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[Cancer Research 65, 2815-2824, April 1, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

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

Incheol Shin1,7, Jeniffer Edl5, Swati Biswas1, P. Charles Lin1,4, Raymond Mernaugh2,5 and Carlos L. Arteaga1,3,6

Departments of 1 Cancer Biology, 2 Biochemistry, 3 Medicine, and4 Radiation Oncology, Vanderbilt University School of Medicine and 5 Molecular Recognition Unit and 6 Breast Cancer Program, Vanderbilt-Ingram Comprehensive Cancer Center, Nashville, Tennessee; 7 Department of Life Sciences, Hanyang University, Seoul, Korea

Requests for reprints: Carlos L. Arteaga, Division of Oncology, Vanderbilt University School of Medicine, 2220 Pierce Avenue, 777 Preston Residence Building, Nashville, TN 37232-6307. Phone: 615-936-3524; Fax: 615-936-1790; E-mail: carlos.arteaga{at}vanderbilt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-Ser473 Akt and GSK-3{alpha}/ß phosphorylation, suggesting that Ser473 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.

Key Words: single-chain antibody • membrane translocating sequence • Akt • phosphatidylinositol-3 kinase • cancer


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antibodies and antibody-based reagents have been used for the treatment of cancer (1, 2). For example, the humanized IgG1 trastuzamab (Herceptin) is an effective treatment for breast cancers that overexpress the HER2/neu (erbB2) proto-oncogene (3). The chimeric IgG2 cetuximab (Erbitux, C225) was recently approved for the treatment of epidermal growth factor (EGF) receptor–positive metastatic colorectal carcinoma (4). Genetic engineering of antibodies can be used to modify and enhance antibody efficacy. For example, mouse monoclonal antibodies can be chimerized by such approaches to prevent the production of human antimurine antibodies (HAMA) when administered to immune-competent humans (5). An alternative strategy is to replace the antibody gene present in mouse B cells with human antibody genes. These modified B cells can then be used to produce hybridoma cell lines that express fully humanized monoclonal antibodies that avoid cross-species immune response (i.e., HAMA) and, in addition, can trigger human host cell effector functions, such as complement fixation (6).

The two variable domains of an antibody binding site can be cloned and reconstituted in a variety of molecular forms and expressed in various hosts from bacteria to transgenic animals and plants (7). Over a decade ago, McCafferty et al. (8) first described that recombinant antibody fragments could be displayed on the tip of M13 bacteriophage, a bacterial virus (phage) than infects Escherichia coli. Some of the advantages of phage-displayed recombinant antibodies over the conventional polyclonal or monoclonal antibodies are quick generation time, cheap production cost, and, importantly, accessibility to the antibody DNA for further genetic manipulations (9).

The serine-threonine kinase protein kinase B or Akt, the cellular homologue of the AKT8 retrovirus oncogene, has been shown to play an important role in cell survival and proliferation (10, 11) . The antiapoptotic functions of Akt are mediated, in part, by phosphorylation and functional inactivation of proapoptotic molecules, such as Bad (12), Forkhead transcription factors (13), and caspase-9 (14) among others. By promoting cell survival and proliferation, Akt signaling contributes to cancer progression (15). Akt1 gene amplification was first described in gastric adenocarcinomas (16). The Akt2 gene is amplified in some ovarian and breast carcinomas (17). High levels of Akt2 protein have been reported in pancreatic adenocarcinoma (18). The potential involvement of Akt activity in cancer progression has suggested its role as a therapeutic target. Inhibition of Akt signaling has been accomplished with LY294002 (19) and Wortmannin (20), two small-molecule inhibitors of phosphatidylinositol-3 kinase (PI3K), a lipid kinase upstream of Akt (21). However, these small molecules are not suitable for use in humans. In addition, because of the many cellular targets of this lipid kinase, use of PI3K inhibitors may be associated with undesirable side effects (22).

