Specific depletion of TIGIThigh CD226- clonally expanded intratumoral Tregs defines safe and effective TIGIT targeting

Background: Global clinical programs based on TIGIT (T cell immunoglobulin and ITIM domain) blockade have been terminated due to inadequate clinical efficacy. New approaches are needed to increase the antitumor activity of anti-TIGIT-based immunotherapy. Methods: Multiomics analyses, including single-cell RNA sequencing (scRNA-seq), single-cell TCR sequencing (scTCR-seq), and The Cancer Genome Atlas bulk RNA-seq, were performed to profile TIGIT and the costimulator CD226 expression and assess intratumoral regulatory T cells (Tregs) clonality, with validation by flow cytometry in murine and patient-derived tumor-infiltrating lymphocytes. The therapeutic efficacy of anti-TIGIT antibodies (αTIGIT), including αTIGIT-IgG1-wild-type (WT), αTIGIT-IgG1-WT with enhanced antibody-dependent cellular cytotoxicity (ADCC) activity (αTIGIT-IgG1-ADCC), αTIGIT-IgG4-WT, and tiragolumab, was evaluated in MC38 tumors-inoculated humanized TIGIT knock-in mice (Tigith/h , Tigith/m ). Tumor microenvironment alterations were analyzed using flow cytometry and scRNA/TCR-seq. Antibody binding affinity and ADCC activity were assessed via biolayer interferometry and in vitro ADCC assays. Safety profiles were examined in a murine immune-related adverse events model through growth monitoring, survival analysis, and histopathological evaluation. Results: Using mouse models and clinical sample analysis, we identified a population of TIGIThigh CD226- clonally expanded intratumoral Tregs as a major barrier limiting the ability of TIGIT blockade to enhance effector cell function. αTIGIT-IgG1-ADCC specifically targeted and eliminated the TIGIThigh CD226- clonally expanded Treg subset, resulting in a marked improvement in antitumor efficacy compared with WT and clinically unsuccessful αTIGIT. Mechanistically, removal of these Tregs through αTIGIT-IgG1-ADCC relieved their suppression of stem-like CD4+ T cells, facilitating their differentiation into T helper cell 1 (Th1) effector cells. Th1-derived interferon-gamma (IFN-γ) further enhanced the functionality of tumor-infiltrating CD8+ T cells. Importantly, the presence of clonally expanded intratumoral Tregs and the suppression of stem-like CD4+ T cells correlated with poor immunotherapy response in patients with non-small cell lung cancer. Conclusions: Targeted elimination of clonally expanded intratumoral Tregs is essential to unlock the full therapeutic potential of αTIGIT. These novel findings provide a key rationale and strategic direction for overcoming the limitations of current αTIGIT-based cancer immunotherapy.