The role of pigmentation in tumor treatment with the investigational virus-like drug conjugate belzupacap sarotalocan in an in vitro and in vivo murine model

Tumor pigmentation is known to be a barrier for applying photodynamic therapy in ocular melanoma. We investigated the role of pigmentation in tumor behavior and compared the anti-tumor efficiency of belzupacap sarotalocan (bel-sar, AU-011) treatment using a pigmented (wt) and non-pigmented (TYR knockout, ko) in an in vitro and in vivo murine melanoma model. A tyrosinase gene knockout B16F10 cell line was developed using CRISPR/Cas9. Following bel-sar binding and light activation (wavelength 690 nm) in vitro, cellular cytotoxicity and damage-associated molecular patterns (DAMPs) were measured by flow cytometry (FACS). Bel-sar treated tumor cells were co-cultured with antigen-presenting cells (APC) to assess phagocytosis and maturation. The B16F10 wt and ko cell lines were grown in syngeneic mice and treated with bel-sar and light. TYR knockout in B16F10 led to the loss of pigmentation which had no effect on in vivo tumor growth. Bel-sar treatment induced near complete cell death, accompanied with exposure of DAMPs, resulting in enhanced phagocytosis and maturation of DCs, regardless of pigmentation level. Bel-sar treatment delayed tumor growth in both tumor models. Pigmented tumors had more infiltrating M1 and less M2 macrophages compared with non-pigmented tumors. Bel-sar treatment stimulated more M1 influx in both models. Following treatment, CD8+T cells were upregulated, particularly in the pigmented tumors, and more mature dendritic cells were detected in the tumor draining lymph nodes. Pigmentation influences the type of infiltrating macrophages in the tumor. Bel-sar treatment induced immunogenic cell death and a positive anti-tumor response in vivo with inhibition of growth in both pigmented and non-pigmented models, and stimulates the influx of M1 stimulating macrophages.