Abstract Introduction: The co-stimulatory receptor CD40 is strongly expressed on B cells, monocytes and antigen-presenting cells (APC). By promoting their maturation, activation and survival, CD40 signaling greatly contributes to anti-tumor responses of the immune system. The HERA-Technology developed by Apogenix is a powerful engineering platform for the production of modular fusion proteins targeting the TNF-receptor superfamily. Structurally, these novel ligands comprise a single-chain arrangement of three TNFSF protomers forming one TNFSF-receptor binding domain (RBD) with specific linkers and the Fc-part of human IgG as dimerization scaffold. The result is a stable and defined hexavalent receptor agonist with high clustering capacity for the cognate receptor. Here we compared the efficacy of different CD40 agonist formats, including the novel hexavalent scCD40L-RBD-Fc (APG1233), and the functional consequences of differential receptor clustering. In addition, the in vivo pharmacokinetics of the fully human APG1233 is compared with a chimeric mouse/human surrogate molecule APG1274. Materials and Methods: Immune cells were isolated from healthy-donor blood samples and profiled by multicolor flow cytometry (MC-FC). Subsequently, immune cells were cultured in growth media containing various forms of CD40 agonists. Upregulation of activation markers on B cells and monocytes (e.g. CD69, CD86, HLA-DR) and T cell-induced killing of tumor cells in direct co-culture was assessed by MC-FC and employing a real-time cell analysis system (xCELLigence), respectively. Secretion of cytokines in response to CD40 ligation and the pharmacokinetic properties of the fully human APG1233 and the chimeric murine/human APG1274 were determined by ELISA. Results: In vivo stability of APG1233 was demonstrated in a single dose mouse PK study revealing a terminal half-life of 84 hours. The human CD40L, however, does not bind murine CD40. Therefore, we used APG1274, a chimeric protein comprising the murine CD40L fused to human IgG and binding to mouse CD40, to determine serum pharmacokinetics. As a result of functional binding of APG1274 to murine CD40, the surrogate molecule is eliminated much quicker (t1/2 of 4 hours) demonstrating the specificity of both compounds. In vitro only the hexavalent APG1233 displayed activity in terms of stimulation of B cells, monocytes and PBMCs. In contrast, neither trimeric CD40L nor an agonistic antibody against CD40 were able to upregulate expression of activation markers. Similarly, the secretion of proinflammatory cytokines such as IL-12, CD95L and IFNγ by PBMCs was only stimulated after exposure to APG1233 and not in the presence of other CD40 agonists. In functional co-culture assays, after exposure to APG1233, in vitro generated M2-macrophages underwent conversion and acquired M1-type surface markers which strongly enhanced proliferation of naïve CD4+ T cells. Induction by M2-macrophages in this setup was much lower. Consistent with these data, only the hexavalent CD40 agonist APG1233 efficiently increased direct cytotoxic activity of immune cells against tumor cells measured by a real-time cell analysis assay. Conclusion: The CD40 agonist APG1233 is a member of a novel class of hexavalent TNFRSF agonists which binds its target with high specificity, exhibits excellent in vivo stability and superior biological activity over other agonistic formats (e.g. agonistic antibodies). Citation Format: Christian Merz, Jaromir Sykora, Meinolf Thiemann, David M. Richards, Thamara Beyer, Stefanie Kühn, Harald Fricke, Christian Gieffers, Oliver Hill. The hexavalent CD40 agonist APG1233 is a superior inducer of immune cell activation compared to trimeric formats or agonistic anti-CD40 antibodies [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 B066.