In this report, we describe the development of a novel recombinant Akt inhibitor that binds to and specifically blocks Akt activity in vitro and in intact cells. Recombinant single-chain antibodies (scFv) from a mouse phage–displayed antibody library that recognized Akt1 were screened for inhibitory activity against the Akt kinase. To generate a fusion protein that can translocate into cells, the gene from the bacterial clone producing the anti-Akt scFv was subcloned into an expression vector containing a membrane-translocating sequence (MTS; refs. 23, 24). We describe, herein, a chimeric glutathione S-transferase (GST)–anti-Akt1–MTS protein that is efficiently imported into intact cells in an MTS-dependent manner, resulting in inhibition of Akt function and, in turn, tumor cell survival in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Expression of glutathione S-transferase–Akt1 fusion protein. The Akt1 plasmid was provided by Rakesh Kumar (University of Texas M. D. Anderson Cancer Center, Houston, TX). Akt1 was PCR-amplified and subcloned into the BamH1 site of the GST expression vector pGEX-3X (Amersham, Piscataway, NJ) via TA cloning vector pGEM-T-Easy (Promega, Madison, WI). The primers used, each containing BamH1 sites at their 5' end, were 5'-CCGGATCCCCATGAGCGACGTGGCTATTGTGAAGGAGGGT-3' and 5'-CCGGATCCCGGCCGTGCTGCTGGCCGAGTAGGAGAACTG-3'. DNA sequencing indicated that the Akt1 gene was inserted in the right orientation and in frame with GST. The resulting pGEX-3X-Akt1 was transformed into E. coli DH5{alpha}. GST-Akt1 fusion protein expression and purification by reduced glutathione (GSH)–agarose affinity chromatography were done as previously described (23).

Panning of phage-displayed single-chain antibody library and antibody purification. One milligram of GST-Akt1 fusion protein was dialyzed against 5,000 volumes of 0.2 mol/L NaCl for 48 hours. The fusion protein was concentrated by ultrafiltration using a PM10 membrane (Amicon, Beverly, MA) and then used for phage selection and ELISA assays as described in Supplementary Methods.

Recombinant Akt1 kinase assay and single-chain antibody kinetic analysis. Preactivated Akt1 [with mitogen-activated protein kinase (MAPK)-activated protein (MAPKAP) kinase 2; Upstate Biotechnology, Lake Placid, NY] was dissolved in assay dilution buffer [ADB; 20 mmol/L MOPS (pH 7.5), 25 mmol/L ß-glycerophosphate, 5 mmol/L EGTA, 1 mmol/L Na3VO4, 1 mmol/L DTT]. E-tag ELISA-positive scFvs (0.15 µg each) were dissolved in 10 µL ADB and incubated with 0.15 µg recombinant Akt1 (rAkt1) for 1 hour at room temperature. An irrelevant scFv (0.15 µg) was used as control. The kinase reaction was started by the addition of 40 µmol/L Akt/serum glucocorticoid-inducible kinase (SGK) substrate peptide (Upstate Biotechnology) and 0.2 µCi [{gamma}-32P]ATP (specific activity 3,000 Ci/mmol, DuPont-NEN, Boston, MA) diluted in 10 µL of 500 µmol/L cold ATP containing 75 mmol/L MgCl2. After a 10-minute incubation at 30°C with continuous shaking, the reaction was quenched with the addition of 20 µL of 40% trichloroacetic acid; 25 µL of the reaction supernatant were spotted onto phosphocellulose paper disks (Upstate Biotechnology). The disks were washed thrice with 0.75% phosphoric acid and once with acetone (5 minutes each). Disk-associated dpm values were measured in a liquid scintillation counter (Beckman, Fullerton, CA).

To determine the kinetics of the rAkt1 kinase reaction, Michaelis-Menten plots and corresponding double-reciprocal Lineweaver-Burk plots were generated. Kinase reactions were done in vitro with Akt/SGK substrate concentrations ranging from 10 to 480 µmol/L in the absence or presence of the anti-Akt1 scFv. rAkt1 (0.15 µg) was preincubated with 0.45 µg scFv (clone N5) for 1 hour before the kinase reaction. Dpm values incorporated onto the Akt/SGK substrate were converted to Ci by using the dpm values in standard samples. The activity in Ci was transformed to mole units using the specific activity of [{gamma}-32P]ATP. Nonlinear Michaelis-Menten regression plots and Lineweaver-Burk plots were generated by GraphPad Prism software version 3.03 (GraphPad Software, San Diego, CA).

