Abstract B026: Development of tumor-targeted agonistic Immunomodulators using the DARPin® technology platform
Authors: Levitsky Victor , Link Alexand , Stump Michael T. , Baur Elmar vom 
Published: February 2019

Abstract
Background: Antibodies against several agonistic immunomodulatory receptors proved to be highly efficacious in preclinical tumor models especially in combination with other immunomodulatory compounds and treatment modalities. However, their clinical application met with limited success and was associated with systemic toxicities which are believed to be driven by Fc-receptor-mediated cross-linking of antibody molecules. We developed a new class of biologics based on the DARPin® technology platform, which allow tumor-localized engagement of immunomodulatory agonists in the absence of Fc-receptors. Preclinical in vitro and in vivo results indicate that DARPin®-based compounds are at least comparable to conventional antibodies in agonistic potency. The localization of their activity to the tumor site relative to the systemic activity of agonistic MAbs should improve the safety and therefore the therapeutic index for drug candidates which exploit this mode of action. Methods: We generated multi-specific DARPin® therapeutics, which bind to human fibroblast activation protein (FAP) and either to 4-1BB, OX40 or CD40 receptors. Each of these receptors can effectively enhance immune responses through a set of distinct and non-redundant mechanisms. Functional properties of these molecules were characterized using appropriate sets of biochemical methods and cell-based assays utilizing reporter cells lines or various subsets of primary immune cells. The FAP-targeted 4-1BB agonist referred to as MP0310, which represents our most advanced drug candidate, was also characterized in vivo in terms of its pharmacokinetics and pharmacodynamics in mice as well as cynomolgus monkeys. The in vivo efficacy of MP0310 was analyzed both in monotherapy and in combination with tumor-specific T-cell engagers using a xenotransplantation human tumor model of immunodeficient mice engrafted with human PBMCs. Results and Conclusions: All analyzed FAP-targeted DARPin®-based agonistic immunomodulators efficiently induced activation of the NFkB pathway in reporter cell lines in a dose dependent manner but only upon crosslinking through binding to FAP. The same pattern of functional activity was exhibited by the molecules in assays with appropriately selected human primary immune cell subsets. We show that MP0310 promotes tumor regression in a dose dependent manner when co-administered with a tumor-specific T-cell engaging bi-specific antibody into tumor-bearing immunodeficient NGS mice engrafted with human PBMCs. The observed effect correlated well with 4-1BB receptor occupancy in blood and an increase of CD8 T lymphocytes in the tumors. Urelumab, a clinically tested 4-1BB agonistic antibody associated with liver toxicity, led to signs of severe graft-versus-host disease (GVHD) and weight loss in the humanized NOG mice. In contrast, mice treated with several doses of MP0310 showed only minor signs of GVHD and marginal weight loss. Thus, tumor targeted DARPin® agonists of co-stimulatory molecules can enhance T-cell activity in a manner independent of Fc-crosslinking and localized primarily to the tumor site, thereby avoiding the systemic toxicity seen with antibodies against the same immunologic targets. This approach with DARPin® therapeutics can be broadly applied to a range of tumor targets and immune receptors.
Citation Format: Victor Levitsky, Alexand Link, Michael T. Stump, Elmar vom Baur. Development of tumor-targeted agonistic Immunomodulators using the DARPin® technology platform [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B026.