Abstract
Leptin plays a central role in the control of energy homeostasis and appetite and, thus, has attracted attention for therapeutic approaches in spite of its limited pharmacological activity owing to the very short circulation in the body. To improve drug delivery and prolong plasma half-life, we have fused murine leptin with Pro/Ala/Ser (PAS) polypeptides of up to 600 residues, which adopt random coil conformation with expanded hydrodynamic volume in solution and, consequently, retard kidney filtration in a similar manner as polyethylene glycol (PEG). Relative to unmodified leptin, size exclusion chromatography and dynamic light scattering revealed an approximately 21-fold increase in apparent size and a much larger molecular diameter of around 18 nm for PAS(600)-leptin. High receptor-binding activity for all PASylated leptin versions was confirmed in BIAcore measurements and cell-based dual-luciferase assays. Pharmacokinetic studies in mice revealed a much extended plasma half-life after ip injection, from 26 min for the unmodified leptin to 19.6 h for the PAS(600) fusion. In vivo activity was investigated after single ip injection of equimolar doses of each leptin version. Strongly increased and prolonged hypothalamic STAT3 phosphorylation was detected for PAS(600)-leptin. Also, a reduction in daily food intake by up to 60% as well as loss in body weight of >10% lasting for >5 days was observed, whereas unmodified leptin was merely effective for 1 day. Notably, application of a PASylated superactive mouse leptin antagonist (SMLA) led to the opposite effects. Thus, PASylated leptin not only provides a promising reagent to study its physiological role in vivo but also may offer a superior drug candidate for clinical therapy.
Highlights
Leptin is primarily secreted from white adipose tissue and plays a key role in energy balance regulation.[1]
These encode leptin, preceded by a bacterial signal peptide, together with an N- or C-terminal His6-tag as well as a short linker comprising a unique SapI recognition site. This restriction site served for subsequent insertion of one or multiple PAS#1.2(200) gene cassettes an improved version of the previously described PAS#1(200) cassette, coding for the same amino acid sequence in the proper reading frame according to a published cloning strategy.[47]
PASylation, which can be accomplished by the convenient fusion of PAS gene cassettes of tunable length with the open reading frame of a therapeutic protein or peptide, may offer a promising opportunity to convert leptin into a viable biotherapeutic with prolonged and enhanced action
Summary
Leptin is primarily secreted from white adipose tissue and plays a key role in energy balance regulation.[1]. During long-term treatment the poor biodegradability can lead to kidney vacuolization and tissue accumulation of the synthetic polymer, in particular in the choroid plexus epithelial cells of the brain, and there is an increasing number of reports on the emergence of neutralizing antibodies against PEGylated biologics.[43−46] To circumvent these caveats, “PASylation” was recently developed as a biological alternative to PEGylation.[47] PASylation involves the genetic fusion of a therapeutic protein (or peptide) with a conformationally disordered polypeptide of defined sequence comprising the small amino acids Pro, Ala, and/or Ser. To circumvent these caveats, “PASylation” was recently developed as a biological alternative to PEGylation.[47] PASylation involves the genetic fusion of a therapeutic protein (or peptide) with a conformationally disordered polypeptide of defined sequence comprising the small amino acids Pro, Ala, and/or Ser This approach offers a superior way to attach a solvated molecular random chain with large hydrodynamic volume to a biologically active compound, showing biophysical properties very similar to those of PEG.[48] PASylation has so far been used to extend the plasma half-life of various biologics such as antibody fragments, growth hormones, and interferons both with regard to therapeutic applications and for in vivo imaging.[47,49,50] Here, we describe the design and biochemical analysis of PASylated murine leptin versions as well as their functional characterization in mice, demonstrating substantially prolonged circulation and much enhanced suppressive effects on food intake in vivo
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