatment of a panel of breast cancer cell lines with varying HER2 expression demonstrated no changes in HLA-A2 or HLA-ABC expression, confirming the increased killing after Tz treatment was caused by specific expression of the peptide/HLA-A2 complex.

Conclusion: HER577 is a class I HER2-derived epitope identified using a novel method for the discovery of immunogenic peptides for vaccine development. Further investigation of combination immunotherapy with therapeutic antibodies and unique peptide vaccines is warranted.

Citation Format: Na Qiao, Akhil Chawla, Gheath Alatrash, Anne Philips, George Peoples, Sathibalan Ponniah, Elizabeth A. Mittendorf. A novel method to identify HER2-derived HLA-A2 epitopes for use in combination immunotherapy with Trastuzumab. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1271. doi:10.1158/1538-7445.AM2013-1271