Unleashing the Power of HERA-CD40L: Superior Biological Activity of a Hexavalent CD40 Agonist in Cancer Immunotherapy

Targeted cancer therapies typically involve antibodies, but natural signaling in the TNF superfamily and their receptors is characterized by trimer clusters. While bivalent antibodies rely on Fc receptor crosslinking for activity, Apogenix's HERA-Technology has created hexavalent fusion proteins that effectively target the TNF-receptor superfamily with enhanced clustering. This study evaluates various CD40 agonist formats, highlighting the novel HERA-CD40L and its impact on receptor clustering.

The CD40 agonists' efficacy was assessed using a modified cell line and flow cytometry to measure IκBα degradation in Ramos B cells. Isolated T lymphocytes and monocytes from buffy coats were analyzed for CD marker expression post-CD40 ligation via multicolor flow cytometry. Cytokine secretion was quantified by ELISA, and T cell-mediated tumor cell killing was monitored in real-time using a cell analysis system.

Findings indicate that the hexavalent HERA-CD40L outperformed bivalent and trivalent CD40L agonists in bioactivity assays, showing full activity without crosslinking. Unlike other formats, HERA-CD40L effectively upregulated B cell activation markers and induced proinflammatory cytokine secretion by PBMCs. It also induced M2-macrophages to adopt an M1 phenotype and enhanced CD4+ T cell proliferation. HERA-CD40L facilitated cytotoxic activity of CD4+ cells against tumor cells in direct co-cultures, which was cell-contact dependent and absent in indirect cultures.

The study concludes that the hexavalent HERA-CD40L, developed through Apogenix's HERA-Technology, effectively triggers CD40 signaling, leading to cytolytic activation and proliferation of CD4+ T cells and a shift towards proinflammatory conditions. It forms highly clustered signaling complexes, surpassing other agonist formats in biological activity without Fc receptor crosslinking.

The research was presented by Christian Merz et al. at the American Association for Cancer Research Annual Meeting in 2017, with the abstract published in Cancer Research.