Tumour-associated macrophages (TAMs) are abundant in many cancers, and often display an immune-suppressive M2-like phenotype that fosters tumour growth and promotes resistance to therapy. Yet macrophages are highly plastic and can also acquire an anti-tumourigenic M1-like phenotype. Here, we show that R848, an agonist of the toll-like receptors (TLRs) TLR7 and TLR8 identified in a morphometric-based screen, is a potent driver of the M1 phenotype in vitro and that R848-loaded β-cyclodextrin nanoparticles (CDNPs) lead to efficient drug delivery to TAMs in vivo. As a monotherapy, the administration of CDNP-R848 in multiple tumour models in mice altered the functional orientation of the tumour immune microenvironment towards an M1 phenotype, leading to controlled tumour growth and protecting the animals against tumour rechallenge. When used in combination with the immune checkpoint inhibitor anti-PD-1, we observed improved immunotherapy response rates, also in a tumour model resistant to anti-PD-1 therapy. Our findings demonstrate the ability of rationally engineered drug–nanoparticle combinations to efficiently modulate TAMs for cancer immunotherapy.

Anti-PD-1 immune checkpoint blockers can induce sustained clinical responses in cancer but how they function in vivo remains incompletely understood. Here, we combined intravital real-time imaging with single cell RNA sequencing analysis and mouse models to uncover anti-PD-1 pharmacodynamics directly within tumors. We showed that effective antitumor responses required a subset of tumor-infiltrating dendritic cells (DCs), which produced interleukin 12 (IL-12). These DCs did not bind anti-PD-1 but produced IL-12 upon sensing interferon γ (IFN-γ) that was released from neighboring T cells. In turn, DC-derived IL-12 stimulated antitumor T cell immunity. These findings suggest that full-fledged activation of antitumor T cells by anti-PD-1 is not direct, but rather involves T cell:DC crosstalk and is licensed by IFN-γ and IL-12. Furthermore, we found that activating the non-canonical NFkB transcription factor pathway amplified IL-12-producing DCs and sensitized tumors to anti-PD-1 treatment, suggesting a therapeutic strategy to improve responses to checkpoint blockade.

Monoclonal antibodies targeting the immune checkpoint Programmed Death-1 (aPD-1 mAbs) have demonstrated impressive benefits for the treatment of some cancers; yet, these drugs are not always effective and we still have a limited understanding of the mechanisms that contribute to their efficacy or lack thereof. Here we employed in vivo imaging to uncover the fate and activity of aPD-1 mAbs in real-time and at subcellular resolution in mice. We show that aPD-1 mAbs effectively bind PD-1+ tumor-infiltrating CD8+ T cells at early time-points after administration. However, this engagement is transient, as aPD-1 mAbs are captured within minutes from the T cell surface by PD-1− tumor-associated macrophages. We further show that macrophage accrual of aPD-1 mAbs depends both on the drug’s Fc domain glycan and on Fcγ-receptors (FcγRs) expressed by host myeloid cells, and extend these findings to the human setting. Finally, we demonstrate that in vivo blockade of FcγRs prior to aPD-1 mAb administration substantially prolongs aPD-1 mAb binding to tumor-infiltrating CD8+ T cells and enhances immunotherapy-induced tumor regression in mice. These investigations yield new insight into aPD-1 target engagement in vivo and identify specific Fc : FcγR interactions that can be modulated to improve checkpoint blockade therapy.