2915 - Radiation-Induced Antigen Expression Benefits Tumor Uptake of ICAM1 Antibody-Drug Conjugates

Y. Xing^1,2, P. J. Li³, and P. Guo^1,2; ¹Tianjin University, Academy of Medical Engineering and Translational Medicine, Tianjin, China, ²Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences, Hangzhou, Zhejiang, China, ³Zhejiang cancer hospital, Hangzhou, Zhejiang, China

Purpose/Objective(s): X-ray irradiation is known to stimulate the expression of certain cell membrane proteins including intercellular adhesion molecule-1 (ICAM1). In this study, we explore the potential of X-ray irradiation to augment the tumoral expression of ICAM1, thereby enhancing tumor uptake of ICAM1 ADCs. By increasing ICAM1 expression, we aim to transform a tumor environment with negligible antigen expression into one with low, but therapeutically relevant, levels of expression. Such modulation is anticipated to improve the uptake and effectiveness of ICAM1 ADCs with enzyme-cleavable linkers, leveraging their bystander-killing effects to treat a broader spectrum of tumors. Materials/

Methods: This study applied flow cytometry to detect protein augmentation after radiotherapy in 293T, HCT116, and SUM190 cells. Radiation exposure varied, with 293T and HCT116 cells receiving doses from 1 to 5 Gy, and SUM190 cells treated with 2 Gy per fraction daily, reaching a total of 6 Gy. We analyzed the expression of ICAM1 and reactive oxygen species (ROS) in these cells, employing fluorescence confocal microscopy to examine ICAM1 antibody endocytosis. The antioxidant N-acetylcysteine (NAC) was utilized to investigate the relationship between ICAM1 and ROS. Furthermore, transmission electron microscopy (TEM) was used to assess the impact of radiation on mitochondrial structures in HCT116 cells. The X-ray irradiation-sensitized cytotoxicity of ICAM1-MMAE and ICAM1-DXd, two enzyme-cleavable ADCs, were determined with the Cell Counting Kit-8 assay. An in vivo study was conducted using a xenograft nude mouse model, divided into control and radiotherapy groups, the latter receiving 2 Gy/fraction radiation for 48 hours followed by an intravenous injection of ATTO 647-labeled ICAM-1 (5mg/kg). IVIS imaging was conducted 24 hours post-injection.
Results: ICAM1 levels in normal and tumor cells following radiotherapy elevated under various irradiation protocols. Notably, at an optimized radiation dose of 2 Gy, ICAM1 expression on the membranes of irradiated HCT116 and SUM190 cells was significantly elevated relative to normal 293T cells, with a prolonged-expression duration (RT: control = 7:5 days). During the ICAM1-mediated endocytosis in tumor cells, the internalization capacity of cells post-irradiation markedly exceeded that of the pre-irradiated cells within the initial 60 minutes. Observations of HCT116 mitochondria indicated swelling and vacuolization, while the alterations in ROS levels were found to positively correlate with ICAM1 expression levels. The enhanced expression of ICAM1 in mice post-radiotherapy corroborates these findings.
Conclusion: Our study demonstrates that X-ray irradiation significantly enhances ICAM1 expression on tumor cells, improving the tumor uptake of ICAM1 ADCs, representing a promising approach to broadening the therapeutic impact of ADCs in antigen-negative tumors.