In vivo imaging reveals a tumor-associated macrophage-mediated resistance pathway in anti-PD-1 therapy

Abstract

Monoclonal antibodies (mAbs) targeting the immune checkpoint anti-programmed cell death protein 1 (aPD-1) have demonstrated impressive benefits for the treatment of some cancers; however, these drugs are not always effective, and we still have a limited understanding of the mechanisms that contribute to their efficacy or lack thereof. We used 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, and 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 before 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 insight into aPD-1 target engagement in vivo and identify specific Fc/FcγR interactions that can be modulated to improve checkpoint blockade therapy.

Figure 1. Anti-PD-1 mAb labeling facilitates tracking of tissue biodistribution

(A) Conjugation of Alexa Fluor 647 via NHS ester linkage to rat anti-mouse PD-1 29F.1A12 clone was confirmed by flow cytometry to not alter the binding tropism to PD-1⁺ EL4 cells (grey). Rat IgG2a isotype control (white). (B) MC38 tumors were equally responsive to single-dose AF647-aPD-1 and unlabeled aPD-1, while tumor areas increased 72 h after control IgG2a treatment. (C) Fluorescence reflectance imaging of 3 tumors compared to draining (dLN) and contralateral (cLN) lymph nodes 24 hours after AF647-aPD-1 treatment (AF647: λ_ex = 620–650 nm, λ_em = 680–710 nm). Scale bars represent 5 mm. (D) Quantified AF647-aPD-1 in each tissue demonstrating tumor accumulation. Values represent SEM and n = 3 unless otherwise noted. ***P < 0.001; unpaired, two-tailed t-test.

Figure 2. In vivo temporal aPD-1 mAb pharmacokinetics reveals drug accumulation in TAMs

(A) Diagram depicting intravital imaging setup with labeled aPD-1, MC38 tumor cells, T cells, and tumor associated macrophages (TAMs). (B) Treatment with single-dose aPD-1 mAb can achieve significant remission in the MC38/H2B-mApple tumor model. Tumors are outlined in grey; scale bars represent 2 mm. (C) Z-projections of an MC38/H2B-mApple tumor in a DPE-GFP mouse injected i.v. with AF647-aPD-1 after 15 minutes (top) or 24 hours (bottom). (D) Micrographs of T cells (magenta outline) identified as GFP⁺ cells and TAMs (yellow outline) identified by Pacific Blue signal. Outlines are overlaid on micrographs of the corresponding AF647-aPD-1 channel; scale bars represent 30 μm. (E) IVM biodistribution studies indicate early aPD-1 binding to T-cells and long-term accumulation in TAMs. Data are representative of 5 independently-treated DPE-GFP mice and normalized to autofluorescent signal.

Figure 3. In vivo imaging reveals aPD-1 mAb transfer from CD8⁺ T cells to TAMs

(A) Quantified AF647 signal on T cells and TAMs from a representative DPE-GFP mouse demonstrates collection of AF647-aPD-1 in T cells at 15 minutes and in TAMs at 24 hours following injection of therapy. (B) Early time-course IVM images acquired in an IFNγ-YFP reporter mouse with an MC38-H2B-mApple tumor. Yellow arrows indicate site of aPD-1 mAb binding to an IFNγ⁺ CD8 T-cell at 6-15 minutes and macrophage internalization at times >21 min. (C) Quantification of aPD-1-mAb on IFNγ-expressing CD8⁺ lymphocytes and TAMs reveals a narrow window of target binding. (D) Flow cytometry histograms pre-gated for 7-AAD⁻/CD45⁺ show AF647-aPD-1 signal (x-axis, logarithmic scale) on immune cell populations at 0.5 hr and 24 hours post administration (blue). Cell populations from untreated control animals (red) were used as reference. (E) aPD-1 mAb binds to CD8⁺ lymphocytes early but accumulates in TAMs at later time points. Scale bars represent 30 μm. **P < 0.01; ****P < 0.0001; unpaired, two-tailed t-test.

Figure 4. aPD-1 mAb transfer to macrophages is mediated by FcγRs

(A) Ex vivo flow cytometry histograms of MC38 tumors stained with PE-aPD-1 show CD8⁺ T cells but not TAMs express cell surface PD-1. (B) Co-culture of bone marrow-derived macrophages (Mø) and AF647-aPD-1 coated EL4 lymphocytes was used to quantify the AF647-aPD-1 puncta in macrophages pre-blocked with FcγR inhibitors or the phagocytosis inhibitor, dynasore. *P < 0.05; ****P < 0.0001; one-way ANOVA. (C) Representative images of macrophages labeled with PKH-red and observed by time-lapse imaging for aPD-1 mAb (yellow) transfer from neighboring lymphocytes (cerulean) after 30 min. FcγRII/III inhibition blocks antibody transfer (highlighted); Scale bars represent 10 μm. (D-E) Flow cytometry was used to estimate AF647-aPD-1 transfer to F4/80⁺ BMDMs in co-culture assays when the mAb was added directly (green), bound to EL4 cells (red) or bound to EL4 cells in the presence of FcγRII/III neutralizing antibody (blue). Results in D are a representative histogram of AF647 signal in macrophages and E is G-MFI value of conditions presented in D. Data are from 3 independent experiments. *P < 0.05; ** P < 0.01; two-way ANOVA with Tukey’s multiple comparisons test. Values represent SEM for three separate experiments.

Figure 5. Nivolumab shares similar glycan patterns to mouse aPD-1 and is transferred to macrophages via FcγRs

(A) HPLC analysis of the glycan patterns found on the mouse aPD-1 mAb and nivolumab shows the G0F isoform to be predominant, but glycosylation is not uniform. (B) AF647-labeled nivolumab (yellow) was used to stain the surface of aCD3 stimulated PKH-green labeled human CD8⁺ T cells (cerulean) co-incubated with PKH-red labeled peripheral blood mononuclear cell-derived macrophages in the presence or absence of Fc Block. Scale bars represent 20 μm. (C) Quantification of AF647⁺ puncta per macrophage confirms that nivolumab is transferred via FcγRs. Values represent SEM for 4 separate experiments.

Figure 6. Disrupting Fc binding affects macrophage uptake of aPD-1 and improves treatment efficacy

(A) PKH-green labeled EL4 cells (cerulean) stained with native AF647-aPD-1 or deglycosylated AF647-aPD-1 co-cultured with PKH-red labeled bone marrow-derived macrophages (Mø). Images are representative of 3 separate experiments. Scale bars represent 10 μm. (B) FACS plots of mouse Mø (gated on F4/80⁺) co-cultured with PD-1⁺ EL4 cells labeled with either AF647-aPD-1 mAb (blue) or deglycosylated AF647-aPD-1 mAb (red). (C) aPD-1-mAb deglycosylation substantially reduces the transfer from EL4 cells to Mø (n = 3). **P < 0.01; Unpaired 2-tailed t test. (D) Intravital images 10 and 120 minutes after AF647-aPD-1 drug extravasation in a representative IFNγ-YFP reporter mouse MC38-H2B-mApple tumor pre-treated with the FcγRII/III blocking antibody (2.4G2). (E) Quantified AF647-aPD-1 from three 2.4G2-treated mice shows prolonged aPD-1 binding to tumor T cells, relative to mice treated with AF647 aPD-1 alone (data repeated from Fig. 3C for comparison). (F) MC38 tumor growth curves of mice (n ≥ 5) treated with isotype control (black), aPD-1 (blue), and aPD-1 plus 2.4G2 (red). *P < 0.05 One-Way ANOVA with Tukey’s multiple comparison test.