Summary

Human epidermal growth factor receptor 2, HER2, is a membrane receptor tyrosine kinase overexpressed in 18–20% of gastric cancers. Gastric tumours are characterized by a heterogeneous pattern of HER2 expression, which can hinder response to HER2-targeted therapy. Beyond the observed efficacy for the HER2-targeting antibody trastuzumab and the antibody-drug conjugate (ADC) trastuzumab deruxtecan, clinical trials failed to demonstrate efficacy for other HER2-targeting antibodies. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) that targets HER2-positive (HER2⁺) cancer cells with a chemotherapeutic agent, emtansine. Emtansine is cytotoxic to HER2⁺ cells by inducing cell arrest and apoptosis through disruption of microtubule function. T-DM1 is used in the clinic for both adjuvant treatment of patients with HER2⁺ breast cancer with residual disease at surgery and those with HER2⁺ metastatic breast cancer. T-DM1 has low efficacy in HER2⁺ gastric cancer. Patients with HER2⁺ gastric cancer have demonstrated poor response to T-DM1 treatment, warranting novel strategies to be explored in preclinical models. The failure of studied agents in gastric cancer may be explained by spatial and temporal heterogeneity of HER2+ expression and differences in HER2 staining patterns between breast cancer and gastric cancer.

This study explored the efficacy of combining drugs known to modulate HER2 internalization to enhance T-DM1 efficacy in gastric cancer. To enhance HER2 membrane density cholesterol-depleting drugs (lovastatin) were used. The irreversible pan-HER tyrosine kinase inhibitor neratinib was used to enhance the internalization of HER2-bound T-DM1. An enhancement of cell surface and internalized HER2 was observed after lovastatin and neratinib incubations, respectively. The combination of lovastatin with neratinib enhanced T-DM1 internalization in cancer cells.

Results from nanoScan PET/CT

Mice in each treatment group were administered with [⁶⁴Cu]Cu-NOTA-trastuzumab through intravenous injection pre-therapy and post-therapy. At 24 h after [⁶⁴Cu]Cu-NOTA-trastuzumab injection, mice were anesthetized and were then imaged using the Mediso nanoScan PET/CT scanner (Mediso) at the preclinical imaging facility of Washington University in St. Louis. The images were analyzed using 3D Slicer software or Imalytics Preclinical software. PET images were calibrated as a percentage of injected dose per mL (%ID/mL) and scaled from 0 (min) to 35 (max). Regions of interest (ROI) were delineated in the tumour, and activity values were obtained as %ID/mL. The values represented in the graph were expressed as mean uptake %ID/mL in the tumour.

As shown in Figure 5A, ⁶⁴Cu-labeled trastuzumab was injected on day 1, followed by pre-therapy PET images. On day 29 after therapy initiation, ⁶⁴Cu-labeled trastuzumab was administered once again, followed by post-treatment PET imaging on day 30. The mice used in PET studies had similar tumour volumes when compared with pre- versus post-therapy and they were between 200 and 250 mm³ so that HER2 levels were monitored by PET imaging before major changes in tumour volume occurred. At day 63 after therapy initiation, tumours were excised and collected for future Western blot analyses.

PET images of T-DM1/neratinib and T-DM1/lovastatin/neratinib groups demonstrated a 1.7–2.4-fold decrease in ⁶⁴Cu-labeled trastuzumab uptake from pre- to post-therapy, indicating decreased HER2 expression in these groups (Figure 5B). No major changes in HER2-PET were observed for the cohorts neratinib or neratinib/lovastatin (Supplementary Figure 19).

Figure 5. A Schematic illustrating 64Cu-labeled trastuzumab PET/CT before and at 30 days after T-DM1/neratinib or T-DM1/lovastatin/neratinib therapy in nu/nu mice bearing NCIN87 tumours. Schematic created using Biorender.com. B Representative PET images of ⁶⁴Cu-labeled trastuzumab pre-therapy and post-therapy in T-DM1/neratinib or T-DM1/statin/neratinib treatment groups. PET images shown here were calibrated as a percentage of injected dose per mL (%ID/mL) and scaled from 0 (min) to 35 (max). Regions of interest (ROI) were delineated in the tumour, and activity values were obtained as %ID/cm3. The values shown here are expressed as mean uptake %ID/mL in the tumour. C Western blot of tumour lysates collected 63 days after therapy showing phosphorylated HER2, total HER2, and β-actin loading control levels in control, statin, neratinib, statin/neratinib, T-DM1, T-DM1/lovastatin, T-DM1/neratinib, T-DM1/lovastatin/neratinib groups. Full-length blots are included in the Supplementary Information document. Scheme made using Biorender.com.

Supplementary Figure 19. Representative PET images of 64Cu-labeled trastuzumab pre-therapy and post-therapy in neratinib and neratinib/statin treatment groups.

Western blot analyses demonstrated a reduction in total HER2 protein, as well as other proteins in the HER pathway in HER2⁺ gastric cancer cells and HER2-low breast cancer cells treated with the triple T-DM1/statin/neratinib combination therapy. There was a greater decrease in expression of proteins involved in HER2 signalling in the triple combination approach in comparison to T-DM1 alone, T-DM1/lovastatin, and T-DM1/neratinib treatments. Preclinical studies showed efficacy in both female and male mice of the T-DM1/lovastatin/neratinib combination therapy , resulting in the least tumour volume growth out of the eight treatment combination groups. Pre-treatment and post-treatment (28 days after the start of therapy) PET/CT imaging using ⁶⁴Cu-labeled trastuzumab confirmed a reduction in HER2 tumoral levels (Fig. 5), demonstrating the ability of ⁶⁴Cu-labeled trastuzumab to noninvasively monitor the response of tumours to HER2-targeted treatment.

Conclusion

• These studies demonstrate the therapeutic enhancement of T-DM1 using combination therapy with lovastatin/neratinib in gastric tumor xenografts in a single dose both in vitro and in vivo.
• ⁶⁴Cu-labeled trastuzumab PET/CT imaging was able to monitor the response of mice to HER2-targeted therapy through pre-treatment and post-treatment noninvasive imaging.
• A decrease in HER2 protein levels and HER2 phosphorylation was detected in cells treated with T-DM1/lovastatin/neratinib when compared to control and T-DM1-only groups.
• PET/CT imaging of mice in the T-DM1/lovastatin/neratinib group showed a decrease in HER2 tumoral expression, which was associated with a decrease in tumour volume and sustained treatment efficacy in the T-DM1/lovastatin/neratinib group.
• T-DM1, lovastatin, and neratinib in combination showed effective suppression of HER2⁺ gastric tumour growth when compared to the other treatment combinations, demonstrating the potential for this drug combination therapy to be used in future clinical trials to enhance ADC efficacy in patients.

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