Beschreibung:
<jats:title>Abstract</jats:title>
<jats:p>CD40 ligand is a member of the TNF superfamily 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 have shown previously, that hexavalent HERA-CD40L is a potent CD40 agonist which is clearly superior over anti-CD40 benchmark antibodies and able to establish single agent anti-tumor immune responses both in vitro and in vivo. Since this compound qualifies as an ideal candidate for combinatorial cancer treatments we have created bispecific molecules by adding antibody derived targeting domains to the HERA-CD40L scaffold. These bispecific fusion proteins combine the potent co-stimulatory CD40-agonist with additional functionalities to enable tumor targeting and/or additional immuno-modulatory activities. To evaluate the different fusion protein formats in principle, the tumor associated antigens CEA and PD-L1 were chosen as targets. In addition to the hexavalent targeted HERA-CD40L, trivalent targeted fusion proteins employing the single-chain CD40L (scCD40L) as building block were created. Anti-CEA-HERA-CD40L, anti-CEA-trivalent scCD40L, anti-PD-L1-HERA-CD40L and anti-PD-L1-trivalent scCD40L were produced in CHO-S cells and purified resulting in highly pure non-aggregating protein lots as demonstrated by SDS-PAGE and HPLC-SEC. ELISA assays confirmed the specific binding to their targets - CD40 and CEA or CD40 and PD-L1, respectively. Employing a CD40 Luciferase reporter gene assay, hexavalent anti-CEA-HERA-CD40L showed a strong agonistic activity which was clearly superior to the anti-CEA-trivalent scCD40L- construct. Similarly, hexavalent anti-PD-L1-HERA-CD40L showed a strong agonistic activity in this assay which also was clearly superior to the anti-PD-L1-trivalent scCD40L construct. A PD-1/PD-L1 Luciferase reporter gene assay assessing the cellular activity of compounds interfering with PD-1/PD-L1 binding showed a clear activity for anti-PD-L1-HERA-CD40L. As expected for an assay assessing antagonistic activities, the activity of hexavalent anti-PD-L1-HERA-CD40L was in the same range as a reference anti-PD-L1 antibody and the anti-PD-L1-trimeric scCD40L- construct. Based on the in vitro data presented, the bispecific molecules combining HERA-CD40L with tumor targeting (anti-CEA) or with a checkpoint-blockade inhibitor (anti-PD-L1) are promising therapeutic approaches to promote anti-tumor immune responses.</jats:p>
<jats:p>Citation Format: Meinolf Thiemann, Katharina Billian-Frey, Matthias Schröder, Christian Merz, Jaromir Sykora, David M. Richards, Mauricio Redondo Müller, Julian P. Sefrin, Karl Heinonen, Christian Gieffers, Oliver Hill. Novel bispecific molecules combining HERA-CD40L with anti-CEA or with anti-PD-L1 for targeting [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4460.</jats:p>