PD-1 endocytosis unleashes the cytolytic potential of checkpoint blockade in tumor immunity.

PD-1 immune checkpoint blockade (ICB) is a key cancer treatment. While blocking PD-1 binding to ligand is known, the role of internalization in enhancing ICB efficacy is less explored. Our study reveals that PD-1 internalization helps unlock ICB's full potential in cancer immunotherapy. Anti-PD-1 induces 50%-60% surface PD-1 internalization from human and mouse cells, leaving low to intermediate levels of resistant receptors. Complexes then appear in early and late endosomes. Both CD4 and CD8 T cells, especially CD8⁺ effectors, are affected. Nivolumab outperforms pembrolizumab in human T cells, while PD-1 internalization requires crosslinking by bivalent antibody. While mono- and bivalent anti-PD-1 inhibit tumor growth with CD8 tumor-infiltrating cells expressing increased granzyme B, bivalent antibody is more effective where the combination of steric blockade and endocytosis induces greater CD8⁺ T cell tumor infiltration and the expression of the cytolytic pore protein, perforin. Our findings highlight an ICB mechanism that combines steric blockade and PD-1 endocytosis for optimal checkpoint immunotherapy.