title: Rational cytokine design for increased lifetime and enhanced potency using pH-activated "histidine switching".
abstract: We describe a method for the rational design of more effective therapeutic proteins using amino acid substitutions that reduce receptor binding affinity in intracellular endosomal compartments, thereby leading to increased recycling in the ligand-sorting process and consequently resulting in longer half-life in extracellular medium. We demonstrate this approach for granulocyte colony-stimulating factor by using computationally predicted histidine substitutions that switch protonation states between cell-surface and endosomal pH. Molecular modeling of binding electrostatics indicates two different single-histidine mutants that fulfill our design requirements; experimental assays demonstrate that each mutant indeed exhibits an order-of-magnitude increase in medium half-life along with enhanced potency due to increased endocytic recycling.
authors.affiliation: Department of Chemical Engineering, Biotechnology Processing Center, Massachusetts Institute of Technology, Cambridge, MA 02139-4307, USA.
authors.forename: Casim A
authors.initials: CA
authors.lastname: Sarkar
authors.forename: Ky
authors.initials: K
authors.lastname: Lowenhaupt
authors.forename: Thomas
authors.initials: T
authors.lastname: Horan
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Boone
authors.forename: Bruce
authors.initials: B
authors.lastname: Tidor
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Lauffenburger
mesh_terms: D003198:Computer Simulation; D016207:Cytokines / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005812:Genes, Switch; D016179:Granulocyte Colony-Stimulating Factor / Q000138:chemical synthesis* / Q000235:genetics* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D055672:Static Electricity
chemical_list: D016207:Cytokines; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D016179:Granulocyte Colony-Stimulating Factor; D006639:Histidine