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Background Immune checkpoint inhibitors (ICIs) targeting PD-1/PD-L1 have transformed cancer therapy, but resistance remains a major clinical challenge. Anti-TGF-β/PD-L1 bispecific antibodies (BsAbs), initially developed as next-generation PD-L1 blockers to alleviate TGF-β-mediated immune exclusion, have shown limited efficacy in later-phase clinical trials. Our preliminary findings indicate that resistance to anti-TGF-β/PD-L1 BsAbs may result from insufficient innate immune activation, which could be reversed through pharmacologic stimulation of the STING pathway.Methods We evaluated the therapeutic efficacy of combining the STING agonist MSA-2 with the anti-TGF-β/PD-L1 BsAb YM101 in multiple murine tumor models. Multi-omics approaches, including bulk and single-cell RNA sequencing (scRNA-seq), flow cytometry, and proteomics, were used to profile the tumor immune microenvironment. Functional importance of the CXCL16-CXCR6 axis was examined using CXCL16-neutralizing antibodies, conditional Cxcr6 and Stat1 knockout mice, and adoptive transfer of CXCR6+ CD8+ T cells. To translate this combination into a single-agent therapeutic, we developed an antibody-drug conjugate (ADC), Y101S, by conjugating MSA-2 to YM101 via a cleavable linker (figure 1). Antitumor activity and systemic safety of Y101S were assessed in vivo.Results MSA-2 combined with YM101 significantly inhibited tumor growth and improved survival in EMT-6, CT26, and 4T1 models, outperforming either monotherapy or the STING agonist plus anti-PD-L1. scRNA-seq analysis revealed substantial expansion of CXCR6 + CD8+ T cells and increased CXCL16 expression in tumor-associated macrophages and dendritic cells. Functional blockade or genetic ablation of the CXCL16-CXCR6 axis abolished the therapeutic synergy, confirming its necessity. Mechanistically, STING activation induced CXCL16 expression via the IFN-I-STAT1 pathway, which was suppressed by TGF-β through HDAC4-mediated inhibition of IRF3 phosphorylation. The engineered ADC Y101S effectively reproduced the immune-activating effects of combination therapy, resulting in robust tumor suppression and prolonged survival with no detectable systemic toxicity.Conclusions Our study identifies insufficient innate immune activation as a key mechanism underlying resistance to anti-TGF-β/PD-L1 BsAbs. Activation of the STING pathway restores IFN-I signaling and promotes CXCL16-CXCR6-mediated recruitment of cytotoxic CD8 + T cells, overcoming resistance. Y101S, a rationally designed triple-targeting ADC, delivers this strategy in a single molecule and demonstrates strong translational potential for patients unresponsive to conventional immunotherapy.Abstract 429 Figure 1Schematic depiction of the STING/TGF-β/PD-L1 triple-targeting antibody-drug conjugate