Enhancing the Anticancer Activity of a Carcinoma-Directed Peptide-HLA-I Fusion Protein by Armoring with Mutein IFNα

Previously, we reported on the peptide-HLA-I fusion protein EpCAM-ReTARG TPR, which allows us to redirect the cytotoxic activity of pre-existing anti-CMV CD8 pos T cell immunity to selectively eliminate EpCAM pos cancer cells. EpCAM-ReTARG TPR consists of the CMV pp65-derived peptide TPRVTGGGAM (TPR) fused in tandem with a soluble HLA-B*07:02/β2-microglobulin (β2M) molecule and an EpCAM-directed Fab antibody fragment. To further enhance its anticancer activity, we equipped EpCAM-ReTARG TPR with the immune-potentiating cytokine muteins IL2 (H16A,F42A) and IFNα R149A, respectively. Both cytokines are engineered to have attenuated affinity for their respective cytokine receptors. Compared to EpCAM-ReTARG TPR, in vitro treatment of EpCAM pos carcinoma cell lines with EpCAM-ReTARG TPRvIL2 for 24 h increased the cytotoxic activity of PBMCs containing low levels of TPR-specific CD8 pos T cells by ~15%, whereas EpCAM-ReTARG TPRIFNα R149A induced an increase of ~50%. Moreover, treatment for 120 h with EpCAM-ReTARG TPRIFNα R149A inhibited the proliferative capacity of the cancer cell lines OvCAR3 and PC3M by ~91% without compromising the viability of the TPR-specific CD8 pos T cells and increased their capacity for IFNγ secretion. Importantly, EpCAM-ReTARG TPRIFNα R149A potently induced the elimination of primary EpCAM pos refractory carcinoma cells from a Merkel cell carcinoma (MCC) patient. Taken together, the armoring of the carcinoma-directed peptide-HLA-I fusion protein EpCAM-ReTARG TPR with IFNα R149A potently enhanced the efficacy of pre-existing anti-CMV CD8 pos T cell immunity to selectively eliminate EpCAM pos cancer cells.