Generation of glutathione S-transferase–anti-Akt1–MTS fusion protein. The scFv gene in pCANTAB 5E phagemid clone N5 was PCR amplified. The sequences of degenerative primers used were 5'-CCGGATCCCCATGGCCCAGGTSMARCTGCAGSAGTCWGG-3' and 5'-CCGGATCCCTGCGGCACGCGGTTCCAGCGGATCCGG-3'. The PCR product including the E-tag at the 3' end of VL was first subcloned into pGEM-T-Easy, digested with BamH1, and ligated in BamH1-digested pGEX-3X-MTS2 (a gift from Mauricio Rojas, Emory University, Atlanta, GA; ref. 23). DNA sequence analyses confirmed that GST, scFv, and MTS translational frames were maintained. To test the specificity of GST–anti-Akt1–MTS, an irrelevant control scFv was also subcloned into pGEX-3X-MTS2. The expression and purification of the GST fusion proteins was done as described (23).

Cell lines. 293T human embryonic fibroblasts, BT-474 human breast cancer, and U87-MG human glioma cells were purchased from American Type Culture Collection (Rockland, ME). The PyVmT mouse cell line derived from a mammary tumor arising in a mouse mammary tumor virus/polyomavirus middle T antigen (MMTV/PyVmT) transgenic mouse and has been described previously (25). All cell lines, except BT-474 cells, were maintained in DME/10% FCS. BT-474 cells were kept in IMEM/10% FCS.

Immunofluorescence analysis and confocal microscopy. Subconfluent cell monolayers on cover slips placed in 12-well plates were treated with 10 µmol/L GST–anti-Akt1–MTS or GST-MTS for 6 hours. The cells were washed five times with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton in PBS, and then incubated with 3% milk in PBS for 30 minutes followed by an E-Tag monoclonal antibody (0.5 µg/mL in 1% milk in PBS; Amersham) for 1 hour. After three washes with PBS, the samples were incubated with Oregon green-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR) for 1 hour. The cover slips were mounted on glass slides with Poly/Mount (Polysciences, Warrington, PA) and subjected to Z-axis optical sectioning (1 µm/section) in a Zeiss laser scanning confocal microscope (LSM 510).

Immunoblot analysis. Subconfluent cell monolayers were treated with GST–anti-Akt1–MTS or GST–control scFv–MTS. The cells were washed five times with PBS and then lysed in a buffer containing 20 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% NP40, 10% glycerol, 20 mmol/L NaF, 1 mmol/L Na3VO4, and protease inhibitor cocktail (Roche, Nutley, NJ). Protein extracts (30 µg/lane) were separated by 10% SDS-PAGE and subjected to immunoblot analysis as described (26). Horseradish peroxidase–conjugated secondary antibodies were from Amersham; immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Pierce, Rockford, IL). Akt, p-Ser473 Akt, p-Thr308 Akt, p-p38, GSK-3{alpha}/ß, p-GSK-3{alpha}/ß, cleaved caspase-3, phosphoinositide-dependent kinase 1 (PDK1), and p-Ser241 PDK1 antibodies were from Cell Signaling (Beverly, MA). Antibodies to p-MAPK and total MAPK were from Promega. The monoclonal antibody to p38 was from Santa Cruz Biotechnology (Santa Cruz, CA). The GST and E-tag antibodies were from Amersham. The HER2 and p-Tyr antibodies were from Neomarkers (Freemont, CA) and Upstate Biotechnology, respectively. ZD1839 (Iressa, gefitinib) was provided by Alan Wakeling (AstraZeneca Pharmaceuticals, Macclesfield, United Kingdom).

Expression of Akt isoforms. Hemagglutinin-tagged Akt1DD, Akt2DD, myr-Akt1, myr-Akt2, and myr-Akt3 were provided by Gordon Mills (University of Texas M.D. Anderson Cancer Center). 293T cells on 100-mm dishes were transfected with 5 µg of each plasmids using Fugene 6 (Roche). After 48 hours, the cells were lysed in NP40 buffer (described above). Cell lysates (500 µg) were precipitated with a hemagglutinin antibody (1 µg; Santa Cruz Biotechnology) and protein A-Sepharose (50% slurry) overnight at 4°C. The precipitated hemagglutinin-Akt was washed twice with lysis buffer and twice with (cold) kinase buffer [25 mmol/L Tris (pH 7.5), 5 mmol/L ß-glycerolphosphate, 2 mmol/L DTT, 0.1 mmol/L Na3VO4, 10 mmol/L MgCl2]. Pellets were suspended in 40 µL kinase buffer supplemented with 200 µmol/L ATP and 1 µg of a fusion protein containing a GSK-3ß peptide (Cell Signaling) and incubated for 30 minutes at 30°C. The kinase reaction was terminated by addition of SDS sample buffer and boiling. Reaction products were subjected to SDS-PAGE and immunoblot analyses with total and phospho-GSK-3{alpha}/ß antibodies.

