Abstract
Introduction: Targeted therapeutics for cancer treatment are mostly developed as antibodies, however, the natural signaling complexes of the members of the TNF superfamily and their receptors consist of clusters of trimers. Consequently, most of these bivalent agonistic antibodies depend on Fc receptor mediated crosslinking for biological activity. The HERA-Technology developed by Apogenix generates hexavalent fusion proteins targeting the TNF-receptor superfamily with high clustering capacity for the cognate receptor, which overcomes this disadvantage of antibody-based drugs. Here we compared the efficacy of different CD40 agonist formats, including the novel HERA-CD40L and the functional consequences of differential receptor clustering.
Materials & Methods: Biological activity of CD40 agonists was compared using an engineered reporter cell line and by flow cytometric analysis of CD40-induced IκBα degradation in Ramos B cells. T lymphocytes and monocytes were isolated from buffy coats and expression of CD markers upon CD40 ligation was analysed by multicolor flow cytometry (MC-FC). Secretion of cytokines in response to CD40 ligation was determined by ELISA. Monitoring of T cell-induced killing of tumor cells in direct co-cultures employed a real-time cell analysis system (xCELLigence).
Results: Direct comparison of bivalent CD40 antibodies with trivalent CD40L and the hexavalent HERA-CD40L in two independent bioactivity assay formats demonstrated that only the hexavalent agonist was fully active without additional crosslinking. In contrast to HERA-CD40L, neither the bivalent agonistic CD40 antibody nor the trivalent CD40L were able to upregulate expression of activation markers on B cells or to induce secretion of proinflammatory cytokines such as IL-12 and TNFα by PBMCs. In vitro generated M2-macrophages acquired an M1 phenotype and enhanced proliferation of naïve CD4+ T cells in direct co-culture. Similarly, direct co-culture of CD4+ T cells with Ramos B cells in the presence of HERA-CD40L induced cytotoxic activity of CD4+ cells against tumor cells. The activating effect was dependent on cell-cell contacts and was not observed in indirect co-cultures. Importantly, only the hexavalent HERA-CD40L showed full biological activity without additional crosslinking.
Conclusion: The hexavalent CD40 agonist HERA-CD40L produced by Apogenix HERA-Technology platform triggers CD40 signaling on B cells and cells of the monocytic lineage, leads to direct cytolytic activation and proliferation of CD4+ T cells and shifts the M1/M2 balance towards proinflammatory conditions. Unlike bivalent CD40 antibodies or trivalent CD40L_ based agonists, the hexavalent HERA-CD40L forms highly clustered signaling complexes and thus exhibits superior biological activity over other agonistic formats without the need for Fc receptor mediated crosslinking.
Citation Format: Christian Merz, Jaromir Sykora, Meinolf Thiemann, Viola Marschall, Karl H. Heinonen, Harald Fricke, Christian Gieffers, Oliver Hill. HERA-CD40L: A novel hexavalent CD40 agonist with superior biological activity [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 1688. doi:10.1158/1538-7445.AM2017-1688
- ©2017 American Association for Cancer Research.
Volume 77, Issue 13 Supplement
