Abstract <p>Immune checkpoint inhibitors (ICIs) have significantly advanced the field of cancer immunotherapy. However, clinical data has shown that many patients have a low response rate or even resistance to immune checkpoint inhibitor alone. The underlying reasons for its poor efficacy include the deficiency of immune infiltration and effective CD28/CD80 costimulatory signal in tumor. Discoidin domain receptor 1 (DDR1) has been reported to be negatively related to immune cell infiltration in tumors. Herein, we constructed a soluble fusion protein using CD80, the natural ligand of CD28, in combination with DDR1 inhibitor. Our results demonstrated that CD80-Fc effectively activated T cells and inhibited tumor growth in vivo, even in tumors with poor efficacy of ICIs. Importantly, CD80-Fc fusion protein had a milder affinity against the targets which suggested a potential higher safety than CD28 agonists. Further, in order to promote tumor immune infiltration, we attempted to combine CD80-Fc fusion protein with DDR1 inhibitor for treatment. Our results indicated that using CD80-Fc fusion protein along with DDR1 inhibitor significantly promoted T cell infiltration in tumor microenvironment and more strongly inhibited tumor growth. Therefore, the combination use of CD80 fusion protein and DDR1 inhibitor could become an effective tumor immunotherapy strategy, potentially benefiting a larger number of patients.</p> Key points <p><i>• We successfully constructed, expressed, and purified the recombinant CD80-Fc fusion protein</i></p> <p><i>• We demonstrated that CD80-Fc fusion protein has good safety and anti-tumor activity</i></p> <p><i>• We demonstrated that using CD80-Fc fusion protein along with DDR1 inhibitor can significantly promote immune infiltration of T cells in tumor microenvironment and more strongly inhibit tumor growth</i></p>