APG350 induces superior clustering of TRAIL receptors and shows therapeutic antitumor efficacy independent of cross-linking via Fcγ receptors

• Published in: Molecular cancer therapeutics 2013-12, Vol.12 (12), p.2735-2747
• Main Authors: Gieffers, Christian, Kluge, Michael, Merz, Christian, Sykora, Jaromir, Thiemann, Meinolf, Schaal, René, Fischer, Carmen, Branschädel, Marcus, Abhari, Behnaz Ahangarian, Hohenberger, Peter, Fulda, Simone, Fricke, Harald, Hill, Oliver
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - pharmacology; Antineoplastic Agents - administration & dosage; Antineoplastic Agents - chemistry; Antineoplastic Agents - pharmacology; Cell Line, Tumor; Cell Proliferation - drug effects; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Humans; Mice; Models, Biological; Peptide Fragments - administration & dosage; Peptide Fragments - chemistry; Peptide Fragments - pharmacology; Receptors, IgG - metabolism; Receptors, TNF-Related Apoptosis-Inducing Ligand - agonists; Receptors, TNF-Related Apoptosis-Inducing Ligand - antagonists & inhibitors; Receptors, TNF-Related Apoptosis-Inducing Ligand - metabolism; Recombinant Fusion Proteins - administration & dosage; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - pharmacology; Xenograft Model Antitumor Assays
• ISSN: 1535-7163; 1538-8514
• DOI: 10.1158/1535-7163.MCT-13-0323

Cancer cells can be specifically driven into apoptosis by activating Death-receptor-4 (DR4; TRAIL-R1) and/or Death-receptor-5 (DR5; TRAIL-R2). Albeit showing promising preclinical efficacy, first-generation protein therapeutics addressing this pathway, especially agonistic anti-DR4/DR5-monoclonal antibodies, have not been clinically successful to date. Due to their bivalent binding mode, effective apoptosis induction by agonistic TRAIL-R antibodies is achieved only upon additional events leading to antibody-multimer formation. The binding of these multimers to their target subsequently leads to effective receptor-clustering on cancer cells. The research results presented here report on a new class of TRAIL-receptor agonists overcoming this intrinsic limitation observed for antibodies in general. The main feature of these agonists is a TRAIL-mimic consisting of three TRAIL-protomer subsequences combined in one polypeptide chain, termed the single-chain TRAIL-receptor-binding domain (scTRAIL-RBD). In the active compounds, two scTRAIL-RBDs with three receptor binding sites each are brought molecularly in close proximity resulting in a fusion protein with a hexavalent binding mode. In the case of APG350-the prototype of this engineering concept-this is achieved by fusing the Fc-part of a human immunoglobulin G1 (IgG1)-mutein C-terminally to the scTRAIL-RBD polypeptide, thereby creating six receptor binding sites per drug molecule. In vitro, APG350 is a potent inducer of apoptosis on human tumor cell lines and primary tumor cells. In vivo, treatment of mice bearing Colo205-xenograft tumors with APG350 showed a dose-dependent antitumor efficacy. By dedicated muteins, we confirmed that the observed in vivo efficacy of the hexavalent scTRAIL-RBD fusion proteins is-in contrast to agonistic antibodies-independent of FcγR-based cross-linking events.