Abstract 4774: Synergistic enhancement of immunogenic cell death: Combining AWT020 with antibody-drug conjugates for superior anti-tumor activity

Antibody-Drug Conjugates (ADCs) are targeted cancer therapies delivering cytotoxic agents selectively to cancer cells. Despite clinical success across various malignancies, resistance to ADC therapy limits long-term efficacy. A key ADC mechanism is inducing immunogenic cell death (ICD), releasing damage-associated molecular patterns (DAMPs) and tumor antigens to stimulate an immune response. This provides a rationale for combining ADCs with immunotherapies to overcome resistance and improve survival rates.AWT020 is a novel fusion protein currently in Phase I clinical trials. It combines a humanized antibody targeting programmed cell death protein 1 (PD-1) with a potency attenuated interleukin-2 (IL-2) mutein. This design blocks the PD-1 pathway while preferentially activating IL-2 signaling on PD-1high tumor-infiltrating T cells (TILs), minimizing peripheral PD-1low lymphocyte activation and reducing systemic toxicity. In preclinical tumor models, a murine surrogate of AWT020 (mAWT020) demonstrated superior anti-tumor activity over a PD-1 monoclonal antibody by selectively expanding TILs.Both ADCs and AWT020 selectively target the tumor microenvironment and promote tumor cell killing through distinct mechanisms. We hypothesized that ADC-induced ICD could be further amplified by AWT020. To test this, we developed two syngeneic tumor models, EMT6B7H3 and EMT6Her2, expressing human B7H3 and human Her2, allowing specific ADC targeting. In the EMT6B7H3 model, combination of B7H3 ADC (B7H3-Exa) and mAWT020 eradicated all tumors after a single dose, outperforming monotherapies. Similar synergy between Her2-Exa ADC and mAWT020 was observed in the EMT6Her2 model. In a subsequent tumor rechallenge study, most mAWT020-treated mice rejected the replantation of EMT6B7H3, indicating that mAWT020 had induced long-term immunological memory in these animals. As a comparison, combination of anti-mPD-1 with B7H3-Exa did not enhance the antitumor activity of ADC. Characterization of ICD markers and TIL phenotypes in the EMT6B7H3 model showed that B7H3-Exa treatment significantly upregulated calreticulin (CRT) expression in EMT6B7H3 tumor cells. The combination of B7H3-Exa and mAWT020 further increased CRT expression. Compared to B7H3-Exa monotherapy or the combination of B7H3-Exa and anti-mPD-1, mAWT020 treated groups significantly increased CD3+ T cell infiltration within the tumor, as well as tumor-specific T cells, as detected by ELISpot assay.These preclinical findings demonstrate that ADC-induced ICD is amplified by AWT020, but not by anti-mPD-1, resulting in greater tumor regression and enhanced immunological memory. These results support the continued clinical development of AWT020 and to explore its potential in combination with ADCs, aiming to improve long-term survival and complete response rates in cancer patients. Citation Format: Fan Ye, Jianing Huang, Ella Li, Wen-Chin Huang, Fang Huang, Xiaoli Cheng, Jenny Jiang, Shih Chieh Chen, Botong Hua, Hanna Lin, Joyce Kwan, Eugene Liu, Ziyang Zhong. Synergistic enhancement of immunogenic cell death: Combining AWT020 with antibody-drug conjugates for superior anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4774.