Depleting IL1R2+ Tumor-Infiltrating Regulatory T Cells with an ADCC-Prone Nanobody Construct Boosts the Efficacy of Anti-PD-1 Immunotherapy.

Eliminating immunosuppressive cells, such as regulatory T cells (Treg), is a promising approach to boost immunotherapy success. However, this approach may suffer from systemic autoimmune adverse events, highlighting the need to specifically target tumor-infiltrating Tregs (tiTreg). Based on cellular indexing of transcriptomes and epitopes by sequencing and single-cell RNA sequencing data from mouse models of triple-negative breast cancer (TNBC) and colorectal carcinoma, as well as a meta-analysis of human TNBC and colorectal carcinoma datasets, we obtained a comprehensive overview of the tiTreg heterogeneity and IL1R2 expression. Several IL1R2-expressing tiTreg clusters were identified in mouse and human TNBC and colorectal carcinoma tumors, with some level of conservation. IL1R2 was identified as a surface marker that was most highly expressed by activated and strongly T-cell-suppressive tiTregs in the tumor microenvironment but not by peripheral Tregs. IL1R2 upregulation resulted from T-cell receptor-mediated Treg triggering in a Rel-dependent fashion, but the receptor itself was dispensable for tiTreg abundance and activation and did not influence tumor growth. Accordingly, the blockade of IL1R2, by using an Ab-dependent cell-mediated cytotoxicity (ADCC)-dead anti-IL1R2 nanobody-Fc construct, had no impact on tumor growth. Conversely, anti-IL1R2 nanobody-Fc constructs with an optimized ADCC functionality, mediated by the SDALIE mutation, resulted in the specific depletion of IL1R2+ tiTregs, elicited antitumor immunity, and reduced tumor growth in synergy with anti-PD-1 therapy. Collectively, these findings identify IL1R2 as a marker for highly activated and suppressive tiTregs that is suitable as a target for ADCC-dependent tiTreg depletion, which can synergize with immune checkpoint blockade. SIGNIFICANCE: IL1R2+ Treg depletion using IL1R2-targeting ADCC-prone constructs is a potential cancer therapy to selectively target tumor-infiltrating Tregs and circumvent autoimmune complications caused by systemic Treg depletion.