Abstract 5452: Novel tryptophan-based antibody-drug conjugate (ADC) targeting EGFR-positive lung cancer demonstrates robust antitumor efficacy by triggering immunogenic cell death

Abstract Background and Aim: Antibody-drug conjugates (ADCs), which combine a cytotoxic small molecule drug with an antibody to enable targeted delivery at tumor sites, are emerging as next-generation therapeutic options for non-small cell lung cancer with HER2 mutations. However, clinical development of ADCs for solid tumors has been hindered by hematologic toxicity, such as thrombocytopenia, caused by FcγRIIa-mediated uptake into megakaryocytes (antibody-dependent cellular cytotoxicity: ADCC), as well as the prolonged time and high cost required for linker optimization at cysteine (Cys) or lysine (Lys) residues on antibodies. This study aimed to develop a novel ADC targeting the human EGFR surface antigen, which is expressed in a broad range of lung cancers, through collaborative research in pharmaceutical engineering. Method: We synthesized novel ADCs using our proprietary ABNO linker, which offers high stability through tryptophan (Trp) modification on anti-EGFR IgG4 antibodies. This approach allows for more homogeneous and stable ADC production at a lower development cost compared to conventional linkers. We assessed whether the synthesized ADCs eliminated ADCC activity in co-culture with NK cells and examined their ability to induce immunogenic cell death by evaluating calreticulin exposure on the surface of dying cancer cells. Results: We successfully synthesized a novel ADC (Anti-hEGFR(IgG4)-ABNO-DM-1: E4-AD) by conjugating the cytotoxic drug DM-1 to an anti-human EGFR-IgG4 antibody using the ABNO linker. In CKK8 assays, E4-AD demonstrated potent, concentration-dependent cytotoxic activity against EGFR-positive human lung cancer cell lines, with IC50 values ranging from 2 to 20 nM, while showing no cytotoxicity in EGFR-negative lung cancer cells. Furthermore, E4-AD effectively abolished NK cell-mediated ADCC in vitro and significantly increased calreticulin exposure on cell surface and HMGB-1 release from dying lung cancer cells, indicating the induction of immunogenic cell death. Conclusion: These findings suggest that the newly developed E4-AD, with its innovative tryptophan-based linker technology, holds promise as a safer and more effective ADC for treating EGFR-positive lung cancers. Citation Format: Hiroyuki Inoue, Kounosuke Oisaki, Hiroshi Tagawa, Akira Nakao, Noriyuki Ebi, Takeshi Mori, Masaki Fujita. Novel tryptophan-based antibody-drug conjugate (ADC) targeting EGFR-positive lung cancer demonstrates robust antitumor efficacy by triggering immunogenic cell death [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 5452.