Abstract 1587: Generation and characterization of novel bispecific molecules combining single-chain-CD40L with anti-CEA, anti-CD95L or anti-PD-L1 targeting moieties
Authors: Schroeder Matthias , Billian-Frey Katharina , Sykora Jaromir , Redondo-Mueller Mauricio , Heinonen Karl , Frankish Jamie , Gieffers Christian , Thiemann Meinolf , Hill Oliver 
Published: July 2021

Abstract
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. We developed HERA-CD40L, a construct composed of a trivalent single-chain CD40L-receptor-binding-domain (scCD40L-RBD) linked to a silenced human IgG1-Fc-domain thereby generating a hexavalent molecule. We showed previously that HERA-CD40L mimics the natural ligand, thereby inducing potent agonistic activity which is independent of FcγR mediated crosslinking and superior to anti-CD40 benchmark antibodies. For combinatorial cancer immunotherapy we have created bispecific molecules by combining the HERA-CD40L scaffold with antibody derived domains targeting different classes of tumor associated antigens. These bispecific fusion proteins combine the potent co-stimulatory CD40-agonist with either direct tumor-cell targeting and/or additional immunomodulatory activities in the tumor microenvironment. To evaluate the different fusion protein formats, the tumor associated antigens CEA, PD-L1 and CD95L were chosen as model-targets. In addition to the hexavalent targeted HERA-CD40L, trivalent targeted fusion proteins, employing the scCD40L-RBD as building block, were created as well. All engineering prototypes were produced in CHO-S cells and purified, resulting in highly pure non-aggregating protein lots as demonstrated by SDS-PAGE and HPLC-SEC. Functional binding to their respective targets was shown by ELISA and to proof biological in vitro activity luciferase reporter gene assays were employed. The basic underlying immunological processes have been investigated in vitro. The trivalent CD40L, the trivalent CD40L-bispecifics anti-CD95L-scCD40L-RBD and anti-CEA-scCD40L-RBD as well as anti-CD40, anti-PD-L1 or anti-CD95L antibodies did not activate human monocytes, even if the antibodies were co-administered with trivalent CD40L. In contrast, the hexavalent HERA-CD40L, the hexavalent bifunctional CD40L-constructs and the trivalent anti-PD-L1-scCD40L-RBD-construct induced strong activation/maturation of the monocytes as indicated by CD83, CD86, HLA-DR upregulation which was accompanied by increased chemokine receptor (CD54, CCR7) and PD-L1 expression. When co-cultured with CD3 positive T cells, these pre-activated monocytes led to subsequent activation of CD4 as well as CD8 positive T cells, indicated by increased expression of CD25, CD69 and CD54. This clearly shows that the close proximity of anti-PD-L1 and trivalent CD40L within one molecule makes a huge difference for biological activity. Hence, these novel bispecific constructs are a promising therapeutic approach to promote anti-tumor immune responses.
Citation Format: Matthias Schroeder, Katharina Billian-Frey, Jaromir Sykora, Mauricio Redondo-Mueller, Karl Heinonen, Jamie Frankish, Christian Gieffers, Meinolf Thiemann, Oliver Hill. Generation and characterization of novel bispecific molecules combining single-chain-CD40L with anti-CEA, anti-CD95L or anti-PD-L1 targeting moieties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1587.