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Background Immune checkpoint inhibitors (ICIs) have shown limited efficacy in colorectal cancer (CRC), largely due to immunosuppressive tumor microenvironment (TME) including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Additionally, the off-target effects of ICIs can reduce drug accumulation in tumor tissues and lead to immune-related adverse events, further compromising their clinical utility.Methods Using knob-into-hole technology, we developed ATAPL1, a bispecific antibody targeting both programmed death-ligand 1 (PD-L1) and tumor necrosis factor receptor 2 (TNFR2). In multiple subcutaneous and orthotopic CRC mouse models, ATAPL1 was evaluated for its tumor targeting, antitumor efficacy, immunomodulatory effects within the TME, and potential combinations with standard chemotherapies.Results Compared with αPD-L1 or αTNFR2 monotherapy, ATAPL1 showed enhanced tumor accumulation and significantly improved antitumor efficacy, with no significant toxicity. It effectively modulated the TME by decreasing immunosuppressive Tregs and monocytic MDSCs (M-MDSCs), while promoting CD8 + T-cell activation and macrophage function. ATAPL1 also primed long-term immune surveillance, suggesting durable antitumor activity. Furthermore, ATAPL1 overcame chemotherapy resistance and exhibited superior therapeutic effects when combined with FOLFOX or FOLFIRI, compared with either treatment alone.Conclusion These findings support ATAPL1 as a promising dual-targeted therapeutic strategy for CRC. It offers the potential to overcome immune and chemotherapy resistance, reduce off-target effects, and improve treatment outcomes through enhanced immune activation and tumor targeting.