| Obesity has become a global epidemic, and the research and development of antiobesity drug has high priority for drugmakers. The discovery of fat-regulating hormone leptin reveals the molecular mechanism of energy homeostasis. But the protein performed poorly in its first clinical trial, because of leptin resistance. It was recently demonstrated that CNTF (ciliary neurotrophic factor), which is the natural copycat molecule of leptin, can activate leptin-like pathways and reduce body fat, without cachexia or rebound weight gain, even in leptin-resistant obesity animal model. AX15 is a recombinant human CNTF variant created by Regeneron that has improved biological activity, stability and solubility. AX15 is currently in clinical trials as a therapeutic for obesity associated with diabetes, and has demonstrated efficacy in a phase III clinical trial. But the prospect of being an effective antiobesity drug was hampered by the intrinsic defects of AX15, which is susceptible to proteolytic degradation, prone to aggregation , can cause severe side effects, and is highly immunogenic in human, and it is imperative to further improve the property of AX15.The KEX2 cleavage site in AX15 was identified by N-terminal amino acids sequencing, and was eliminated by site-directed mutagenesis. The arginien at position 13 was replaced with lysine, and a protease resistant mutein AX15(R13K) was constructed. It was demonstrated that the stability of AX15(R13K) improved significantly, as no degradation was detected evenafter 120 hours of induction in shake flask culture. AX15(R13K) was effectively secreted but was shown to exist as large aggregates with molecular weight over several hundred kDa. Once formed, the aggregates could only be disaggregated by high concentration of denaturing agent, such as 6M guanidine-HCl. The denatured AXIS(RISK) was simultaneously refolded and purified to homogeneity by Superdex75 gel filtration chromatography. In vitro and in vivo assay showed AX15(R13K) had equivalent specific activity to AXIS. The protease resistant mutein of AXIS may have greater in vivo stability and thus have superior therapeutic potential.Chimeric human-rat CNTF gene library was constructed by family DNA shuffling, and the library was subjected to 3 rounds of receptor affinity biopanning. Of the selected 5 clones, 3 were AX 15(RISK) parent gene, only 2 encoded chimeric CNTF, which were designated as CH1.1 and CHI.4 respectively. CH1. 1 differs from AX 15 (RISK) in 1 amino acid residue, and has similar expression level and activity. CHI. 4 differs from AXIS (RISK) in 8 amino acid residues. While the specific activity was only 1/5 of that of AXIS(RISK), the expression level of CHI. 4 was about 2 fold of that of AXIS(RISK). It indicates CHI.4 maybe selected because of its higher stability and/or expression efficiency.The Q63R mutation was combined with Q166D/D167H mutation, and a superagnonist mutein DH-AX15(R13K) was constructed. In vitro TF-1 cell survival assay and in wVoantiobesity tests showed DH-AX(RISK) was about 5 fold more potent than AXIS (RISK). It was further demonstrated that the antiobesity effect of DH-AXIS(RISK) was more durable than that of AX15(R13K). The durable effects of DH-AXIS(RISK) is ascribed to its higher specific activity, but not to its prolonged half-life.The C-terminus of HSA was fused to the N-terminus of AXIS(RISK), and HSA-AX15(R13K) fusion protein was constructed. The expression level ofHSA-AX15(R13K) fusion protein was about 5 fold of that of AX15(R13K), but it was severely degraded at the junction region. The degradation was not abolished by the insertion of an 11 amino acid residues linker at the junction region. The aggregation of AXIS(RISK) was prevented by fusion to HSA, as HSA-AX15(R13K) was primarily secreted as monomers. The half-life of AX15 (R13K) was extended by fusion to HSA, as single injection of HSA-AX 15 (R13K) can reduce the body weight of KM mice in the following 3 consecutive days, while the body weight of KM mice received single AX15(R13K) administration will rebound... |
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