title: Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation.
abstract: The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes.
authors.affiliation: University of Wisconsin-Madison, Dept. of Chemical and Biological Engineering, Madison, WI, United States of America.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Tillotson
authors.affiliation: University of Wisconsin-Madison, Dept. of Chemical and Biological Engineering, Madison, WI, United States of America.
authors.forename: Loukas I
authors.initials: LI
authors.lastname: Goulatis
authors.affiliation: University of Wisconsin-Madison, Dept. of Chemical and Biological Engineering, Madison, WI, United States of America.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Parenti
authors.affiliation: University of Wisconsin-Madison, Dept. of Chemical and Biological Engineering, Madison, WI, United States of America.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Duxbury
authors.affiliation: University of Wisconsin-Madison, Dept. of Chemical and Biological Engineering, Madison, WI, United States of America.
authors.forename: Eric V
authors.initials: EV
authors.lastname: Shusta
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space / Q000378:metabolism*; D016296:Mutagenesis; D011485:Protein Binding; D015202:Protein Engineering; D021381:Protein Transport; D011990:Receptors, Transferrin / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism
chemical_list: D011990:Receptors, Transferrin; D057127:Single-Chain Antibodies