Abstract Background Trastuzumab deruxtecan (T-DXd) administration improves response for patients with HER2-positive metastatic breast cancer (HER2+ BC). Unfortunately, 50% of patients relapse after 2 years. T-DXd resistance mechanisms are being explored. For trastuzumab we have shown that mucin 4 (MUC4) expression is an independent predictor of poor response in HER2+BC patients. MUC4 is upregulated by soluble TNF (sTNF) secreted by the tumor, confers resistance to trastuzumab by hiding its epitope on the HER2, reducing its binding and decreasing anti-tumor phagocytic function. In preclinical models of de novo trastuzumab-resistant tumors, combination of a sTNF blocking agent INB03, (DN), with T-DXd decreases tumor growth compared to T-DXd alone. To disclose the underlying mechanism, we studied whether DN improved internalization of T-DXd in tumor cells and modified the innate immune response to enhance T-DXd antitumor effects in a multiple HER2-targeted therapy-resistant model. Methods Nude mice bearing HER2+MUC4+ JIMT-1 tumor, primary resistant to trastuzumab, pertuzumab and lapatinib, were treated with IgG 5 mg/kg, T-DXd 5 mg/kg (T-DXd 5), 2.5 mg/kg (T-DXd 2.5) or 1.25 mg/kg (T-DXd 1.25), DN 10 mg/kg or the combined therapies. T-DXd and IgG were administered i.v. on days 0, 7 and 14. DN was administered i.p. twice a week for 3 weeks. Tumor growth was monitored. Mitotic figures/field (mean) were analyzed in H&E tumor sections. Tumor-infiltrating macrophages were studied by flow cytometry. IFNƔ was determined in tumor extracts by ELISA. Internalization of T-DXd in JIMT-1 cells was studied in a S1 Incucyte along 18h by Fab-rhodo red labeling. Results T-DXd dose-response curves showed inhibition in tumor growth of 83%, 61% and 37% for T-DXd 5, 2.5 and 1.25 mg/kg treatment respectively compared with IgG-treated tumors. DN alone exhibited no antitumor activity. T-DXd+DN increased the antitumor effect by 10%, 33% and 97% for T-DXd dose of 5, 2.5 and 1.25 mg/kg respectively. Tumor growth inhibition of T-DXd 5 was similar to combination of T-DXd1.25+DN. Addition of DN did not have toxicity. DN increases IFNƔ production in the TME of T-DXd treated tumors vs T-DXd alone and promoted macrophage recruitment to the tumor bed and polarization to the antitumor M1-like phenotype. Histopathological analysis of the tumor showed a significant decrease in proliferation in all the combined and in the T-DXd 5mg/kg (2.8-3.7 mitotic figures/field) vs IgG (6.0 mitotic figures/field). In vitro, JIMT-1 cells treated with DN internalized 40% more T-DXd than its vehicle-treated counterparts. Conclusions Neutralizing sTNF with DN enhances T-DXd effect in a multiple HER2 targeted therapy resistant model of MUC4 expressing HER2+ BC. Combination of DN with T-DXd increases tumor response at all dose levels tested. The largest effect was seen at lower doses (T-DXd 1.25+DN), which mimicked the effect of 4 times the dose of T-DXd alone, suggesting significant synergy with DN as dose of T-DXd decreases. Combination of DN with T-DXd in MUC4 expressing HER2+ BC improves response to T-DXd alone by increasing T-DXd internalization and improving anti-tumor innate immune responses in the TME without increasing toxicity. The results suggest this combination should be investigated in clinical trials in patients who have MUC4 expressing tumors when T-DXd is started or become resistant to T-DXd therapy. Citation Format: Sofia Bruni, Florencia Mauro, Sofia Naveiro, Rosalia Cordo-Russo, Agustina Dupont, Mercogliano María Florencia, Roxana Schillaci. Blocking soluble TNF to Improve potency of trastuzumab deruxtecan by increasing internalization and antitumor innate immune response in a resistant HER2-positive breast cancer model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-18-05.

Clinical trials showed that trastuzumab deruxtecan (T-DXd) provides durable responses for patients with HER2+ and HER2 low metastatic breast cancer (BC), determined by immunohistochemistry. Approximately 50% of patients with HER2+ metastatic BC were still alive and progression-free at 24 months (DESTINY-Breast03). We proved that mucin 4 (MUC4) expression is an independent predictor of poor response to trastuzumab in HER2+ BC patients. In JIMT-1 tumors we proved that soluble TNFα (sTNFα) upregulates MUC4, conferring trastuzumab resistance by hiding its epitope on the HER2 molecule and reducing its binding. Here, we study whether sTNFα blockade with INB03 (DN) plays a role in regulation of innate immunity to enhance T-DXd antitumor effects in a multiple HER2-targeted therapy-resistant model. Nude mice bearing HER2+MUC4+ JIMT-1 tumor, primary resistant to trastuzumab, pertuzumab and lapatinib, were treated with IgG 5 mg/kg, T-DXd 5 mg/kg (T-DXd 5), 2.5 mg/kg (T-DXd 2.5) or 1.25 mg/kg (T-DXd 1.25), DN 10 mg/kg or the combined therapies. T-DXd and IgG were administered i.v. on days 0, 7 and 14. DN was administered i.p. twice a week for 3 weeks. Tumor growth was monitored. Mitotic index was analyzed in H&E tumor sections. The tumor-infiltrating innate cells, macrophages, NK cells and myeloid-derived suppressor cells (MDSCs), were studied by flow cytometry. T-DXd dose-response curves exhibited tumor growth inhibitions of 83% (T-DXd 5), 61% (T-DXd 2.5) and 37% (T-DXd 1.25) vs IgG-treated tumors. DN alone had no antitumor effect. T-DXd+DN reinforced the antitumor effect, as tumor growth inhibition escalated to 98% (T-DXd 5+DN), 81% (T-DXd 2.5+DN) and 73% (T-DXd 1.25+DN). A reduced number of mitotic figures were observed in T-DXd 5, T-DXd 1.25+DN and T-DXd 5+DN. Combining DN with T-DXd 1.25 and 5 enhanced the infiltration of resident macrophages and promoted polarization to the M1-like phenotype The tumor associated macrophages (TAMs) were similar among treatments. However, the combination T-DXd 1.25+DN showed an increase in M1-like tumor associated macrophages (TAMs) and a decrease in M2-like TAMs vs T-DXd 1.25 alone. T-DXd 1.25+DN treatment mimics the increase of infiltrating NK cells observed in the T-DXd 2.5 and 5 doses Finally, adding DN to T-DXd 2.5 and 5 diminishes MDSCs infiltration. Combination therapies were well tolerated without evidence of toxicity. Our results suggest that sTNFα blockade enhances T-DXd effect in a multiple HER2-targeted therapy resistant model. Adding DN allows to lower T-DXd doses to induce a reinforced antitumor innate immune response, reduced tumor cell mitosis and achieve similar tumor inhibition. Since sTNFα and MUC4 expression proved to be important variables in the response to T-DXd, neutralizing this cytokine may open new therapeutic strategies to treat patients with MUC4 expressing tumors or have progression on T-DXd therapy. Citation Format: Sofia Bruni, Florencia L. Mauro, Sofia Naveiro, Maria F. Mercogliano, Roxana Schillaci. Soluble TNFα blockade improves effectiveness of trastuzumab deruxtecan and boosts antitumor potential of macrophages in a HER2+ tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2273.