ABSTRACT
Interleukin-15 (IL-15), a 114-amino acid cytokine related to IL-2, regulates immune homeostasis and the fate of many lymphocyte subsets. We reported that, in the blood of mice and humans, IL-15 is present as a heterodimer associated with soluble IL-15 receptor α (sIL-15Rα). Here, we show efficient production of this noncovalently linked but stable heterodimer in clonal human HEK293 cells and release of the processed IL-15·sIL-15Rα heterodimer in the medium. Purification of the IL-15 and sIL-15Rα polypeptides allowed identification of the proteolytic cleavage site of IL-15Rα and characterization of multiple glycosylation sites. Administration of the IL-15·sIL-15Rα heterodimer reconstituted from purified subunits resulted in sustained plasma IL-15 levels and in robust expansion of NK and T cells in mice, demonstrating pharmacokinetics and in vivo bioactivity superior to single chain IL-15. These identified properties of heterodimeric IL-15 provide a strong rationale for the evaluation of this molecule for clinical applications.
Subject(s)
Interleukin-15 Receptor alpha Subunit/metabolism , Interleukin-15/metabolism , Multiprotein Complexes/metabolism , Amino Acid Sequence , Animals , Binding Sites/genetics , Cell Proliferation/drug effects , Chromatography, High Pressure Liquid , Female , Glycosylation , HEK293 Cells , Humans , Immunoblotting , Interleukin-15/chemistry , Interleukin-15/genetics , Interleukin-15 Receptor alpha Subunit/chemistry , Interleukin-15 Receptor alpha Subunit/genetics , Killer Cells, Natural/cytology , Killer Cells, Natural/drug effects , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Multiprotein Complexes/administration & dosage , Multiprotein Complexes/pharmacokinetics , Protein Binding , Protein Multimerization , Proteolysis , Solubility , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , T-Lymphocytes/cytology , T-Lymphocytes/drug effectsABSTRACT
The purpose of this study was to investigate the influence of molecular shape, conformability, net surface charge and tissue interaction on transscleral diffusion. Unfixed, porcine sclera was clamped in an Ussing chamber. Fluorophore-labelled neutral albumin, neutral dextran, or neutral ficoll were placed in one hemi-chamber and the rate of transscleral diffusion was measured over 24 h using a spectrophotometer. Experiments were repeated using dextrans and ficoll with positive or negative net surface charges. Fluorescence recovery after photobleaching (FRAP) was undertaken to compare transscleral diffusion with diffusion through a solution. All molecules were 70 kDa. With FRAP, the diffusion coefficient (D) of neutral molecules was highest for albumin, followed by ficoll, then dextran (p < 0.0001). Positive dextrans diffused fastest, followed by negative, then neutral dextrans (p = 0.0004). Neutral ficoll diffused the fastest, followed by positive then negative ficoll (p = 0.5865). For the neutral molecules, transscleral D was highest for albumin, followed by dextran, then ficoll (p < 0.0001). D was highest for negative ficoll, followed by neutral, then positive ficoll (p < 0.0001). By contrast, D was highest for positive dextran, followed by neutral, then negative dextran (p = 0.0021). In conclusion, diffusion in free solution does not predict transscleral diffusion and the molecular-tissue interaction is important. Molecular size, shape, and charge may all markedly influence transscleral diffusion, as may conformability to a lesser degree, but their effects may be diametrically opposed in different molecules, and their influence on diffusion is more complex than previously thought. Each variable cannot be considered in isolation, and the interplay of all these variables needs to be tested, when selecting or designing drugs for transscleral delivery.
Subject(s)
Dextrans/pharmacokinetics , Ficoll/analogs & derivatives , Fluorescein-5-isothiocyanate/analogs & derivatives , Multiprotein Complexes/pharmacokinetics , Sclera/metabolism , Serum Albumin/pharmacokinetics , Animals , Dextrans/chemistry , Diffusion , Diffusion Chambers, Culture , Ficoll/chemistry , Ficoll/pharmacokinetics , Fluorescein-5-isothiocyanate/chemistry , Fluorescein-5-isothiocyanate/pharmacokinetics , Laser-Doppler Flowmetry , Light , Molecular Weight , Multiprotein Complexes/chemistry , Permeability , Protein Conformation , Scattering, Radiation , Serum Albumin/chemistry , Spectrometry, Fluorescence , SwineABSTRACT
Standard genetic approaches allow the production of protein composites by fusion of polypeptides in head-to-tail fashion. Some applications would benefit from constructions that are genetically impossible, such as the site-specific linkage of proteins via their N or C termini, when a remaining free terminus is required for biological activity. We developed a method for the production of N-to-N and C-to-C dimers, with full retention of the biological activity of both fusion partners and without inflicting chemical damage on the proteins to be joined. We use sortase A to install on the N or C terminus of proteins of interest the requisite modifications to execute a strain-promoted copper-free cycloaddition and show that the ensuing ligation proceeds efficiently. Applied here to protein-protein fusions, the method reported can be extended to connecting proteins with any entity of interest.
