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Background Regulatory T cells (Tregs) are key drivers of immune suppression within the tumor microenvironment (TME). Clinically approved anti-CTLA-4 antibodies, such as ipilimumab and tremelimumab, target Tregs to exert anti-tumor effects. However, their use remains largely confined to melanoma and NSCLC due to systemic Treg depletion and related peripheral toxicity. TIGIT, an inhibitory receptor on T and NK cells, has been studied in clinical trials, but limited efficacy of anti-TIGIT monotherapy has prompted combinatorial approaches with other checkpoint targets.Recent studies show intratumoral Tregs highly co-express CTLA-4 and TIGIT, distinguishing them from peripheral Tregs. CTLA-4 recycling antibodies can reduce systemic toxicity by preserving CTLA-4 on peripheral Tregs.To overcome CTLA-4 blockade toxicity and limited anti-TIGIT efficacy, we identified a CTLA-4 antibody with recycling function and developed a bispecific antibody targeting CTLA-4 and TIGIT. This strategy aims to enhance intratumoral Treg selectivity while minimizing off-tumor immune-related adverse events.Methods We screened an anti-human TIGIT monoclonal antibody and an anti-human CTLA-4 recycling monoclonal antibody using phage display screening method. In vitro, we observed pH independent binding ability of the anti-TIGIT mAb and anti-CTLA-4 mAb, also, degradation and recycling of CTLA-4 in HEK293 CTLA-4 cells.To enable selective intratumoral Treg targeting while avoiding peripheral depletion, we designed a CTLA-4 recycling × TIGIT bispecific antibody. To evaluate the efficacy of anti-TIGITxCTLA-4 BsAb, in vitro, we established HEK293-CTLA-4 cells representing peripheral Tregs and HEK-293-TIGIT/CTLA-4 cells representing tumor-associated Tregs. Also, we assessed binding ability upon different pH, antibody dependent cellular cytotoxicity and immunomodulatory efficacy using human PBMC. In vivo, anti-TIGITxCTLA-4 BsAb efficacy was evaluated in various preclinical mouse models.Results According to single-cell sequencing data, co-expression of TIGIT and CTLA-4 is markedly enriched in tumor-associated Tregs relative to peripheral Tregs. We confirmed that the anti-TIGIT mAb enhance cytokine secretion from immune cells and downregulate TIGIT expression upon binding. We also observed that the anti-CTLA-4 antibody induces recycling of the CTLA-4 antigen after binding. Anti-TIGITxCTLA-4 BsAb showed potent anti-cancer effects through pH independent binding ability, high antibody dependent cellular cytotoxicity and increased immunomodulatory effect in various cancers compared to combination treatment or other anti-TIGIT mAb and anti-CTLA-4 mAb monotherapy.Conclusions These findings suggest that the CTLA-4 recycling × TIGIT bispecific antibody enables selective depletion of tumor-infiltrating regulatory T cells, offering a promising strategy to improve tumor specificity while minimizing systemic effects.