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Background CCR4 + regulatory T cells (Tregs), which are widely present in peripheral circulation and tumor microenvironment (TME), promote tumor immune evasion by infiltrating human epidermal growth factor receptor 2-positive (HER2+) tumors in a chemokine-driven manner. However, therapies targeting CCR4 (eg, mogamulizumab) for systemic Treg depletion risk significant toxicity and have far been confined to hematological malignancies. Notably, tumor-chemotactic peripheral Tregs—key precursors to tumor-infiltrating Tregs (tumor-infiltrating lymphocyte (TIL)-Tregs)—remain overlooked.Methods We designed and expressed four candidate anti-HER2×CCR4 DVD-Ig bispecific antibodies with varying degrees of anti-CCR4 domain masking, while leaving anti-HER2 domains fully exposed, to preferentially deplete tumor-associated Tregs, including TIL-Tregs and tumor-chemotactic peripheral Tregs. Stability and antitumor activity were assessed in vitro. In human immune system-reconstituted NOG mice, we systematically: (1) conducted a comprehensive dose-response evaluation of the XL-11 to characterize pharmacological efficacy and potential systemic toxicity, (2) assessed the immune memory, (3) studied the synergy of XL-11 with programmed cell death protein-1/programmed death-ligand 1 (PD-1/L1) blocker, and (4) analyzed pharmacokinetic profile (PK).Results The lower affinity for CCR4 compared with HER2 enables anti-HER2×CCR4 DVD-Igs to priority target TIL-Tregs, and reduce binding to peripheral Tregs compared with mogamulizumab. Anti-HER2×CCR4 DVD-Igs inhibit Treg chemotaxis to TME, and killing Tregs and HER2 + tumor cells through antibody-dependent cellular cytotoxicity. Among the candidates, XL-11, which had the greatest exposure of the anti-CCR4 domain, was selected for in vivo evaluation due to its superior stability and potent antitumor activity. In vivo models of gastric cancer and breast cancer, XL-11, by reducing TIL-Tregs and increasing CD8+/Tregs ratios, induces potent antitumor activity even in advanced stages, with no evidence of metastasis. Concurrently, XL-11 specifically depletes tumor-chemotactic peripheral Tregs, further enhancing antitumor immunity while avoiding reducing Tregs throughout the body even at a high dose (10 mg/kg). There was no increase in memory T cells. In addition, XL-11 enhances the antitumor activity of anti-PD-1 antibodies and shows superior PK properties.Conclusions XL-11 mediates potent antitumor immunity in advanced HER2 + tumors while avoiding reducing Tregs throughout the body. XL-11 also acts synergistically with anti-PD-1 therapy, and exhibits favorable stability and PK supporting clinical translation. This work advances Treg-targeted therapies in HER2+ tumors and overcomes the therapeutic limitations of mogamulizumab.