Single-Chain TNF, a TNF Derivative with Enhanced Stability and Antitumoral Activity

Abstract

The inflammatory and proapoptotic cytokine TNF possesses a compelling potential as an antitumoral therapeutic agent. Possible target cells include the malignant cells themselves, the tumor vasculature, or the immune system. As the clinical use of TNF is limited by systemic toxicity, targeting strategies using TNF-based fusion proteins are currently used. A major obstacle, however, is that homotrimeric TNF ligands are prone to activity loss due to dissociation into their monomers. In this study, we report the construction of single-chain TNF molecule, a TNF mutant consisting of three TNF monomers fused by short peptide linkers. In comparison to wild-type TNF, single-chain TNF was found to possess increased stability in vitro and in vivo, displayed reduced systemic toxicity yet slightly enhanced antitumoral activity in mouse models. Creation of single-chain variants is a new approach for improvement of functional activity of therapeutics based on TNF family ligands.

Tumor necrosis factor receptor-associated factor-1 enhances proinflammatory TNF receptor-2 signaling and modifies TNFR1–TNFR2 cooperation

“…The TNFR2-specific TNF mutant was expressed in form of a Flag-tagged single chain trimer (Flag-scTNF(143N/145R)). Thus, three TNF domains representing soluble TNF monomers were fused by genetic engineering using two short peptide linkers (Krippner-Heidenreich et al, 2008). In contrast to a His-tagged version of this molecule (Krippner-Heidenreich et al, 2008) heterotrimerization of TNF domains of different single chain TNF molecules of Flag-scTNF(143N/145R) was observed.…”

Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format

“…19 Thus, 4-1BBL resembles a three-bladed propeller, while in the case of OX40L and GITRL, they are constituted by a shortened THD, presenting a more expanded, blooming flower-like organization. 20 So far, generation of single-polypeptide chain variants had only been reported for apoptotic members of the conventional family, where improved stability was shown for TNF 25 and TRAIL. 26,27 Here, we report in the antibody-fusion protein context the generation of the single-chain format for TNFSF members of the divergent family, comparing binding and activation potential with corresponding recombinant wild-type proteins.…”

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

“…33 This scTRAIL molecule (TR3) displayed increased stability, confirming earlier results for a single-chain TNF molecule showing that physical connection of the 3 subunits increases stability and bioactivity in vitro and in vivo. 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.…”

“…18,20,21,26,27 In addition to a high specific activity, clinically useful recombinant protein therapeutics must also have suitable protein stability and pharmacokinetic properties. 24,28,29 Toward this end, we here describe singlechain variants of the apoptosis-inducing TRAIL with improved stability and superior product quality in the form of a tumortargeted fusion protein, resulting in high anti-tumor activity in a xenograft tumor model in vivo.…”