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.

Therapeutic nanoparticles (TNPs) aim to deliver drugs more safely and effectively to cancers, yet clinical results have been unpredictable owing to limited in vivo understanding. Here we use single-cell imaging of intratumoral TNP pharmacokinetics and pharmacodynamics to better comprehend their heterogeneous behavior. Model TNPs comprised of a fluorescent platinum(IV) pro-drug and a clinically-tested polymer platform (PLGA-b-PEG) promote long drug circulation and alter accumulation by directing cellular uptake toward tumor associated macrophages (TAMs). Simultaneous imaging of TNP vehicle, its drug payload, and single-cell DNA damage response reveals that TAMs serve as a local drug depot that accumulates significant vehicle from which DNA damaging Pt payload gradually releases to neighboring tumor cells. Correspondingly, TAM depletion reduces intratumoral TNP accumulation and efficacy. Thus, nanotherapeutics co-opt TAMs for drug delivery, which has implications for TNP design and for selecting patients into trials.

Therapeutic nanoparticles (TNPs) have shown heterogeneous responses in human clinical trials, raising the question of whether imaging should be used to identify patients with a higher likelihood of nanoparticle accumulation, and thus therapeutic response. Despite extensive debate about the enhanced permeability and retention (EPR) effect in tumors, it is increasingly clear that EPR is extremely variable yet little experimental data exists to predict its clinical utility. Based on the hypothesis that an FDA-approved 30-nm magnetic nanoparticle (MNP) could predict co-localization of therapeutic nanoparticles by MRI, we performed single-cell resolution imaging of fluorescently labeled MNPs and TNPs and studied their intratumoral distribution. We visualized MNPs circulating in tumor microvasculature and found sustained uptake into cells of the tumor microenvironment within minutes. MNPs could predictably demonstrate areas of co-localization for a model TNP [poly(D,L-lactic-co-glycolic acid)-b-polyethylene glycol; PLGA-b-PEG] within the tumor microenvironment (> 85% accuracy) and circulating within the microvasculature (>95% accuracy) despite their markedly different sizes and compositions. Computational analysis of NP transport enabled predictive modeling of TNP distribution based on imaging data, and identified key parameters governing intratumoral NP accumulation and macrophage uptake. Finally, MRI imaging accurately predicted initial treatment response and drug accumulation in a therapeutic study testing for the efficacy of paclitaxel-encapsulated nanoparticle. These approaches yield valuable insight into the in vivo kinetics of NP distribution and suggest that clinically-relevant imaging can be used to select patients with high EPR for treatment with TNPs.