Apoptosis assay. Cells (5 x 105/well) were seeded in six-well plates in triplicate in serum-containing medium. The following day, the medium was changed to serum-free medium with 10 µmol/L GST–control scFv–MTS or GST–anti-Akt1–MTS. Both floating cells and adherent cells were collected 72 hours later. Pooled cells were washed with PBS and then subjected to terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) analysis with the use of an Apo-bromodeoxyuridine (BrdUrd) assay kit (Phoenix Flow Systems, San Diego, CA) according to the manufacturer's protocol. TUNEL+ cells were quantitated in a FACSCalibur Flow Cytometer (BD Biosciences, Mansfield, MA).

Tumor window chamber model. This model has been described previously (27). Briefly, a small metal frame was surgically implanted in a dorsal skin flap in FVB mice. An 8-mm-diameter hole was dissected in the surface of the skin flap, which was next retracted away from the s.c. dermis. The underlying fascia was dissected away until a facial plane with associated vasculature was exposed. An ~0.5 mm3 fragment from an MMTV/PyVmT transgenic mammary tumor (25) was implanted onto the facial plane of the window. Sterile saline solution (100 µL) was added and the window was sealed with glass cover slips to protect it from the outside environment. The resulting tissue plane within the window is ~200 µm thick and is semitransparent. Mice bearing the dorsal chambers were housed individually in pathogen-free units in compliance with Vanderbilt University Institutional Animal Care and Use Committee regulations. Upon the formation of vascularized tumors in the window chambers after 7 days, mice were randomly assigned to receive anti-Akt scFv or control antibody, respectively. The first treatment was injected locally into the window chamber (120 µg/mouse in a 100 µL volume), followed by two similar doses delivered i.p. every other day. Tumor neovascularization was scored using an Olympus PROVIS AX70 microscope (Olympus, New York, NY) on days 3 and 7. The tumor vascular length density as a functional index of tumor neovascularization was calculated from the tumor images within the window chambers using previously described methods (27). After 7 days of treatment, tumor tissues were harvested and subjected to H&E staining and TUNEL analysis as described (25). To obtain tumor size, H&E-stained sections representing the largest cross-sectional areas were photographed and both tumor thickness and diameter were used to calculate tumor volume in mm3 as described (27).

Statistical analysis. Results were reported as mean ± SD for both tumor volume and vascular density for each group. A two-tailed Student's t test was used to analyze statistical differences between them. Differences were considered to be statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anti-Akt1 single-chain antibodies competitively inhibit Akt kinase in vitro. A GST-Akt1 fusion protein was utilized to pan a large (~2.9 x 109 members) mouse phage–displayed scFv library. More than 50 scFv clones interacted with GST-Akt1 as determined by E-Tag ELISA. To screen for scFv-mediated inhibition of Akt activity, we developed an in vitro kinase assay utilizing rAkt1 and a synthetic Akt/SGK peptide. In dose-dependent experiments, 0.15 µg rAkt1 showed kinase activity within a linear range (Fig. 1A). Using this concentration of rAkt1, maximal incorporation of [32P]ATP onto the SGK substrate occurred after 40 minutes with 50% incorporation being achieved after only 10 minutes of reaction time (Fig. 1B). Anti-Akt1 scFv phage, which bound to GST-Akt1, was screened for its inhibitory activity against Akt in the kinase reaction. Of >50 scFv tested, two clones (H7 and N5) exhibited a dose-dependent inhibitory effect against Akt in vitro (Fig. 1C).



