CD40 ligand is a member of the TNF superfamily (TNF-SF) and a key regulator of the immune system. Its cognate receptor CD40 is expressed on antigen-presenting cells and on many tumor types, and has emerged as an attractive target for immunological cancer treatment.

Effective signaling for CD40/CD40L depends on the formation of a defined ligand/receptor complex triggered by interaction of a trimeric CD40L with three CD40 receptor chains allowing correct assembly of intracellular signaling complexes and respective signal transduction. Trimerization is a hallmark of the TNF-SF and has pivotal implications for the generation of respective TNFR-SF agonists in particular. However, ignoring the underlying trimeric structural concept bivalent antibodies are still the main agonistic biotherapeutic development candidates to address TNFR-SF members including CD40. Such bivalent antibodies are inherently associated with limited agonistic activity that requires Fc/FcγR interactions or potentially show increased toxicity caused by super-clustering of endogenous ligand receptor pairs.

To overcome the inadequacies of antibodies, we have developed HERA-CD40L composed of three receptor binding domains in a single chain arrangement, linked to an Fc-silenced human IgG1 thereby generating a hexavalent molecule. HERA-CD40L mimics the natural ligand, induces potent agonistic activity and, importantly, does not require FcγR mediated crosslinking. Comparison of HERA-CD40L to anti-CD40 benchmark antibodies (including CP-870,893) revealed superiority for HERA-CD40L in all assays tested. (i) In contrast to antibodies, HERA-CD40L showed strong activation of NFkB signaling upon treatment of B cells. (ii) HERA-CD40L treatment, but not clinical benchmark antibodies, converts immature phagocytic macrophages into mature/professional APCs and promoted differentiation towards the M1 spectrum macrophages. (iii) Furthermore, HERA-CD40L treated PBMCs stimulate potent allogeneic anti-tumor T cell response that was not detectable for CD40-antibodies.

In vivo, a murine surrogate of HERA-CD40L stimulated clonal expansion of OT-I specific murine CD8+ T cells without affecting non-specific immune cells. In the syngeneic CT26wt mouse model mHERA-CD40L treatment converts cold into hot tumors by increasing infiltration of CD8+ and CD4+ T cells. In addition mHERA-CD40L showed single agent anti-tumor activity in the CD40-negative syngeneic MC38-CEA mouse model, suggesting an involvement of the immune system in controlling tumor growth.

In summary HERA-CD40L is a potent agonist able to establish single agent anti-tumor immune responses. Comparison to bivalent benchmark antibodies showed superior biological activity of HERA-CD40L and qualifies this molecule as an ideal candidate for combinatorial cancer treatments.

Citation Format: Christian Gieffers, Jaromir Sykora, Christian Merz, Mauricio Redondo Müller, David M. Richards, Julian Sefrin, Katharina Billian-Frey, Karl Heinonen, Viola Marschall, Matthias Schröder, Harald Fricke, Meinolf Thiemann, Hill Oliver. HERA-CD40L a hexavalent CD40 agonist induces T cell mediated anti-tumor immune response and shows superior activity in direct comparison to benchmark agonistic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5015.