Proapoptotic activity of cell-permeable anti-Akt single-chain antibodies.
We developed anti-Akt1 single-chain antibodies (scFv) by panning a mouse phage-displayed scFv recombinant antibody library. Recombinant scFv that bound glutathione S-transferase (GST)-Akt1 were screened for their ability to inhibit Akt activity in vitro 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 scFv 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(473) Akt and GSK-3alpha/beta phosphorylation, suggesting that Ser(473) 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 in vitro 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 in vitro and in vivo.
Shin
Incheol
I
Department of Cancer Biology, Vanderbilt University School of Medicine, Vanderbilt-Ingram Comprehensive Cancer Center, Nashville, Tennessee 37232-6307, USA.
Edl
Jeniffer
J
Biswas
Swati
S
Lin
P Charles
PC
Mernaugh
Raymond
R
Arteaga
Carlos L
CL
D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000276:immunology; D002460:Cell Line; D045744:Cell Line, Tumor; D005982:Glutathione Transferase / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D006801:Humans; D051379:Mice; D019151:Peptide Library; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology*; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology
D000906:Antibodies; D019151:Peptide Library; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; C494918:AKT1 protein, human; C494921:AKT2 protein, human; C494922:Akt2 protein, mouse; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt