APG350 Induces Superior Clustering of TRAIL Receptors and Shows Therapeutic Antitumor Efficacy Independent of Cross-Linking via Fcγ Receptors

Abstract

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 TRAILreceptor-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 FcgR-based cross-linking events.

Enhancing the antitumor efficacy of a cell-surface death ligand by covalent membrane display

“…A number of approaches to increase oligomerization of Apo2L/ TRAIL have been reported (21,22). However, a key caveat with these is the addition of significant exogenous peptide sequences likely to cause immunogenicity in humans.…”

RG7386, a Novel Tetravalent FAP-DR5 Antibody, Effectively Triggers FAP-Dependent, Avidity-Driven DR5 Hyperclustering and Tumor Cell Apoptosis

“…Conatumumab-coated nanoparticles encasing a cytotoxic drug that allow a sufficient density of antibody paratopes at the tumor site to activate DR5 are currently under investigation (29). APG350 is a hexavalent scTRAIL-RBD (single-chain TRAIL receptor-binding domain), which in contrast with previous agonistic antibodies triggers multimer formation independently of FcgRs (30).…”

An optimized antibody-single-chain TRAIL fusion protein for cancer therapy

“…26 In another study, 3 TRAIL subunits comprising aa 121-281 were connected with glycine/serine linkers of 8 aa length comprising one N-glycosylation site (GSGSGNGS). 34 Both studies did not report on the thermal stability of these scTRAIL molecules. By defining Val122 as the N-terminus of the TRAIL THD, the molecules described by Spitzer et al 33 and Gieffers et al 34 comprise 14 and 9 aa residues, respectively, that functionally serve as linker.…”

“…34 Both studies did not report on the thermal stability of these scTRAIL molecules. By defining Val122 as the N-terminus of the TRAIL THD, the molecules described by Spitzer et al 33 and Gieffers et al 34 comprise 14 and 9 aa residues, respectively, that functionally serve as linker. Our results suggest that glycine/serine linkers of 10 or 12 residues have no or only marginal positive effects on the thermal stability of a scTRAIL, whereas a linker of 6 or fewer residues is required to significantly increase thermal stability, demonstrated most obviously for scTRAIL-FLVGGVA.…”