Abstract 3268: Costimulatory T-cell engagement by PRS-342, a GPC3/4-1BB bispecific molecule, leads to activation of T-cells and tumor growth inhibition in a HCC humanized mouse model

Background: Increasing evidence shows that 4-1BB is a key costimulatory immunoreceptor and a highly-promising therapeutic target in cancer. Current antibody-based approaches showed immune cell activation not only in tumor tissues, but also in the periphery, which is associated with dose-limiting on-target toxicity. To overcome this limitation, we generated PRS-342, a GPC3/4-1BB bispecific molecule based on the Anticalin technology. This molecule is designed to promote 4-1BB clustering by bridging 4-1BB-positive T cells with GPC3-expressing tumor cells. GPC3 is an oncofetal protein with high tumor selectivity and high expression in not only hepatocellular carcinomas, but also in a variety of other tumors with high medical need. Methods: Anticalin therapeutics are 18 kD proteins derived from human lipocalins. We utilized phage display to generate an Anticalin protein binding to 4-1BB with high affinity and specificity. The PRS-342 bispecific construct was generated by genetic fusion of a 4-1BB-specific Anticalin protein to a humanized high affinity GPC3-targeting monoclonal antibody with an engineered IgG4 backbone. Results: We show that the bispecific molecule PRS-342 retains its ability to bind both targets (4-1BB and GPC3) with similar affinity to the parental building blocks and is capable of binding both targets simultaneously. Biophysical characterization of PRS-342 reflect its good drug-like properties. Using in vitro assays based on mixed culture of human pan-T cells and GPC3-expressing tumor cell lines, PRS-342 induced T-cell costimulation leading to increase production of IL-2 with EC50 in the sub-nanomolar range. In contrast, monospecific binding to GPG3 or 4-1BB by benchmarks or single building blocks was not able to activate T cells in this assay. These data demonstrate the ability of PRS-342 to bind both targets simultaneously, which is necessary for clustering of 4-1BB. PRS-342 was also evaluated for activity in a HepG2 mouse xenograft engrafted with human PBMCs with results supporting its differentiated MoA compared to relevant benchmark controls. Conclusion: PRS-342 was designed to elicit 4-1BB costimulatory effects in a tumor-localized manner. Here we report potent T-cell activation that is strictly dependent on the presence of GPC3-positive tumor cells. Collectively our in vitro and in vivo data support the continued development of PRS-342. Citation Format: Birgit Bossenmaier, Corinna Schlosser, Rachida Siham Bel Aiba, Christian Barthels, Benjamin Weiche, Timo Eichner, Michelle Yegres, Shane A. Olwill. Costimulatory T-cell engagement by PRS-342, a GPC3/4-1BB bispecific molecule, leads to activation of T-cells and tumor growth inhibition in a HCC humanized mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3268.