Abstract 1760: The hexavalent CD40 agonist HERA-CD40L augments multi-level crosstalk between T cells and antigen-presenting cells
Authors: Merz Christian , Sykora Jaromir , Marschall Viola , Richards David M. , Thiemann Meinolf , Fricke Harald , Hill Oliver , Gieffers Christian 
Published: August 2018

Abstract
Introduction: HERA-CD40L is a novel hexavalent CD40 agonist engineered with the HERA-Technology developed by Apogenix. We have previously shown that the natural binding mode via the receptor/ligand binding domains and the high clustering capacity for the cognate receptor clearly distinguish HERA-CD40L from other, e.g. antibody-based, CD40-targeting compounds. Here, we report on the effects of HERA-CD40L on crosstalk between T cells and antigen presenting cells (APC) and the functional consequences in vitro. Materials & Methods: Biological activity of CD40 agonists was analyzed using co-cultures of primary T cells with B cells or monocytes/macrophages. All primary cells were isolated by negative selection using magnetic sorting from healthy donor buffy coats. Expression of CD markers upon CD40 ligation on B cells and monocytes was analyzed by flow cytometry (FC). Monitoring of T cell-induced killing of tumor cells primed in direct co-cultures with APC was done on a real-time cell analysis system (xCELLigence). For analysis of phagocytosis, we developed an FC-based assay employing primary monocytes/macrophages and Jurkat A3 cells. Results: Treatment of primary B cells and monocytes with HERA-CD40L induced expression of co-stimulatory molecules, like CD86, and promotes M1 maturation of naïve (M0) monocytes. In vitro, treatment of alternatively activated M2 macrophages with HERA-CD40L induced an M2 to M1 phenotype switch (re-programming) which concurs with CD16 downregulation and a dose-dependent decrease of phagocytic activity of re-programmed macrophages compared to M0 or M2 macrophages. Primary B cells and M1 macrophages enhanced the proliferation and cytotoxic activity of naïve T cells in direct co-cultures in the presence of HERA-CD40L. The activating effect on T cells required direct cell-cell contact with APC and was not observed in indirect co-cultures. Functionally, neutralization of either MHC-I or CD80/CD86 in direct co-cultures inhibited full activation of the T cells in vitro as shown by kill assays with various tumor cell lines. Conclusion: The hexavalent CD40 agonist HERA-CD40L produced by the Apogenix HERA-Technology is a potent immune-regulator acting on B cells and myeloid cells. HERA-CD40L promotes activation of B cells, maturation of APC and induces an M2 to M1 phenotype switch which inhibits tolerance-inducing phagocytic activity of the repolarized macrophages in vitro. In response to CD40 ligation on APC, an efficient anti-tumor response is conferred to primary T cells through cell-cell interactions via MHC-I and CD80/CD86.
Citation Format: Christian Merz, Jaromir Sykora, Viola Marschall, David M. Richards, Meinolf Thiemann, Harald Fricke, Oliver Hill, Christian Gieffers. The hexavalent CD40 agonist HERA-CD40L augments multi-level crosstalk between T cells and antigen-presenting cells [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 1760.