Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation.
Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation.
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DOI：
10.1371/journal.pone.0145820
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Shusta EV
中科院分区：
文献类型：
--
作者：
Tillotson BJ;Goulatis LI;Parenti I;Duxbury E;Shusta EV
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.
