TROP2靶向抗体药物偶联物datopotamab deruxtecan（Dato-DXd）在乳腺癌细胞系中与DNA损伤反应相关的抗肿瘤作用

Background: Trophoblast cell surface antigen-2 (TROP2), Tumor-associated calcium signal transducer 2 (TACSTD2) is a transmembrane glycoprotein expressed in various cancer types. Datopotamab deruxtecan (Dato-DXd) is an antibody-drug conjugate (ADC) composed of an anti-TROP2 antibody (Datopotamab) linked via a cleavable peptide linker to DXd, a Topoisomerase-I inhibitor. Topoisomerase-I maintains DNA topological stress by resolving DNA supercoils during replication and transcription. We sought to determine whether this DNA damage mechanism classically induced by topoisomerase I inhibitors emerges when delivered through an ADC, and to further elucidate the previously uncharacterized molecular mechanisms of DNA damage response (DDR) and cell death by Dato-DXd. Methods: Five established human breast cancer cell lines and one patient-derived breast cancer (PDC) cell line (SNU-3171) were used in this study. MCF7 and SNU-3171 were luminal breast cancer cell lines, and the others were triple-negative breast cancer (TNBC) cell lines. Sensitivity of each cell to Dato-DXd was assessed by colony formation assay (CFA) for 14 days (0.25-5 nM). Internalization and cell cycle analysis were performed by flow cytometry. Apoptosis was detected by Annexin V assay. The protein expression of TROP2, DNA damage, and repair molecules was analyzed by Western blotting and immunofluorescence. Results: Based on CFA results, HCC1806, HCC70, and SNU-3171 were identified as sensitive cells (IC 50 <0.5 nM), whereas MCF7, MDA-MB-157, and HCC1395 as less sensitive cells (IC 50 >2.5 nM) to Dato-DXd. Dato-DXd was internalized within 2 hours in TROP2-positive cells. Following internalization, topoisomerase-I cleavage complexes (Top1ccs) were formed in all TROP2-positive cells; however, co-localization of Top1ccs and ɣ-H2AX was observed only in sensitive cells. Dato-DXd induced G2/M arrest in all TROP2-positive cells. In sensitive cells, the expression of TDP1, XRCC1, DNA Ligase III, and PNKP was decreased, while phospho-Chk1 (S345) and ɣ-H2AX were increased, indicating a reduction in single-strand DNA damage repair capacity and activation of DDR. Moreover, increased doses of Dato-DXd were associated with higher number of Annexin V-positive cells and sub-G1 populations, as well as higher expression of cleaved PARP, caspase-3, and caspase-7 in sensitive cells. Conclusion: Dato-DXd demonstrated efficient internalization in TROP2-positive breast cancer cells including PDC, and impaired DNA single-strand break (SSB) repair, which consequently induce apoptosis in sensitive breast cancer cells. In particular, formation of Top1ccs and G2/M phase accumulation are induced in most of TROP2-positive cells. Further experiments are required to elucidate how impaired SSB repair contributes to the induction of DNA double-strand breaks and apoptosis.