Radiotherapy Shows Synergistic Anti-Tumor Efficacy with Treg Depletion by Reprogramming the Immune-Excluded Tumor Microenvironment

Abstract

Tumor-infiltrating Treg cells (TITR) negatively regulates anti-tumor immune response and promotes tumor progression. Increased levels of TITR cells have been associated with poor prognosis in multiple cancer indications which led to the development of immunotherapies that target tumor Tregs. CCR8 antibody is one of the best-in-class therapies in clinical development for depleting TITR cells. It has demonstrated potent efficacy in tumors that are responsive to checkpoint inhibitors but failed in cold tumors that lack immune infiltration and activation. Radiotherapy (RT) has been shown to induce pro-inflammatory responses in tumors, but still failed to show consistent synergy with checkpoint inhibitors. Here we test the hypothesis that RT synergizes with Treg depletion to improve anti-tumor efficacy by reprogramming the immune-excluded tumor microenvironment. The CCR8 + RT combination therapy was tested in syngeneic mouse tumor models: 4T1 breast, LL2 lung, and B16F10 melanoma, all of which present a cold tumor phenotype. Tumor-bearing mice (n = 10 per group) received a single dose of focal irradiation by SARRP, 4 doses of anti-CCR8 depleting antibody or a combination of the two. Tumor size was measured 3 times per week, survival was monitored until day 42, and tumor metastasis was evaluated at endpoint by necropsy. Mouse tumors, spleens, draining lymph nodes, and blood were collected 1 day after the last dose of CCR8 therapy and analyzed by multi-color flow cytometry and RNA-sequencing for pharmacodynamics and mechanism studies. In the syngeneic mouse models, CCR8+RT combination therapy displayed significantly improved tumor growth inhibition, metastasis control, and prolonged survival compared with either treatment alone, without increase in toxicity. Flow cytometry showed reduced TITR population and increased infiltration and activation of CD8+ cytotoxic T cells in the tumors of CCR8 + RT treated mice. Increased activation of CD8+ T cells was also observed in the blood and draining lymph nodes in mice treated with the combination therapy. Moreover, transcriptomic analysis identified up-regulation of pathways associated with TNF-α and IFN-γ response in the combination-treated tumors, suggesting the creation of a pro-inflammatory tumor microenvironment. Interestingly, these changes were only induced by the combination therapy, but not either treatment alone. Immunotherapies targeting Treg/TITR cells have so far failed to show promise in clinical trials. Our data suggest that TITR depletion is not sufficient to treat tumors with minimal immune infiltration. Our animal data demonstrate that combining RT with CCR8 therapy significantly inhibited tumor growth and prevented metastasis by enriching the tumor microenvironment with pro-inflammatory cytokines and activated cytotoxic T cells. These findings have clinical relevance to apply TITR depletion therapy in combination with RT.