Abstract
TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) has long been considered a tantalizing target for cancer therapy because it mediates activation of the extrinsic apoptosis pathway in a tumor-specific manner by binding to and trimerizing its functional receptors DR4 or DR5. Despite initial promise, both recombinant human TRAIL (native TRAIL) and dimeric DR4/DR5 agonist monoclonal antibodies (mAbs) failed in multiple human clinical trials. Here we show that in-frame fusion of human C-propeptide of α1(I) collagen (Trimer-Tag) to the C-terminus of mature human TRAIL leads to a disulfide bond-linked homotrimer which can be expressed at high levels as a secreted protein from CHO cells. The resulting TRAIL-Trimer not only retains similar bioactivity and receptor binding kinetics as native TRAIL in vitro which are 4–5 orders of magnitude superior to that of dimeric TRAIL-Fc, but also manifests more favorable pharmacokinetic and antitumor pharmacodynamic profiles in vivo than that of native TRAIL. Taken together, this work provides direct evidence for the in vivo antitumor efficacy of TRAIL being proportional to systemic drug exposure and suggests that the previous clinical failures may have been due to rapid systemic clearance of native TRAIL and poor apoptosis-inducing potency of dimeric agonist mAbs despite their long serum half-lives.
Highlights
TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) has long been considered a tantalizing target for cancer therapy because it mediates activation of the extrinsic apoptosis pathway in a tumor-specific manner by binding to and trimerizing its functional receptors DR4 or DR5
To produce highly pure and sufficient amounts of TRAIL-Trimer fusion protein for functional analyses, we began by screening for high-titer production clones of TRAIL-Trimer vector-transfected CHO cells via MTX-mediated gene amplification; the resulting leading clone was adapted to serum free media and grown under fed-batch cell culture process in a bioreactor, which led to high-level expression of TRAIL-Trimer (Fig. 2a)
Dimeric agonist monoclonal antibodies (mAbs) exhibit low activity in inducing apoptosis, and while native TRAIL is highly potent, it is trimerized via weak noncovalent interactions and rapidly eliminated in vivo[15,26]
Summary
TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) has long been considered a tantalizing target for cancer therapy because it mediates activation of the extrinsic apoptosis pathway in a tumor-specific manner by binding to and trimerizing its functional receptors DR4 or DR5. Despite initial promise, both recombinant human TRAIL (native TRAIL) and dimeric DR4/DR5 agonist monoclonal antibodies (mAbs) failed in multiple human clinical trials. Because DR4 and DR5 receptors must be trimerized upon ligand binding in order to initiate the extrinsic apoptosis pathway, the clinical failures of all agonist mAbs tested to-date is unsurprising, given their inherently dimeric antibody structure. Trimerization via covalent bond-linkage may stabilize TRAIL/Apo2L trimeric structure essential for its biological activity as well as increase its molecular weight in order to extend half-life for improved antitumor efficacy in vivo
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