Abstract 534: Trastuzumab increases uptake and cross-presentation of HER2-derived antigens by dendritic cells
Authors: Gall Victor A. , Philips Anne V. , Qiao Na , Dwyer Karen C. , Perakis Alexander A. , Zhang Mao , Sukhumalchandra Pariya , Molldrem Jeffrey J. , Alatrash Gheath , Mittendorf Elizabeth A. 
Published: July 2016

Abstract
Introduction:
The purpose of this study was to identify the effect of trastuzumab on the uptake and presentation of HER2-derived peptides by dendritic cells (DCs). Clinical trials for HER2-derived peptide vaccines have shown improved outcomes for patients receiving a combination of vaccine and trastuzumab suggesting potential synergy between the two. We hypothesized that antigen processing and cross-presentation would be enhanced via trastuzumab facilitating antigen presenting cell (APC) uptake of HER2 shed from breast cancer cells.
Methods:
An ELISA assay was used to evaluate HER2 shedding in multiple cancer cell lines (SKBR3, SKOV3, BT474, MDA-MB-231). To look for uptake of HER2 shed from breast cancer cells into culture media by APCs, DCs were generated by incubating healthy donor monocytes in media supplemented with GM-CSF, IL-4, and TNF-α. These DCs were co-incubated with SKBR3, BT474, and SKOV3 (high HER2 expressers) as well as MDA-MB-231 (low HER2 expresser) in the presence of trastuzumab. Rituximab was used as an isotype control to determine trastuzumab specificity and an Fc blocker was used to evaluate the importance of the Fc receptor in the uptake mechanism. Cells were then stained for DC markers, including CD11c and HLA-DR, permeabilized and stained for HER2 to detect uptake, and then analyzed using flow cytometry. To evaluate antigen cross-presentation, antigen-specific T cells were expanded by co-culturing healthy donor PBMCs with DCs that have been pulsed with SKBR3 cells or trastuzumab-treated SKBR3 cells. DCs pulsed with E75 peptide (HER2, aa: 369-377) were used as a control. After T cell activation and expansion, the number of E75-specfic CD8+ T cells was enumerated using an E75-dextramer assay.
Results:
When compared to SKOV3 and MBA-MB-231 cells, SKBR3 and BT474 cells shed a significantly higher concentration of HER2 into the growth media. DCs took up HER2 when co-cultured with cell lines with higher levels of HER2 shedding (SKBR3, BT474). Trastuzumab treatment of SKBR3 resulted in a 50% increase in HER2 uptake by DCs at 24 hours (p = 0.0014). This finding persisted among all trastuzmab doses. Treatment with rituximab showed no significant increase in HER2 uptake when compared to untreated SKBR3. The effect of trastuzumab on uptake was abrogated by adding an Fc blocking agent. DCs treated with SKBR3 and trastuzumab also led to an increase in the generation of E75-CTLs as measured using a dextramer assay.
Conclusions:
Trastuzumab treatment leads to increased uptake of soluble HER2 by DCs in vitro. This effect is specific to trastuzumab and is Fc receptor mediated. Increased HER2 uptake led to increased E75-CTL generation secondary to increased cross-presentation of E75 by DCs. These data have global implications for HER2-targeting vaccine approaches. Specifically, by enhancing antigen presentation, trastuzumab can augment the immune response initiated by a peptide vaccine.
Citation Format: Victor A. Gall, Anne V. Philips, Na Qiao, Karen C. Dwyer, Alexander A. Perakis, Mao Zhang, Pariya Sukhumalchandra, Jeffrey J. Molldrem, Gheath Alatrash, Elizabeth A. Mittendorf. Trastuzumab increases uptake and cross-presentation of HER2-derived antigens by dendritic cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 534.