The PD-L1/4-1BB bispecific Antibody-Anticalin fusion protein PRS-344/S095012 elicits strong T-cell stimulation in a tumor-localized manner

While patients responding to checkpoint blockade often achieve remarkable clinical responses, there is still significant unmet need due to resistant or refractory tumors. A combination of checkpoint blockade with further T-cell stimulation mediated by 4-1BB agonism may increase response rates and durability of response. A bispecific molecule that blocks the PD-1/PD-L1 axis and localizes 4-1BB co-stimulation to a PD-L1-positive tumor microenvironment (TME) or tumor draining lymph nodes could maximize antitumor immunity and increase the therapeutic window beyond what has been reported for anti-4-1BB monoclonal antibodies (mAbs). We generated and characterized the PD-L1/4-1BB bispecific molecule PRS-344/S095012 for target binding and functional activity in multiple relevant in vitro assays. Transgenic mice expressing human 4-1BB were transplanted with human PD-L1-expressing murine MC38 cells to assess in vivo antitumoral activity. PRS-344/S095012 bound to its targets with high affinity and efficiently blocked the PD-1/PD-L1 pathway, and PRS-344/S095012-mediated 4-1BB co-stimulation was strictly PD-L1 dependent. We demonstrated a synergistic effect of both pathways on T-cell stimulation with the bispecific PRS-344/S095012 being more potent than the combination of mAbs. PRS-344/S095012 augmented CD4 and CD8 T cells effector functions and enhanced antigen-specific T-cell stimulation. Finally, PRS-344/S095012 demonstrated strong antitumoral efficacy in an anti-PD-L1 resistant mouse model in which soluble 4-1BB was detected as an early marker for 4-1BB agonist activity. The PD-L1/4-1BB bispecific PRS-344/S095012 efficiently combines checkpoint blockade with a tumor-localized 4-1BB mediated stimulation burst to antigen-specific T cells, more potent than the combination of mAbs, supporting the advancement of PRS-344/S095012 towards clinical development.