A novel anti-HER2 anthracycline-based antibody-drug conjugate induces adaptive anti-tumor immunity and potentiates PD-1 blockade in breast cancer

Abstract

Increasing evidence suggests that antibody-drug conjugates (ADCs) can enhance anti-tumor immunity and improve clinical outcome. Here, we elucidate the therapeutic efficacy and immune-mediated mechanisms of a novel HER2-targeting ADC bearing a potent anthracycline derivate as payload (T-PNU) in a human HER2-expressing syngeneic breast cancer model resistant to trastuzumab and ado-trastuzumab emtansine. Mechanistically, the anthracycline component of the novel ADC induced immunogenic cell death leading to exposure and secretion of danger-associated molecular signals. RNA sequencing derived immunogenomic signatures and TCRβ clonotype analysis of tumor-infiltrating lymphocytes revealed a prominent role of the adaptive immune system in the regulation of T-PNU mediated anti-cancer activity. Depletion of CD8 T cells severely reduced T-PNU efficacy, thus confirming the role of cytotoxic T cells as drivers of the T-PNU mediated anti-tumor immune response. Furthermore, T-PNU therapy promoted immunological memory formation in tumor-bearing animals protecting those from tumor rechallenge. Finally, the combination of T-PNU and checkpoint inhibition, such as α-PD1, significantly enhanced tumor eradication following the treatment. In summary, a novel PNU-armed, HER2-targeting ADC elicited long-lasting immune protection in a murine orthotopic breast cancer model resistant to other HER2-directed therapies. Our findings delineate the therapeutic potential of this novel ADC payload and support its clinical development for breast cancer patients and potentially other HER2 expressing malignancies.

Keywords: Anthracycline; Antibody-drug conjugates; Checkpoint inhibitor combination therapy; HER2-positive breast cancer.

Fig. 1

T-PNU strongly affects tumor growth in hHER2 orthotopic breast cancer model unresponsive to T-DM1 treatment. a, Therapeutic response of EMT6-hHER2 tumor-bearing mice following treatment with T-PNU (1 mg/kg, 2x) and T-DM1 (15 mg/kg, 2x). Mice were treated when tumors reached an average tumor volume of 80 mm³ and were euthanized at 1200 mm³. Pooled data from at least three independent experiments (n = number of cured mice out of treated animals as indicated). b, Overall survival curves showing results of a. c, Therapeutic response of EMT6-WT tumor-bearing mice following treatment with T-PNU (1 mg/kg, 2x). Pooled data from two independent experiments (n = number of cured mice out of treated animals as indicated. d, Overall survival curves showing results of c

Fig. 2

T-PNU displays immunogenic cell death properties. a, EMT6-hHER2 cell death kinetics induced by T-PNU. Percentage of early (annexin V⁺/DAPI⁻) and late (annexin V⁺/DAPI⁺) apoptotic cells and necrotic cells (annexin V⁻/DAPI⁺) was determined by flow cytometry. Data presented as mean ± SD for 3 independent experiments. b, Representative dot plots of flow cytometry analysis of cell death induced by T-PNU, idarubicine and UVB radiation at indicated time points. c, T-PNU induction of HSP70, HSP90 and CRT on EMT6-hHER2 cells. UVB treated cells are used as controls. Experiments were performed in triplicates. d, Representative immunofluorescence staining of HSP70, HSP90 and CRT on EMT6-hHER2 following T-PNU treatment. The presence of indicated markers on the cell surface was verified by confocal microscopy. e, T-PNU induces oxidative stress and production of reactive oxygen species (ROS). ROS were stained by CellRox Deep Red reagent and detected by flow cytometry. Experiments were performed in triplicates. f, Quantification of intracellular or extracellular ATP levels in EMT6-hHER2 cell line exposed to T-PNU, idarubicine and UVB. Data presented as mean ± SD for 3 independent experiments. g, HMGB1 release into EMT6-hHER2 culture supernatants induced by T-PNU. The compiled results of a total of 3 experiments are shown

Fig. 3

T-PNU induced tumor control involves immune responses. a, Experimental design of RNA-sequencing of CD45⁺ immune cells after treatment: Four cohorts of EMT6-hHER2 tumor-bearing mice (n = 6) received following treatments: (1) untreated, (2) trastuzumab (20 mg/kg, 1x), (3) T-DM1 (15 mg/kg, 1x), and (4) T-PNU (1 mg/kg, 1x). Tumors were isolated when reached an average of 30 mm³ and CD45-positive cells were isolated with magnetic beads selection. Extracted RNA was subjected to RNA-sequencing. b, Heatmap of a customized immune-specific gene panel. c, Heatmap of an immune phenotype gene panel including adaptive and innate immune response. Asterisks denote low-responding T-PNU samples

Fig. 4

The T-PNU anti-tumor response consists of infiltrating and activated T cells. a, Gene set enrichment analysis of gene sets involved in T cell activation. Depicted is T-PNU vs. untreated. FDR: false discovery rate. b, Network cluster of gene sets with overlapping genes with TCR pathway function. The network includes 22 gene sets and 5448 genes (see Additional file 1: Table S1). c-e, Heatmaps of selected BIOCARTA THELPER, IL12 and TH1TH2 pathway gene sets, respectively (see Additional file 1: Figure S5 and S6 for more gene set heatmaps). f, MiXCR TCRβ CDR3 clonotype analysis. TCRβ CDR3 diversity (unique clones) plotted against TCRβ CDR3 abundance (read count). Donut plots depict clonotype distribution for one selected sample per cohort

Fig. 5

T-PNU anti-tumor protection is CD8 T cell-dependent. a, Tumor growth curves of EMT6-hHER2 tumor-bearing animals upon treatment with T-PNU (1 mg/kg, 2x), α-CD8 (10 mg/kg), or α-CD8 followed by T-PNU treatment, P-value < 0.0001 *** (Gehan-Breslow-Wilcoxon Test). b, Overall survival curves of a

Fig. 6

T-PNU promotes long-lasting immune protection. a, Outline of a representative rechallenge experiment. b, EMT6-hHER2 tumor-free animals after T-PNU treatment were rechallenged with EMT6-hHER2, EMT6-WT and TS/A Thy1.1 cancer cells. P-value < 0.0005 *** (Gehan-Breslow-Wilcoxon Test). c, Tumor growth of untreated controls. d, Overall survival curves of b and c

Fig. 7

T-PNU treatment in combination with checkpoint inhibitor therapy. a, Therapeutic response of EMT6-hHER2 animals with a tumor volume of 150–200 mm³ following treatment with T-PNU (1 mg/kg, 2x) alone, α-PD1 alone (12.5 mg/kg) or their combination (T-PNU + α-PD1) p-value< 0.0001*** (Gehan-Breslow-Wilcoxon Test) in between all the survival curves. b, Survival curves of a