Treatment with an agonist anti-OX40 antibody (aOX40) boosts anti-tumor immunity by providing costimulation and driving effector T cell responses. However, tumor-induced immune suppression contributes significantly to poor response rates to aOX40 therapy, thus combining aOX40 with other agents that relieve tumor-mediated immune suppression may significantly improve outcomes. Once such target is galectin-3 (Gal-3), which drives tumor-induced immunosuppression by increasing macrophage infiltration and M2 polarization, restricting TCR signaling, and inducing T cell apoptosis. A wide-variety of tumors also upregulate Gal-3, which is associated with poor prognosis. Tumor-bearing (MCA-205 sarcoma, 4T1 mammary carcinoma, TRAMP-C1 prostate adenocarcinoma) mice were treated with a Gal-3 inhibitor (belapectin; GR-MD-02), aOX40, or combination therapy and the extent of tumor growth was determined. The phenotype and function of tumor-infiltrating lymphocytes was determined by flow cytometry, multiplex cytokine assay, and multiplex immunohistochemistry. Gal-3 inhibition synergized with aOX40 to promote tumor regression and increase survival. Specifically, aOX40/belapectin therapy significantly improved survival of tumor-bearing mice through a CD8 + T cell-dependent mechanism. Combination aOX40/belapectin therapy enhanced CD8 + T cell density within the tumor and reduced the frequency and proliferation of regulatory Foxp3 + CD4 + T cells. Further, aOX40/belapectin therapy significantly reduced monocytic MDSC (M-MDSCs) and MHC-II hi macrophage populations, both of which displayed reduced arginase 1 and increased iNOS. Combination aOX40/belapectin therapy alleviated M-MDSC-specific functional suppression compared to M-MDSCs isolated from untreated tumors. Our data suggests that Gal-3 inhibition plus aOX40 therapy reduces M-MDSC-meditated immune suppression thereby increasing CD8 + T cell recruitment leading to increased tumor regression and survival.
T cells upregulate the immune checkpoint, LAG-3, which is associated with reduced T cell effector function following repeated antigen exposure. Further, high LAG-3 expression on Tregs has been reported in patients with varying cancer types; thus, we hypothesized that the combination of aLAG-3 with another common ICB, aPD-1, would synergize to potently reinvigorate T cells to promote tumor regression and increase survival. Combined aPD-1/aLAG-3 immunotherapy significantly improved the survival of CT26 (BALB/c; colon carcinoma)and MCA-205 (C57BL/6; sarcoma) tumor-bearing mice compared to monotherapy. In-depth analysis of tumor-infiltrating lymphocytes (TIL) revealed that aPD-1/aLAG-3 therapy significantly increased the overall percentage, cytotoxicity (granzyme A), and cytokine production (TNF-a and IFN-g) of CD8+ TIL, and increased CD4+ Teff/Treg ratios compared to aPD-1 or aLAG-3 alone. These effects were enriched in responders to aPD-1/aLAG-3 therapy, which were designated as those exhibiting decreased tumor size on the day of harvest compared to maximum tumor growth post-implant. Surprisingly, Tregs isolated from tumors responding to aPD-1/aLAG-3 displayed increased proliferation, secretion of pro-inflammatory cytokines (TNF-a and IFN-g), and increased LAG-3 expression. In summary, these data suggest that aPD-1/aLAG-3 immunotherapy increased CD8+ TIL exhibiting enhanced effector function, increased CD4+ Teff/Treg ratios, and induced Treg pro-inflammatory responses. Together, these positive immunological changes led to a more immune stimulatory tumor microenvironment capable of supporting tumor regression and significantly improved tumor-free survival.
Only a subset of patients durable clinical responses to aPD-1 and/or aCTLA-4 immunotherapies, thus, developing new therapeutic agents to increase the proportion of responding patients is a priority. Combining aPD-1 with aLAG-3 has shown promising results; however, lack of mechanistic understanding of aPD-1/aLAG-3 synergy remains a barrier for its optimal clinical use. Here, we examined the mechanism of aPD-1/aLAG-3 synergy in multiple mouse models using flow cytometry and single cell RNA sequencing. Combined aPD-1/aLAG-3 immunotherapy significantly improved the survival of CT26 (BALB/c; colon carcinoma) and MCA-205 (C57BL/6; sarcoma) tumor-bearing mice compared to monotherapy. Regulatory T cells (Tregs) suppressed response to this therapy, as in the absence of CD4+ T cells, 100% of mice responded. To understand how responders overcome Treg suppression, we performed an in-depth analysis of tumor-infiltrating lymphocytes (TIL) comparing mice that responded to treatment (decreased tumor size post-treatment) to non-responders (same tumor growth trajectory as control). Responders had reduced Foxp3+ CD4+ Tregs in comparison to non-responders and, in addition, those Tregs had a ‘fragile’ phenotype, including a pro-inflammatory cytokine profile (TNF-a; IFN-g), increased LAG-3, and decreased NRP1 expression. Within responders, CD8+ TIL exhibited increased frequency, effector cytokine production (TNF-a; IFN-g), and LAG-3 expression as compared to non-responders. Together, these data suggest that aPD-1/aLAG-3 can reduce Treg frequency and function leading to expansion of active tumor-specific CD8+ T cells capable of supporting tumor regression and improved survival. Supported by the Providence Portland Medical Foundation and GlaxoSmithKline
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.