Background. Preclinical and clinical data suggest that 4-1BB (CD137), a costimulatory immunoreceptor mainly expressed by cytotoxic cells, represents a promising therapeutic target in cancer. A combination of checkpoint blockade with further T-cell activation mediated by 4-1BB co-stimulation may increase response rates and durability of response. However, the efficacy of systemic 4-1BB stimulation is limited by on target peripheral toxicity events. PRS-344/S095012 has been designed to provide the potential of a combinatorial therapy in one molecule and favor the localized stimulation of antigen-specific T cells in the tumor microenvironment, potentially reducing peripheral toxicity. Methods. Anticalin® proteins are 18 kDa protein therapeutics derived from human lipocalins. We utilized phage display technologies to generate an Anticalin protein that binds to 4-1BB with high affinity and specificity. PRS-344/S095012 was generated by recombinant fusion of two 4-1BB-specific Anticalin proteins to a PD-L1-targeting monoclonal antibody with a modified IgG4 backbone that avoids interaction with Fc gamma receptors and restricts 4-1BB agonism to PD-L1 positive tissues. The activity and potency of PRS-344/S095012 were investigated in binding assays, functional in vitro assays with human primary immune cells, as well as in a human 4-1BB knock in mouse model. Results. The bispecific fusion protein PRS-344/S095012 is capable of binding 4-1BB and PD-L1 simultaneously. We show that the bispecific compound retains its ability to block PD-1/PD-L1 receptor-ligand interaction with similar potency to the parental PD-L1 antibody. In relevant in vitro cell-based assays, PRS-344/S095012 enhances T cell effector functions only in the presence of PD-L1 positive cells, in line with the desired mechanism of action. In vitro, we show that PRS-344/S095012 activity is superior to PD-L1 antibodies, and to the combination of clinically relevant 4-1BB and PD-L1 benchmark antibodies. In a human 4-1BB knock in mouse model, subcutaneously implanted with a human PD-L1 expressing tumor, PRS-344/S095012 showed clear superiority to anti-PD-L1 alone and a robust antitumor response that leads to the complete regression of implanted tumors and extension of survival. Conclusion. We report potent costimulatory T cell engagement of the immunoreceptor 4-1BB in a PD-L1-dependent manner, utilizing the PD-L1/4-1BB bispecific compound PRS-344/S095012. This approach has the potential to provide a localized costimulation of the immune system with high efficacy and reduced peripheral toxicity. Furthermore, dual mechanism of action combining PD-L1-dependent 4-1BB agonist activity with simultaneous PD-1/PD-L1 pathway blockade provides an additional therapeutic benefit in preclinical models. Taken together our in vitro and in vivo data outlines proof of concept functionality of PRS-344/S095012 and supports further development of this promising compound.

Citation Format: Aizea Morales-Kastresana, Marina Pavlidou, Janet Peper, Lucia Pattarini, Christian Barthels, Eva-Maria Hansbauer, Rachida Bel Aiba, Birgit Bossenmaier, Alix Scholer-Dahirel, Thomas Jaquin, Catherine Gallou, Véronique Blanc, Christine Rothe, Shane Olwill. Simultaneous costimulatory T-cell engagement and checkpoint inhibition by PRS-344/S095012, a PD-L1/4-1BB bispecific compound for tumor localized activation of the immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB135.