Superior serum half life of albumin tagged TNF ligands
Introduction
Ligands and receptors of the tumor necrosis factor (TNF) family are crucially involved in the regulation of the immune system, but are also of relevance in development and tissue homeostasis [1]. Deregulated activities of these molecules have been identified in a variety of pathophysiological situations as pivotal disease driving mechanisms. Not at least due to their good extracellular accessibility, the ligands and receptors of the TNF family are straight forward therapeutic targets. Indeed, TNF-specific neutralizing antibodies and a soluble TNFR2 derivate represent highly efficient and established treatment options for the therapy of rheumatoid arthritis, Morbus Crohn and other chronic inflammatory diseases. Moreover, corresponding molecules that inhibit the activity of FasL, BAFF/Blys or RANKL are currently under consideration for the treatment of ischemia, systemic lupus erythematosus and cancer associated bone destruction [2], [3], [4]. However, not only the inhibition of a particular TNF/TNF receptor system is a valuable therapeutic approach, but also its controlled exogenous stimulation. In particular, the immune stimulating and apoptosis inducing ligands of the TNF family have attracted huge interest as effector molecules for the treatment of cancer [5], [6], [7].
Stimulation of TNF receptors can be achieved by various means, including agonistic TNF receptor-specific antibodies and gene therapy with ligand encoding vectors, and, more directly, with recombinantly produced forms of soluble TNF ligands. A general practical disadvantage of recombinant soluble TNF ligands is their small size causing rapid clearance from circulation with half life times significantly below 1 h [8], [9], [10]. Thus, the maintenance of effective concentrations of soluble TNF ligands in vivo at a time scale of hours or even days requires application of high amounts of the corresponding protein and repeated injections or continuous infusion. Means that improve the serum half life of soluble TNF ligands should therefore be of benefit for their potential therapeutic application. One approach that has been successfully used to improve the pharmacokinetics of small proteins was the chemical or genetic conjugation with long lasting circulating serum proteins, such as albumin. For example, reduced immunogenicity and better pharmacokinetic properties have been demonstrated for albumin fusion proteins of hormones such as insulin as well as for type I interferons, interleukin-2 (IL2) and antibody fragments [11], [12], [13], [14], [15].
In this study, we investigated fusion proteins of human serum albumin (HSA) with three TNF ligand family members of clinical interest, namely TNF-related apoptosis inducing ligand (TRAIL), TNF-related weak inducer of apoptosis (TWEAK) and the name giving TNF. We found that HSA-tagging does barley interfere with the activity of the TNF ligands, but strongly enhances the in vivo retention of these molecules in the blood circulation. Accordingly, we observed in a xenotransplantation model that HSA-tagging lowers the amounts of TRAIL necessary to achieve a significant anti-tumoral effect.
Section snippets
Plasmids
Expression vectors encoding the various TNF ligand variants were derived from a modified pCR3 plasmid (Invitrogen, Karlsruhe, Germany) kindly provided by Pascal Schneider (University of Lausanne, Epalinges, Switzerland), which encodes an immunoglobulin signal peptide followed by a Flag tag. To obtain plasmids encoding Flag-TNF and Flag-TWEAK, amplicons of human TNF and TWEAK corresponding to the amino acids indicated in Fig. 1A including a stop codon were inserted in frame 3′ to the Flag tag by
Expression and purification of recombinant TNF ligand variants
By genetic engineering the recombinant human serum albumin (HSA) TNF ligand fusion proteins HSA-Flag-TNC-TRAIL, HSA-Flag-TWEAK and HSA-Flag-TNF were generated (Fig. 1A). It is evident from a variety of studies that protein domains located N-terminally to the TNF homology domain (THD) of TNF ligands do not interfere with receptor binding and receptor activation [7]. In the HSA-TNF ligand fusion proteins the C-terminus of HSA was therefore fused to the N-terminus of the corresponding TNF ligand
Discussion
The data presented in this study show that genetic fusion of human serum albumin to TNF ligands has a strong impact on the pharmacokinetic of such fusion proteins and, as shown for a HSA-TRAIL variant, considerably improved its anti-tumoral activities in vivo. To evaluate the influence of HSA fusion on the biological activity of cytokines of the TNF ligand family, we tested corresponding fusion proteins of TNF, TRAIL and TWEAK in a sampling of in vitro assays. We observed no significant
Acknowledgments
The project was supported by Deutsche Forschungsgemeinschaft (Wa 1025/18-1) and Deutsche Krebshilfe (Projects 108267 and 107034-226).
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