In Vitro Testing of the Virus-Like Drug Conjugate Belzupacap Sarotalocan (AU-011) on Uveal Melanoma Suggests BAP1-Related Immunostimulatory Capacity

Abstract

Purpose The virus-like drug conjugate belzupacap sarotalocan (AU-011), currently under clinical investigation for first-line treatment of primary uveal melanoma (UM), shows enhanced tumor specificity by targeting heparan sulfate proteoglycans (HSPG). Such a treatment may potentially lead to systemic immune responses. We studied the potential of AU-011 treatment to induce immunogenic cell death as the first step to induce systemic immunity. Methods We determined binding and uptake of AU-011 in ten primary and metastatic UM cell lines. The subcellular location of AU-011 was assessed by fluorescence microscopy. Following light activation (wavelength 690 nm) of AU-011, the half-maximal effective concentration (EC 50 ) of AU-011 treatment and exposure of damage-associated molecular patterns (DAMPs) were assessed using flow cytometry. DAMPs were measured by RNAseq. Results Fluorescence microscopy revealed most of the AU-011 was present in the cytoplasm. AU-011 binding and uptake by UM cells increased over time, with a lower uptake in BAP1-negative than in BAP1-positive cell lines. AU-011 activation induced cell death across all UM cell lines with EC 50 values at picomolar concentrations. The AU-011 concentration and total light dose (J/cm 2 ) were the most important parameters for the observed cytotoxicity. Finally, light-activated AU-011 induced exposure of DAMPs calreticulin (CRT) and HSP90. CRT exposure by light-activated AU-011 as well as CRT RNA exposure were lower in BAP1-negative compared to BAP1-positive UM cell lines. Conclusions AU-011 treatment at low picomolar range induces immunogenic cell death in all 10 UM cell lines. The in vitro cytotoxicity was accompanied by exposure of DAMPs (HSP90 and CRT), suggesting AU-011 may contribute to the development of systemic immunity and be a suitable candidate for combination with immunotherapy in vivo. AU-011 treatment was more effective against BAP1-positive cell lines, with a lower EC 50 and higher CRT exposure.

Recent Advances in Molecular and Genetic Research on Uveal Melanoma

“…It is distinctive due to its tumor-targeting capabilities via HSPGs (heparan sulfate proteoglycans), that are overexpressed on tumor cells [ 275 ]. Preliminary AU-011 research showed promise in triggering immunogenic cell death in various UM cell lines [ 276 ]. This treatment appears to work as an immuno-stimulant by increasing the exposure of DAMPs (damage-associated molecular patterns) on the cell membrane, specifically CRT (calreticulin) and HSP90 (heat shock protein 90) [ 276 ].…”