ATG-101 is a tetravalent PD-L1×4-1BB bispecific antibody that stimulates anti-tumor immunity through PD-L1 blockade and PD-L1-directed 4-1BB activation

Immune checkpoint inhibitors (ICI) have transformed cancer treatment. However, only a minority of patients achieve a profound response. Many patients are innately resistant while others acquire resistance to ICIs. Furthermore, hepatotoxicity and suboptimal efficacy have hampered the clinical development of agonists of 4-1BB, a promising immune stimulating target. To effectively target 4-1BB and treat diseases resistant to ICIs, we engineered ATG-101, a tetravalent "2+2" PD-L1×4-1BB bispecific antibody. ATG-101 bound PD-L1 and 4-1BB concurrently, with a greater affinity for PD-L1, and potently activated 4-1BB+ T cells when crosslinked with PD-L1+ cells. ATG-101 activated exhausted T cells upon PD-L1 binding, indicating a possible role in reversing T-cell dysfunction. ATG-101 displayed potent antitumor activity in numerous in vivo tumor models, including those resistant or refractory to ICIs. ATG-101 greatly increased the proliferation of CD8+ T cells, the infiltration of effector memory T cells, and the ratio of CD8+ T/Treg cells in the tumor microenvironment (TME), rendering an immunologically "cold" tumor "hot". Comprehensive characterization of the TME after ATG-101 treatment using single-cell RNA-sequencing further revealed an altered immune landscape that reflected increased antitumor immunity. ATG-101 was well-tolerated and did not induce hepatotoxicity in non-human primates. According to computational semi-mechanistic pharmacology modeling, 4-1BB/ATG-101/PD-L1 trimer formation and PD-L1 receptor occupancy were both maximized at around 2 mg/kg of ATG-101, providing guidance regarding the optimal biological dose for clinical trials. In summary, by localizing to PD-L1-rich microenvironments and activating 4-1BB+ immune cells in a PD-L1 crosslinking-dependent manner, ATG-101 safely inhibits growth of ICI resistant and refractory tumors.