Abstract 3098: Linker cleavable enzymes determine anti-tumor activity of trastuzumab deruxtecan in HER2-positive lung cancer and HER2-low breast cancer

Abstract HER2 genetic aberrations are found across various cancer types. Trastuzumab deruxtecan (T-DXd), an anti-HER2 antibody-drug conjugate (ADC), has been approved for the treatment of HER2-amplified breast and gastric cancers, HER2-mutant lung cancer, and HER2-overexpressing solid tumors. The approval of T-DXd has recently been expanded to include patients with HER2-low breast cancer. In addition, a clinical trial in lung cancer has demonstrated that the efficacy of T-DXd is not correlated with HER2 expression or specific genetic mutations. These findings suggest the presence of factors beyond HER2 expression that affect T-DXd sensitivity. To identify novel determinants of T-DXd efficacy in HER2-positive cancers, we first evaluated its anti-tumor activity in in vitro and in vivo using patient-derived HER2-positive lung cancer cell lines, JFCR-007 and JFCR-381. Interestingly, these cells showed significant shrinkage only by a single dose of T-DXd when implanted in BALB/c-nu mice, they were resistant to T-DXd in a regular 2D culture condition. Therefore, we hypothesized that tumor microenvironment affected T-DXd sensitivity. Cancer cells in patient tumor tissue are surrounded by non-cancerous cells, so we used a three-dimensional (3D) layered co-culture model with fibroblasts and extracellular matrix (ECM). We developed a 3D-in model, in which cancer cells are mixed with fibroblasts and ECM. We also made 3D-on, in which cancer cells were layered on the top of approximately 30 layers of fibroblasts, and 3D-out, in which cancer cells were seeded outside the chamber insert filled with 30 layers fibroblasts. Strikingly, the two lung cancer cell lines became sensitive to T-DXd in the 3D-in and the 3D-on state compared to the 3D-out model. Next, this co-culture model was applied to the HER2-low breast cancer, for which T-DXd has been shown to significantly prolong progression-free survival (PFS) in more than half of cases. We therefore established HER2-knockout and -low expressing breast cancer cell lines (HCC1954 and MDA-MB-453) using the CRISPR/Cas9 system followed by sorting with cell sorter. These HER2-low and -null cells were resistant to T-DXd in 2D culture state compared to parental cells. However, they became sensitive to T-DXd when cultured in 3D-in and 3D-on state. To identify the mechanism by which cancer cells become T-DXd-sensitive in 3D co-culture systems, we focused on cathepsins, lysosomal peptidases that cleave the T-DXd linker. The cathepsin subtypes capable of cleaving the amino acid sequence in the T-DXd linker were knocked down and examined the T-DXd sensitivity. Notably, cancer cells exhibited resistance to T-DXd when some cathepsin subtypes were individually knocked down. Collectively, our results suggest that certain types of cathepsins in the tumor microenvironment may play a critical role in the anti-tumor effects of T-DXd. Citation Format: Akari Shiraishi, Asumi Iesato, Yuki Takahashi, Ken Uchibori, Tomo Osako, Reo Maruyama, Makoto Nishio, Ryohei Katayama. Linker cleavable enzymes determine anti-tumor activity of trastuzumab deruxtecan in HER2-positive lung cancer and HER2-low breast cancer [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 3098.