Abstract 3642: Tumor-antigen expression-dependent activation of the CD137 costimulatory pathway by bispecific DART® proteins
Authors: Liu Liqin , Lam Chia-Ying K. , Long Vatana , Widjaja Lusiana , Yang Yinhua , Shah Kalpana , Smith Doug , Pan Joanna , Johnson Syd , Bonvini Ezio , Moore Paul 
Published: July 2017

Abstract
Introduction: CD137 (4-1BB) is a co-stimulatory molecule expressed by activated T and NK cells that, upon interaction with its CD137 ligand, further supports cell activation, proliferation and survival. Activation via CD137 holds great promise for cancer immunotherapy; however, current CD137 agonistic interventions are associated with systemic safety concerns. To develop a therapeutic modality that reduces the potential for systemic CD137 effects, we applied the DART® bispecific platform to generate proteins that can induce tumor-antigen dependent T-cell activation.
Methods: DART molecules were constructed containing anti-CD137 variable regions together with either anti-HER2 or anti-EphA2 variable regions. DART binding properties were evaluated by ELISA or flow cytometry; signaling responses assessed using a NF-κB luciferase reporter cell line expressing CD137. Co-stimulatory activity was characterized with primary human T cells in the presence or absence of tumor target antigen-expressing cells.
Results: ELISA and flow cytometry analysis demonstrated that both HER2 x CD137 and EphA2 x CD137 DART molecules bind their respective target antigens. Co-culturing of a CD137/NF-κB reporter cell line with tumor lines expressing HER2 or EphA2 revealed tumor antigen-dependent CD137 pathway activation by HER2 x CD137 and EphA2 x CD137 DART molecules, respectively. To evaluate the effects of HER2 x CD137 and EphA2 x CD137 DART molecules on T-cell responses, co-stimulation T-cell assays were performed. In the presence of the relevant antigen-positive cell line, each respective DART molecule was able to promote T-cell proliferation and cytokine release in a HER2 or EphA2-dependent manner. No T-cell co-stimulation was observed by either DART molecule in the absence of antigen-expressing tumor cells. Furthermore, the level of tumor antigen-dependent co-stimulation supported by the DART molecules correlated with the level of tumor target expression. Consistent with the preferential induction of CD137 by the CD8 T cell subset, CD137-based DART proteins induced a substantial increase in the fraction of CD8+ central memory and effector memory T cells in the presence of the proper tumor antigen expressing cells.
Conclusions: HER2 x CD137 and EphA2 x CD137 DART proteins promote T-cell co-stimulation in a tumor antigen-dependent manner and may provide an opportunity to target the CD137 co-stimulatory pathway for cancer immunotherapy, while limiting systemic T-cell activation and related side effects.
Citation Format: Liqin Liu, Chia-Ying K. Lam, Vatana Long, Lusiana Widjaja, Yinhua Yang, Kalpana Shah, Doug Smith, Joanna Pan, Syd Johnson, Ezio Bonvini, Paul Moore. Tumor-antigen expression-dependent activation of the CD137 costimulatory pathway by bispecific DART® proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3642. doi:10.1158/1538-7445.AM2017-3642