Preparation and evaluation of a new releasable PEGylated tumor necrosis factor-α （TNF-α） conjugate for therapeutic application

To design a releasable PEGylated TNF-α （rPEG-TNF-α, a cathepsin B-sensitive dipeptide （Val-Cit moiety） was inserted into conventional PEG-modified TNF-α（PEG-TNF-α, facilitating its clinical use for anti-tumor therapy. Comparative pharmaco- kinetic and pharmacodynamic studies showed that the half-lives of both PEGylated forms of TNF-α were -60-fold greater than that of unmodified TNF-α. In addition, the in vitro bioactivity of rPEG-TNF-α was greater than that of PEG-TNF-α with the same degree of PEG modification. Release of TNF-αfrom rPEG-TNF-α in vitro was dependent on the presence of cathepsin B and was inhibited by a cathepsin B inhibitor. Despite the potent cytotoxicity of unmodified TNF-α against normal cells, its PEGylated forms at higher TNF-α concentrations showed low cytotoxic activity against these cells. In contrast, both forms of PEGylated TNF-α showed potent cytotoxic activity against the B16 and L929 cell lines, with rPEG-TNF-α being 5- and 9- fold more potent, respectively, than PEG-TNF-α. Moreover, rPEG-TNF-α was a more potent in vivo antitumor agent than PEG-TNF-α.