Trastuzumab Increases HER2 Uptake and Cross-Presentation by Dendritic Cells

Abstract

Early-phase clinical trials evaluating CD8⁺ T cell-eliciting, HER2-derived peptide vaccines administered to HER2⁺ breast cancer patients in the adjuvant setting suggest synergy between the vaccines and trastuzumab, the mAb targeting the HER2 protein. Among 60 patients enrolled in clinical trials evaluating the E75 + GM-CSF and GP2 + GM-CSF vaccines, there have been no recurrences in patients vaccinated after receiving trastuzumab as part of standard therapy in the per treatment analyses conducted after a median follow-up of greater than 34 months. Here, we describe a mechanism by which this synergy may occur. Flow cytometry showed that trastuzumab facilitated uptake of HER2 by dendritic cells (DC), which was mediated by the Fc receptor and was specific to trastuzumab. In vitro, increased HER2 uptake by DC increased cross-presentation of E75, the immunodominant epitope derived from the HER2 protein, an observation confirmed in two in vivo mouse models. This increased E75 cross-presentation, mediated by trastuzumab treatment, enabled more efficient expansion of E75-specific cytotoxic T cells (E75-CTL). These results demonstrate a mechanism by which trastuzumab links innate and adaptive immunity by facilitating activation of antigen-specific T cells. On the basis of these data, we conclude that HER2-positive breast cancer patients that have been treated with trastuzumab may experience a more robust antitumor immune response by restimulation of T cells with the E75 peptide vaccine, thereby accounting for the improved disease-free survival observed with combination therapy. Cancer Res; 77(19); 5374-83. ©2017 AACR.

Figure 1

Surface expression and shedding of HER2 by cancer cell lines. A, surface expression of HER2 measured by flow cytometry in multiple breast cancer cell lines Results indicate high HER2 surface expression by the breast cancer cell lines SKBR3 and BT474, and the SKOV3 ovarian cancer cell line. MDA-MB-231 cells, express low levels of HER2. B, levels of soluble HER2 shedding by multiple cancer cell lines as determined by ELISA. SKBR3, BT474, SKOV3, and MDA-MB-231 cells were cultured for 48 hours in serum free media after which the supernatant was assayed for HER2 concentration by ELISA. SKBR3 and BT474 cells shed greater amounts of HER2 into the culture media compared with SKOV3 and MDA-MB-231 cells.

Figure 2

Trastuzumab enhances HER2 uptake by DCs. A, mature DCs were co-cultured with SKBR3 breast cancer cells. Cells were then stained for DC markers (CD11c⁺, HLA-DR⁺) and intracellular HER2 and analyzed using Amnis® imaging flow cytometry which demonstrated uptake of HER2 by DCs. B, mature DCs were co-cultured with SKBR3, SKOV3, BT474, or MDA-MB-231 cells for 24 hours +/− trastuzumab (10 µg/ml). Cells were then stained for DC markers (CD11c⁺, HLA-DR⁺) and intracellular HER2 and analyzed by flow cytometry. Results are expressed as average HER2 MFI for DCs cultured with each cancer cell line +/− trastuzumab. Our results show increased HER2 uptake when DCs were in culture with SKBR3 and BT474 cells (high HER2 shedding) when compared with SKOV3 and MDA-MB-231 cells (low HER2 shedding). *** indicates P <0.0001

Figure 3

Effect of treatment time and dose of trastuzumab on HER2 uptake by DCs. A, mature DCs were co-cultured with SKBR3 cells +/− trastuzumab at increasing durations up to 24 hours. Cells were then surface-stained for DC markers (CD11c⁺, HLA-DR⁺) and intracellular HER2 and analyzed using flow cytometry. HER2 uptake by DCs is expressed using average HER2 MFI. Increasing HER2 uptake is observed over time with trastuzumab contributing significantly to uptake at 24 hours of treatment. B, mature DCs were co-cultured with SKBR3 cells at various doses of trastuzumab. Cells were stained for DC markers (CD11c⁺, HLA-DR⁺) and intracellular HER2 and analyzed by flow cytometry. HER2 uptake by DCs is expressed using fold change in average HER2 MFI. All comparisons were made to DCs co-cultured with SKBR3 cells alone. A 50% increase in HER2 uptake is noted with trastuzumab treatment at all doses tested with little difference between low (10 µg/mL) and high (80 µg/mL) dose treatment. * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.0001

