SB-743921

Ewing sarcoma (EWS) is an aggressive pediatric malignancy of the bone and soft tissues in need of novel therapeutic options. To identify potential therapeutic targets, we focused on essential biological pathways that are upregulated by EWS-FLI1, the primary oncogenic driver of EWS, including mitotic proteins such as Aurora kinase A (AURKA) and kinesin family member 15 (KIF15) and its binding partner, targeting protein for Xklp2 (TPX2). KIF15/TPX2 cooperates with KIF11, a key mitotic kinesin essential for mitotic spindle orientation. Given the lack of clinical-grade KIF15/TPX2 inhibitors, we chose to target KIF11 (using SB-743921) in combination with AURKA (using VIC-1911) given that phosphorylation of KIF15^S1169 by Aurora A is required for its targeting to the spindle. In vitro, the drug combination demonstrated strong synergy (Bliss score ≥ 10) at nanomolar doses. Colony formation assay revealed significant reduction in plating efficiency (1-3%) and increased percentage accumulation of cells in the G2/M phase with the combination treatment (45-52%) upon cell cycle analysis, indicating mitotic arrest. In vivo studies in EWS xenograft mouse models showed significant tumor reduction and overall effectiveness: drug combination vs. vehicle control (p ≤ 0.01), SB-743921 (p ≤ 0.01) and VIC-1911 (p ≤ 0.05). Kaplan-Meier curves demonstrated superior overall survival with the combination compared to vehicle or monotherapy arms (p ≤ 0.0001).

Many clinical trials of kinesin spindle protein (KSP) inhibitors have failed due to issues such as high toxicity and a short circulation half-life in vivo. To address the limitations of current KSP inhibitors and thus broad its use in antitumor therapy, this study applied antibody-drug conjugate (ADC) technology to the KSP inhibitor SB-743921, which was coupled with the HER2-specific antibody trastuzumab using a cathepsin B-dependent valine-alanine (Val-Ala, VA) dipeptide-type linker to generate H2-921. Ex vivo and in vivo analyses of H2-921 showed an increased half-life of SB-743921 and prolonged contact time with tumor cells. Furthermore, H2-921 induced apoptosis and incomplete autophagy in HER2-positive cells. In the in vivo analyses, H2-921 had significant tumor-targeting properties, and tumor inhibition by H2-921 was greater than that by traditional KSP inhibitors but similar to that by the positive control drug T-DM1. In conclusion, this study describes a novel application of ADC technology that enhances the antitumor effects of a KSP inhibitor and thus may effectively address the poor clinical efficacy of KSP inhibitors.