Introduction: Initiation and perpetuation of anti-tumor responses of the immune system are in the focus of current approaches to fight cancer. Manipulation of the tumor microenvironment, e.g. shifting the M1/M2 ratio towards M1 is one goal to support immunologic cancer treatment. Here we examined and compared the functional effects of bivalent agonistic anti-CD40 antibodies, homo-trimeric trivalent CD40-Ligand and the novel hexavalent CD40-agonist APG1233 on the maturation and differentiation of primary monocytes, B cells and T cell mediated tumor cell killing.

Materials and Methods: Following treatment with various CD40 agonists, cytokine secretion by monocytes, PBMCs and T cells from blood samples was assessed by ELISA. Monocytes isolated from healthy-donor blood samples were differentiated in vitro into either M1- or M2-type macrophages, or dendritic cells (DC) which was confirmed by multicolor flow-cytometry (MC-FC). Subsequently, we analyzed the respective M1- and M2-type macrophages and DCs regarding their ability to induce proliferation in a direct allogenic co-culture system with naïve CD4-positive T cells by a flow cytometry-based CFSE-assay. Macrophage plasticity, e.g. re-polarisation of M2-like to M1-like macrophages upon exposure to CD40 agonists was assessed by MC-FC. A real-time cell analysis system (Roche xCelligence RTCA DP) was used to attest T cell activation by CD40 stimulated B cells, resulting in killing of tumor cells in direct co-cultures.

Results: Stimulation of CD40 on PBMCs, T cells and monocytes increased secretion of cytokines (e.g. IL-12, TNFαa, CCL4) dependent on the agonist format and moreover was strictly dependent on Fc-crosslinking when using agonistic anti-CD40 mAb. In vitro, when the hexavalent APG1233 was added to the cytokine cocktail during the in vitro differentiation process, the appearance of M1-type macrophages was substantially increased. Moreover, M2-macrophages underwent conversion and acquired M1-type surface markers after exposure to APG1233. Finally, in direct co-culture of the in vitro differentiated cell populations with naïve CD4+ T cells, M1-macrophages induced strong lymphocyte proliferation, while the induction by monocytes and M2-macrophages was low. On a functional level, T lymphocytes co-cultured with M1 or DC acquired direct cytotoxic activity against tumor cells in a real-time cell analysis assay. Similarly, induction of cytolytic activity of purified T cells in vitro required the presence of both CD40 expressing B cells and APG1233.

Conclusion: Stimulation of CD40 on immune cells triggers development of anti-tumor responses, but efficacy of various agonist formats dramatically varies. Compared to trimeric CD40L formats and agonistic anti-CD40 mAbs, the novel hexavalent CD40 agonist APG1233 emerging from Apogenix HERA Technology platform excels as a strong inducer of B cell activation, M1-type macrophage differentiation and M2->M1 conversion. M1-macrophages generated in vitro are functional and enhance proliferation of naïve CD4+ T cells. CD40 stimulation on a CD40 expressing B cell line enhances its ability to activate T cells and trigger an anti-tumor response.

Citation Format: Christian Merz, Jaromir Sykora, Thamara Beyer, Stefanie Knorn, Harald Fricke, Christian Gieffers, Oliver Hill, David M. Richards. Structure to function: comparison of CD40 agonist formats reveals superior immune-modulating properties of hexavalent scCD40L-RBD-Fc fusion protein APG1233. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B48.