A novel proteolysis-targeting chimera strategy targeting multiple immune checkpoints containing ITIMs enhances antitumor immunity

Immune checkpoint inhibitors (ICIs) have significantly advanced and revolutionized cancer treatment over the past decade; however, their clinical benefits have been limited to a subset of cancer patients. While ICI-based combinations have emerged as promising strategies, they risk broader toxicities and significant cost burdens. This highlights the critical need for the development of inhibitors that target multiple immune checkpoints. In this study, we developed a peptide that emulates the conserved sequence of the Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) C-terminal Src homology 2 (C-SH2) domain, which is capable of binding to immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic tails of multiple immune inhibitory receptors. By utilizing this peptide as the protein of interest (POI) ligand and coupling it with the von Hippel‒Lindau (VHL) ligand via a peptide linker, a proteolytic targeting chimera (PROTAC) named PROTAC of ITIM-targeting inhibitory peptide (PITIP) was constructed. PITIP effectively induced the degradation of multiple immune inhibitory receptors in a proteasome-dependent manner, thereby attenuating immunosuppressive signaling within T cells, natural killer (NK) cells, and macrophages. <i>In vivo</i> investigations demonstrated that PITIP elicited a robust antitumor immune response in xenograft and allograft tumor model mice, including those resistant to αPD-1 therapy. Moreover, the encapsulation of PITIP within liposomes conjugated with anti-CD45 antibodies enhanced the targeting of immune cells by PITIP, thereby improving the therapeutic efficacy of the antibodies. This study reports, for the first time, a universal strategy targeting the common structural motifs of immunosuppressive receptors, which facilitates broader and more extensive immune activation through the ubiquitination-mediated degradation of multiple immune checkpoints.