Abstract B016: Costimulatory T-cell engagement by PRS-343, a CD137 (4-1BB)/HER2 bispecific, leads to tumor growth inhibition and TIL expansion in humanized mouse model
Authors: Hinner Marlon J. , Bel Aiba Rachida-Siham , Schlosser Corinna , Jaquin Thomas , Allersdorfer Andrea , Berger Sven , Wiedenmann Alexander , Matschiner Gabriele , Schüler Julia , Moebius Ulrich , Rothe Christine , Olwill Shane A. 
Published: November 2016

Abstract
Background: CD137 (4-1BB) is a key costimulatory immunoreceptor and a member of the TNF-receptor (TNFR) superfamily. While multiple lines of evidence show that CD137 is a highly promising therapeutic target in cancer, current mAb-based approaches are not designed to achieve a tumor-target driven activation and may display toxicity and a limited therapeutic window due to peripheral T cell and NK cell activation. To overcome this limitation, we generated PRS-343, a CD137/HER2 bispecific that is designed to promote CD137 clustering by bridging CD137-positive T cells with HER2-positive tumor cells, thereby providing a potent costimulatory signal to tumor antigen-specific T cells.
Methods: Anticalin® proteins are 18 kD protein therapeutics derived from human lipocalins. We utilized phage display to generate an Anticalin protein binding to CD137 with high affinity and specificity. PRS-343 was obtained by genetic fusion of the CD137-specific Anticalin protein to a variant of the HER2-targeting monoclonal antibody trastuzumab with an engineered IgG4 backbone. We have shown previously that the bispecific fusion PRS-343 targets CD137 and HER2 in a bispecific manner and efficiently activates T cells ex vivo in the presence of HER2-positive cells. Here, in vivo proof of concept data is presented utilizing a humanized mouse model in immunocompromised mice and the SK-OV-3 cell line as a HER2-positive xenograft. When tumors had reached a predefined size, mice received human PBMC via an intravenous route and weekly intraperitoneal injections of PRS-343 for three weeks. An IgG4 isotype antibody served as the negative control, while a CD137-targeting benchmark antibody and trastuzumab with an engineered IgG4 backbone (“tras-IgG4”) served as controls for monospecific targeting of CD137 and HER2, respectively.
Results: PRS-343 activity was investigated at four different weekly doses of PRS-343 (4μg, 20μg, 100μg and 200μg). We found that PRS-343 dose-dependently led to strong tumor growth inhibition compared to treatment with the isotype control, and that the tumor response was accompanied by a significantly higher tumor infiltration with human lymphocytes (hCD45+). Interestingly, the anti-CD137 benchmark neither displayed tumor growth inhibition nor enhanced lymphocyte infiltration into tumors compared to isotype. The tras-IgG4 control was also devoid of lymphocyte infiltration into the tumor, but displayed a tumor growth inhibition comparable to PRS-343. Taken together, these data show that PRS-343 provided dual activity by both increasing the frequency of tumor-infiltrating lymphocytes by bispecific targeting of CD137 and HER2 as well as mediating direct tumor growth inhibition by the direct, monospecific targeting of HER2. Notably, the tumor growth inhibition provided by targeting HER2 did not require any antibody directed cellular cytotoxicity (ADCC) as both PRS-343 and the tras-IgG4 control lack the ability to interact with Fc-gamma receptors on NK cells that ADCC would require. The animal model also allowed investigating the potential safety of PRS-343: While the anti-CD137 benchmark accelerated the onset of graft-versus-host-disease and led to stronger expansion of CD8+ T cells in the peripheral blood compared to the isotype control group, both of these effects were absent for PRS-343. The data therefore support the envisaged mode of action of selective, tumor-localized costimulatory T cell activation, as well as the concept that such an approach may lead to higher efficacy and reduced systemic toxicity compared to conventional anti-CD137 mAbs.
Conclusion: We report potent costimulatory T-cell engagement of the immunoreceptor CD137 in a HER2-dependent manner, utilizing the CD137/HER2 bispecific PRS-343. In a humanized mouse model, PRS-343 displays dual activity based on monospecific HER2-targeting and bispecific, tumor-localized costimulation of CD137. Compared to known CD137-targeting antibodies in clinical development, this approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity. The direct, monospecific HER2-targeting activity may provide an additional therapeutic benefit and work in synergy with local CD137 costimulation. The positive functional ex vivo and in vivo data of PRS-343 as well as the excellent developability profile support investigation of its anti-cancer activity in clinical trials.
Citation Format: Marlon J. Hinner, Rachida-Siham Bel Aiba, Corinna Schlosser, Thomas Jaquin, Andrea Allersdorfer, Sven Berger, Alexander Wiedenmann, Gabriele Matschiner, Julia Schüler, Ulrich Moebius, Christine Rothe, Shane A. Olwill. Costimulatory T-cell engagement by PRS-343, a CD137 (4-1BB)/HER2 bispecific, leads to tumor growth inhibition and TIL expansion in humanized mouse model [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B016.