Data from Proapoptotic Activity of Cell-Permeable Anti-Akt Single-Chain Antibodies

Abstract

<div>Abstract<p>We developed anti-Akt1 single-chain antibodies (scFv) by panning a mouse phage–displayed scFv recombinant antibody library. Recombinant scFv that bound glutathione <i>S</i>-transferase (GST)-Akt1 were screened for their ability to inhibit Akt activity <i>in vitro</i> in a kinase reaction containing human recombinant Akt1 and an Akt/serum glucocorticoid-inducible kinase (SGK) substrate. Michaelis-Menten analysis of kinase inhibition by a selected scFv was consistent with scFv-mediated competition with enzyme's substrate for the catalytic site of Akt. To generate a membrane-permeable version of the anti-Akt1 scFv, the <i>scFv</i> gene was subcloned into a GST expression vector carrying a membrane-translocating sequence (MTS) from Kaposi fibroblast growth factor. A purified GST–anti-Akt1–MTS fusion protein accumulated intracellularly in 293T, BT-474, and PyVmT cells in a dose- and time-dependent fashion. Intracellular accumulation correlated temporally with inhibition of p-Ser<sup>473</sup> Akt and GSK-3α/β phosphorylation, suggesting that Ser<sup>473</sup> is an Akt autophosphorylation site. Phosphorylated (activated) phosphoinositide-dependent kinase 1, mitogen-activated protein kinase, p38, and HER2 (erbB2) were not affected, supporting Akt kinase specificity for the inhibitory scFv. Exogenously expressed constitutively active Akt2 and Akt3 were also inhibited <i>in vitro</i> by the anti-Akt1 fusion protein. Furthermore, GST–anti-Akt1–MTS induced apoptosis in three cancer cell lines that express constitutively active Akt. Finally, systemic treatment with the anti-Akt scFv reduced tumor volume and neovascularization and increased apoptosis in PyVmT-expressing transgenic tumors implanted in mouse dorsal window chambers. Thus, GST–anti-Akt1–MTS is a novel cell-permeable inhibitor of Akt, which selectively inhibits Akt-mediated survival in intact cells both <i>in vitro</i> and <i>in vivo</i>.</p></div>