title: Genetically engineering transferrin to improve its in vitro ability to deliver cytotoxins.
abstract: We previously demonstrated that decreasing the iron release rate of transferrin (Tf), by replacing the synergistic anion carbonate with oxalate, increases its in vitro drug carrier efficacy in HeLa cells. In the current work, the utility of this strategy has been further explored by generating two Tf mutants, K206E/R632A Tf and K206E/K534A Tf, exhibiting different degrees of iron release inhibition. The intracellular trafficking behavior of these Tf mutants has been assessed by measuring their association with HeLa cells. Compared to native Tf, the cellular association of K206E/R632A Tf and K206E/K534A Tf increased by 126 and 250%, respectively. Surface plasmon resonance studies clearly indicate that this increase in cellular association is due to a decrease in the iron release rate and not to differences in binding affinity of the mutants to the Tf receptor (TfR). Diphtheria toxin (DT) conjugates of K206E/R632A Tf and K206E/K534A Tf showed significantly increased cytotoxicity against HeLa cells with IC(50) values of 1.00 pM and 0.93 pM, respectively, compared to a value of 1.73 pM for the native Tf conjugate. Besides further validating our strategy of inhibiting iron release, these Tf mutants provide proof-of-principle that site-directed mutagenesis offers an alternative method for improving the drug carrier efficacy of Tf.
authors.affiliation: Department of Bioengineering, University of California, Los Angeles, 90095, USA.
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Yoon
authors.forename: David S H
authors.initials: DS
authors.lastname: Chu
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Ng
authors.forename: Edward A
authors.initials: EA
authors.lastname: Pham
authors.forename: Anne B
authors.initials: AB
authors.lastname: Mason
authors.forename: David M
authors.initials: DM
authors.lastname: Hudson
authors.forename: Valerie C
authors.initials: VC
authors.lastname: Smith
authors.forename: Ross T A
authors.initials: RT
authors.lastname: MacGillivray
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Kamei
mesh_terms: D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt / Q000737:chemistry; D019943:Amino Acid Substitution; D001667:Binding, Competitive; D049109:Cell Proliferation / Q000187:drug effects; D003603:Cytotoxins / Q000008:administration & dosage* / Q000737:chemistry / Q000494:pharmacology; D004167:Diphtheria Toxin / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology; D004337:Drug Carriers / Q000737:chemistry / Q000378:metabolism; D004492:Edetic Acid / Q000737:chemistry; D004705:Endocytosis / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D011990:Receptors, Transferrin / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014168:Transferrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D003603:Cytotoxins; D004167:Diphtheria Toxin; D004337:Drug Carriers; D011990:Receptors, Transferrin; D014168:Transferrin; D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; D004492:Edetic Acid; D007501:Iron