Abstract 3754: The novel hexavalent human CD137/4-1BB agonist HERA-CD137L promotes anti-cancer immunity by activating CD8 T cells while regulatory T cells are not affected
Authors: Thiemann Meinolf , Sykora Jaromir , Richards David M. , Merz Christian , Marschall Viola , Mueller Mauricio Redondo , Sefrin Julian P. , Heinonen Karl , Fricke Harald , Gieffers Christian , Hill Oliver 
Published: August 2018

Abstract
CD137/4-1BB is an inducible costimulatory receptor mainly expressed on immune cells following activation. Binding of the cognate ligand CD137L/4-1BBL leads to receptor trimerization and activation of signaling cascades involved in expansion and survival of T cells and myeloid cells as well as memory formation and protection against autoimmunity. Apogenix´ HERA technology is based on trivalent single-chain molecular mimics of the TNF-SF receptor binding domains fused to a silenced human IgG1-Fc-domain which serves exclusively as a dimerization scaffold. This results in hexavalent agonists replicating the natural receptor binding mode. Due to their inherent high receptor clustering capacity, HERA compounds are true agonists and their biological activity is, in contrast to bivalent anti-TNFR-SF antibodies, independent of FcR-mediated crosslinking events. HERA-CD137L was produced in CHO-S cells and purified resulting in highly pure non-aggregating protein lots. PK studies in mice demonstrated a terminal half-life of 75 hours indicating excellent in vivo stability. To study the effects on immune cells in vitro, T cells were isolated from healthy-donor buffy coats and stimulated with anti-CD3 antibody alone or in combination with HERA-CD137L. Using multicolor flow cytometry, we confirmed that expression of CD137 increased on CD8+ T cells following stimulation with anti-CD3 antibody. In accord with upregulation of the activation markers CD25 and CD69 and the memory marker CD45RO, treatment with HERA-CD137L enhanced proliferation of both CD4+ and CD8+ T cells, as determined by CFSE analysis. Intracellular accumulation of IFN-γ, TNF-α, Granzyme B and Perforin upon CD137 ligation was observed in CD8+ but not CD4+ T cells. HERA-CD137L treatment of THP-1 monocytes co-cultured with primary T cells also increased their cytotoxic activity against multiple tumor cell lines, including colorectal and mammary, as shown in a real-time live cell analysis (RTCA) assay. Primary human monocytes express low levels of CD137 and differentiation to macrophages in vitro did not increase expression. However, pro-inflammatory cytokines such as TNF-α and the chemokine CCL4 were secreted after stimulation with HERA-CD137L. RTCA assays further demonstrated enhanced antigen-specific killing of MDA-MB231 tumor cells by HERA-CD137L treated T cells. HERA-CD137L conveys its activity through effector T cell proliferation while regulatory T (Treg) cell proliferation or production of anti-inflammatory cytokines are not altered in Treg cell cultures. In contrast, HERA-CD137L prevented Treg-mediated suppression of effector T cells. Based on the in vitro data presented, HERA-CD137L is a promising candidate to promote anti-tumor immune responses either as single agent or in combination with other IO-compounds.
Citation Format: Meinolf Thiemann, Jaromir Sykora, David M. Richards, Christian Merz, Viola Marschall, Mauricio Redondo Mueller, Julian P. Sefrin, Karl Heinonen, Harald Fricke, Christian Gieffers, Oliver Hill. The novel hexavalent human CD137/4-1BB agonist HERA-CD137L promotes anti-cancer immunity by activating CD8 T cells while regulatory T cells are not affected [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3754.