Agonistic hexavalent CD40 ligand HERA-CD40L is superior to agonistic antibodies in improving antigen presentation and tumor killing
Viola Marschall, Christian Merz, Jaromir Sykora, Meinolf Thiemann, David M. Richards, Karl H. Heinonen, Mauricio Redondo-Müller, Harald Fricke, Oliver Hill and Christian Gieffers
Apogenix AG, Heidelberg, Germany
The co-stimulatory receptor CD40 is an attractive therapeutic target, as CD40 signaling triggers anti-tumor responses of the immune system. We compared the efficacy of different human CD40 agonist formats, including the novel hexavalent HERA-CD40L.
Direct comparison of bivalent anti-CD40 antibodies, a trivalent CD40L and the hexavalent HERA-CD40L demonstrated that only the hexavalent agonist was fully active without additional crosslinking. Biological activity of different CD40 agonists was compared by flow cytometric (FC) analysis of IκBα degradation in Ramos B cells. Human T lymphocytes and monocytes were isolated from buffy coats following analysis of CD marker expression and cytokine secretion in response to CD40 ligation. In contrast to HERA-CD40L, neither the bivalent agonistic anti-CD40 antibody nor the trivalent CD40L were able to upregulate expression of activation markers on macrophages, DCs and B cells or to induce secretion of proinflammatory cytokines such as IL-12 and TNFα. HERA-CD40L induced cytotoxic activity in co-cultures of CD4+ T cells, Ramos B cells and tumor cells, as assessed by real-time cell analysis. Importantly, HERA-CD40L showed in vivo anti-tumor efficacy as single agent in a subcutaneous syngeneic MC38-CEA colon cancer model in mice.
The immuno-oncology field is currently dominated by bivalent antibodies, however, the natural signaling complexes of the TNF superfamily and their receptors consist of clusters of trimers. This suggests that the induction of physiological CD40 signaling depends on the ability of the agonist to cluster multiple receptor chains. The hexavalent CD40 agonist HERA-CD40L is a member of a novel class of TNF receptor superfamily agonists which shows superior biological activity over other agonistic formats without the need for Fc receptor-mediated crosslinking.
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