Figure 4

Trastuzumab’s enhancing effect on HER2 uptake by DCs is specific to trastuzumab and is mediated by the Fc receptor. A, mature DCs were co-cultured with SKBR3 cells and trastuzumab (10 µg/mL) +/− human FcR binding inhibitor. Cells were stained for DC markers (CD11c⁺, HLA-DR⁺) and intracellular HER2 and analyzed with flow cytometry. HER2 uptake by DCs is expressed as fold change in average HER2 MFI when compared to DCs alone. Data indicate that FcR inhibition abrogates the effect of trastuzumab on HER2 uptake by DCs. B, mature DCs were co-cultured with SKBR3 cells +/− trastuzumab or rituximab for 24 hours. Rituximab (10 µg/mL) was used as an antibody isotype control. Cells were stained for DC markers (CD11c⁺, HLA-DR⁺) and intracellular HER2 and analyzed with flow cytometry. Results are represented by average HER2 MFI. No significant increase in HER2 uptake was noted in DCs cultured with SKBR3 cells + rituximab compared to SKBR3 cells alone. * indicates P <0.05, *** indicates P <0.0001

Figure 5

Trastuzumab increases cross-presentation of E75 by DCs. A, mature DCs were cultured with SKBR3 cells +/− trastzumab (10 µg/mL) for 24 hours after which the cell surface was stained for DC markers (CD11c⁺, HLA-DR⁺) as well as an Fab targeting E75 peptide/HLA-A2. Cells were analyzed with flow cytometry and results are expressed as average E75/HLA-A2 Fab MFI. DCs cultured with SKBR3 cells + trastuzmab resulted in increased E75 cross-presentation compared to DCs cultured with SKBR3 cells alone. B, C mature DCs were cultured with SKBR3 cells +/− trastuzumab (10–40 µg/mL) to activate and expand E75-CTLs from HLA-A2⁺ PBMCs. After 1 week of co-culture, enumeration of E75-CTLs was performed using an E75/HLA-A2 dextramer. Results are expressed as percent CD8⁺/E75⁺ cells from a parent gating of Live/CD16⁻/CD19⁻/CD56⁻/CD14⁻/CD3⁺/CD8⁺ cells. DCs that had been cultured with SKBR3 cells + trastuzumab resulted in more efficient expansion of E75-CTLs from donor PBMCs. B, representative scatter plots. C, aggregate results of three experiments. * indicates P <0.05, ** indicates P <0.01

Figure 6

Treatment with 7.16.4 mAb increases expansion of antigen-specific CTLs in vivo. MMTV- neu⁺/H-2Kd⁺ female mice or Balb/C mice implanted with TUBO tumor cells were treated with 7.16.4 mAb (4 mg/kg) or mouse IgG2a isotype control (4 mg/kg) twice a week for one week during which pretreatment tail vain blood and tail vein blood after 1 week of treatment was sampled and stained for TYLPANASL-specific CTLs utilizing TYLPANASL/H-2Kd tetramer. A, a representative scatter plot from one mouse is shown to demonstrate gating strategy. FSC, forward scatter; SSC, side scatter. B, representative scatter plots demonstrating an increase in TYLPANASL-specific CTL after treatment with 7.16.4 mAb. Isotype antibody was used as a treatment control. C, Aggregate representation of the expansion of TYLPANASL-CTL from mice treated with 7.16.4 mAb (n=3 for each tumor model). Isotype antibody was used as a treatment control (n=2 for each experiment). * indicates P <0.05