FIGURE 2 from Estrogen Receptor Mutations as Novel Targets for Immunotherapy in Metastatic Estrogen Receptor–positive Breast Cancer

Kinetics of peptide: HLA-A*0201 dissociation. T2 cells were pulsed with indicated peptides for 8–12 hours, washed, and then stained for surface HLA-A*0201 expression at indicated timepoints for wildtype-1 and E380Q (A), wildtype-2 and Y537S (B), wildtype-2 and D538G (C). MFI for each timepoint was normalized to initial staining (time = 0). Linear regression analysis was used to plot lines of best fit for each individual peptide, and half-life for each peptide is shown in Table 2. Data are pooled from three independent experiments performed in triplicate.</p

FIGURE 4 from Estrogen Receptor Mutations as Novel Targets for Immunotherapy in Metastatic Estrogen Receptor–positive Breast Cancer

Expanded CD8+ T cells demonstrate antigen-specific cytotoxicity. T2 cells were pulsed with corresponding peptides and labeled with calcein-AM. Cytotoxicity was determined by a standard calcein-AM release assay. Wildtype-1 (A), E380Q (B), Wildtype-2 (C), Y537S (D), D538G (E). Non-pulsed T2 cells were used as negative controls. Statistical significance was determined via unpaired Student t test. Data represent the average of three to four experiments from separate healthy female donors run in triplicate. *, P P < 0.001.</p

TABLE 1 from Estrogen Receptor Mutations as Novel Targets for Immunotherapy in Metastatic Estrogen Receptor–positive Breast Cancer

Identification of candidate ESR1-derived peptides</p

Supplementary Table S4 from Estrogen Receptor Mutations as Novel Targets for Immunotherapy in Metastatic Estrogen Receptor–positive Breast Cancer

Frequency of most common ESR1 mutations in key clinical studies</p

FIGURE 3 from Estrogen Receptor Mutations as Novel Targets for Immunotherapy in Metastatic Estrogen Receptor–positive Breast Cancer

Peptide-specific expansion of CD8+ T cells. PBMCs from healthy donors were isolated and expanded as described in the Materials and Methods. Tetramer staining was performed pre- and post-expansion to quantify frequencies of peptide-specific CD8+ T cells. A, Wildtype-1, E380Q. B, Wildtype-2, Y537S, D538G. Data represents three independent experiments, from 3 separate donors, performed in triplicate. Statistical significance was determined via comparison of pre-expansion versus post-expansion CD8+tetramer+ population using unpaired Student t test. *, P < 0.05.</p

TABLE 2 from Estrogen Receptor Mutations as Novel Targets for Immunotherapy in Metastatic Estrogen Receptor–positive Breast Cancer

Stability of candidate peptides to HLA-A*0201</p

Supplementary Figure S1 from Estrogen Receptor Mutations as Novel Targets for Immunotherapy in Metastatic Estrogen Receptor–positive Breast Cancer

Kinetics of Overlapping peptide</p

Supplementary Figure S2 from Estrogen Receptor Mutations as Novel Targets for Immunotherapy in Metastatic Estrogen Receptor–positive Breast Cancer

Expression of ESR1 in various Healthy and Malignant Tissues</p

FIGURE 5 from Estrogen Receptor Mutations as Novel Targets for Immunotherapy in Metastatic Estrogen Receptor–positive Breast Cancer

Peptide-specific CTLs effectively kill ER+ breast tumor cells. MCF7 cells were pulsed with corresponding peptides and labeled with calcein-AM. Cytotoxicity was determined by a standard calcein-AM release assay. MCF7 cells were pulsed with corresponding peptides. CG1-pulsed MCF7 cells and non-pulsed MCF7 cells were used as negative controls. Statistical significance was determined via one-way ANOVA. Data show percent cytotoxicity at the 10:1 effector:target ratio, and represent the average cytotoxicity of five experiments (run in triplicate) from different healthy female donors. *, P P < 0.001.</p

FIGURE 1 from Estrogen Receptor Mutations as Novel Targets for Immunotherapy in Metastatic Estrogen Receptor–positive Breast Cancer

Peptides bind to HLA-A*0201. T2 cells were pulsed with indicated peptide for 4 hours and surface HLA-A*0201 was stained. MFI of pulsed T2 cells were normalized to experimentally matched non-pulsed T2 cells, and data expressed as fold change in MFI. Dashed horizontal line indicates non-pulsed T2 HLA-A*0201. FLU peptide was used as a positive control HLA-A*0201 peptide known to bind with high affinity. Data represent three independent experiments performed in triplicate. Statistical significance was determined via comparison of pulsed MFI versus non-pulsed raw MFI using unpaired Student t test. ***, P < 0.001.</p