Abstract 1560: Selection of a bispecific trivalent HER2 x CD137 TRIDENT format providing optimal tumor-anchored immune co-stimulation
Authors: Liu Liqin , Lam Chia-Ying K. , Alderson Ralph , Long Vatana , Yang Yinhua , Burns Robert , Widjaja Lusiana , Li Jonathan , Wolf Christina , Ciccarone Valentina , Tamura James , Diedrich Gundo , Bonvini Ezio , Johnson Syd , Moore Paul A. 
Published: August 2019

Abstract
Introduction: CD137 (4-1BB) signaling provides co-stimulation of CD8 or NK cells following antigen or FcγR engagement, respectively. Efforts to leverage CD137 co-stimulation via agonistic monoclonal antibodies (mAbs) have been thwarted by limited clinical efficacy or unacceptable toxicity. Bispecific targeting strategies linking CD137 activation to a tumor-targeting moiety provides an approach to localize CD137 activation to the tumor microenvironment. Here we evaluate a panel of Fc-bearing HER2 x CD137 bispecific molecules incorporating different valency and geometry to define the format providing optimal CD137 co-stimulation in a tumor-cell anchor-dependent manner.
Methods: An anti-HER2 mAb specificity that does not cross compete with margetuximab, trastuzumab or pertuzumab and a proprietary anti-CD137 mAb were utilized to assemble a set of HER2 x CD137 bispecific molecules in bivalent and tetravalent DART® or trivalent TRIDENTTM configurations. The resulting molecules were compared in binding, signaling and co-stimulation assays in the presence or absence of tumor cells expressing HER2. Combination studies were performed in vitro and in immune deficient mice reconstituted with human PBMCs.
Results: TRIDENT molecules bearing bivalent CD137 and monovalent HER2 binding achieve optimal HER2-dependent tumor-cell anchored CD137 immune cell co-stimulation. CD137 co-stimulation increases proportionally with the level of HER2 expression as observed with HER2 1+ (MCF7 breast), HER2 2+ (JIMT1 breast) and 3+ (N87 gastric) tumor cells and was paralleled with increased HER2+/CD137+ cell association. No CD137 activation is observed in the absence of HER2-expressing tumor cells. HER2 x CD137 bispecifics enhance NK-cell proliferation and IFN-γ release induced by margetuximab, an Fc-optimized anti-HER2 mAb that up-regulates CD137 expression on NK-cells concomitant with enhanced ADCC against HER2-positive cells. Similarly, HER2 x CD137 bispecific molecules enhanced the in vitro activity of orlotamab, a B7-H3 x CD3 bispecific DART molecule that up-regulates CD137 during T-cell redirected killing. Finally, in vivo mouse studies demonstrate the ability of HER2 x CD137 molecules to expand tumor-associated CD8 cells when co-administered with a tumor targeted CD3 bispecific molecule and support enhance anti-tumor activity.
Conclusion: An optimal HER2 x CD137 bispecific format providing maximal CD137 activation in a HER2- dependent manner was identified as a trivalent TRIDENT molecule bearing bivalent CD137 and monovalent HER2 binding. Combinatorial activity with HER2 x CD137 bispecifics was observed with both a HER2-directed therapeutic mAb and a CD3-engaging tumor-targeted bispecific molecule. HER2 x CD137 TRIDENT molecules have therapeutic potential and provide a structural template for incorporating alternate tumor- and/or co-stimulatory-targeting arms.
Citation Format: Liqin Liu, Chia-Ying K. Lam, Ralph Alderson, Vatana Long, Yinhua Yang, Robert Burns, Lusiana Widjaja, Jonathan Li, Christina Wolf, Valentina Ciccarone, James Tamura, Gundo Diedrich, Ezio Bonvini, Syd Johnson, Paul A. Moore. Selection of a bispecific trivalent HER2 x CD137 TRIDENT format providing optimal tumor-anchored immune co-stimulation [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 1560.