Selective Targeting of Immune Checkpoints HLA‐G and CD47 Using Novel Dual Signaling Protein DSP216 Promotes Innate Anticancer Immunity

ABSTRACT Immunotherapy has significantly improved treatment outcomes for cancer patients within the past decade, with breakthrough results using immune checkpoint inhibitors (ICIs), most notably those targeting the PD‐1/PD‐L1 inhibitory axis. Nevertheless, many patients and tumor types do not respond to current ICIs, and next‐generation drugs are urgently needed. Dual Signaling Protein 216 (DSP216) is a new ligand‐based immunotherapeutic—a dual HLA‐G and CD47 ICI. DSP216 was designed to exclusively bind to cells co‐expressing the immune checkpoints CD47 and HLA‐G, thereby mitigating ICI activity towards normal cells expressing only CD47 or HLA‐G and associated side effects. Computational chemistry was used to optimize DSP216 affinity to HLA‐G with the aim to achieve the desired binding mode and DSP216 binding to CD47 + /HLA‐G + and CD47 + /HLA‐G − cancer cells, PBMCs, and RBCs was tested. Functional blocking of the CD47 and HLA‐G axis was investigated in macrophage polarization and phagocytosis assays and NK cytotoxicity assays. DSP216 with an ‘active’ (DSP216a), but not with an ‘inactive’ Fc (DSP216i), triggered CD16‐signaling in a reporter cell line, and the combination of checkpoint blockade and ADCC by DSP216a potentiated NK‐mediated cytotoxicity. These encouraging findings support the continued preclinical evaluation of DSP216.