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  		Figure 1. Akt kinase assay and inhibitor screening. A, Akt in vitro kinase assay. Increasing amounts of rAkt1 were added to a reaction mixture containing Akt/SGK substrate and [{gamma}-32P]ATP as indicated in Materials and Methods. After stopping the reaction with trichloroacetic acid, reaction products were spotted onto phosphocellulose paper disks. The disks were washed with 0.75% phosphoric acid and acetone and their associated radioactive counts (cpm) were measured in a liquid scintillation counter. B, time dependence of Akt kinase reaction using 0.15 µg rAkt. C, inhibition of Akt kinase in vitro by scFv. Increasing concentrations of the indicated anti-Akt scFvs were preincubated for 1 hour with rAkt (0.15 or 0.45 µg) or 0.45 µg of an irrelevant scFv (control) and then added to the Akt kinase reaction described in A for 10 minutes at 30°C. D, Akt kinase assays were conducted with substrate concentration ranging from 10 to 480 µmol/L in the absence ({bullet}) or presence ({blacktriangledown}) of 0.45 µg anti-Akt1 scFv (clone N5). Nonlinear Michaelis-Menten regression and double-reciprocal Lineweaver-Burk plots of the results were generated by using GraphPad Prism software. The Vmax and Km for the reaction were 30.03 fmol/min (Vmax values are expressed as femtomole of 32P-phosphate transferred per 0.15 µg of rAkt1/min) and 24.81 µmol/L in the absence of anti-Akt scFv and 32.88 fmol/min and 75.84 µmol/L in its presence, respectively.

 
Akt activity can be regulated by site-specific phosphorylation. Thr308 and Ser473 phosphorylation have been shown to be required for full activation of Akt1 (10). Therefore, if the scFvs inhibit Akt by binding to either of these sites and blocking their phosphorylation, we proposed that the antibodies will act as allosteric inhibitors and the kinetics of enzyme inhibition will be noncompetitive with the kinase substrate. An alternative inhibitory mechanism would be binding of the scFv to the catalytic site of Akt1. In this scenario, the inhibitor should compete with the enzyme substrate for the active site of Akt1, thus showing kinetics of competitive inhibition. To elucidate the mechanism of scFv-mediated inhibition of Akt, Michaelis-Menten enzyme kinetics and double-reciprocal Lineweaver-Burk plots were generated using varying concentrations of enzyme substrate (Fig. 1D). The anti-Akt1 scFv inhibited rAkt1 activity at SGK substrate concentrations ranging from 10 to 480 µmol/L, suggesting that the scFv can compete with the enzyme's substrate for binding to Akt1. Lineweaver-Burk plots revealed a Vmax for the kinase reaction of 30.03 and 32.88 fmol 32P/min in the presence and absence of the anti-Akt1 scFv, respectively. Consistent with the competitive inhibition of rAkt1, the Km (the concentration of substrate showing half-maximal velocity) of the reaction was 24.81 versus 75.84 µmol/L in the absence and presence of the anti-Akt1 scFv, respectively.

Anti-Akt1 single-chain antibodies localize intracellularly and bind to and inhibit Akt. To deliver the anti-Akt1 scFv intracellularly, we generated a fusion protein that also contained a MTS derived from the hydrophobic region of the signal peptide of Kaposi fibroblast growth factor (24). This modified 12 amino acid sequence (AAVLLPVLLAAP) functions as a cellular import signal. Therefore, we subcloned the anti-Akt1 scFv gene from clone N5 phagemid DNA (Fig. 2A) into pGEX-3X-MTS2 by PCR with degenerative primers. A PCR product of ~670 bp containing both antibody DNA heavy chain ({approx}340 bp) and light chain ({approx}325 bp) chains and linker DNA (9) was identified on agarose gels (Fig. 2B). The resulting 60 kDa fusion recombinant scFv containing GST on its amino terminus and MTS on its carboxyl terminus was expressed in E. coli and purified by GSH-agarose affinity chromatography (Fig. 2C).



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  		Figure 2. PCR amplification of scFv gene from pCANTAB 5E. A, schematic map of the pCANTAB 5E plasmid containing the scFv gene library. B, agarose gel showing the PCR-amplified scFv gene from the N5 clone. C, pGEX-3X-MTS2 containing the scFv gene was transformed in E. coli (BL21). Expression of the GST fusion protein was induced by 0.1 mmol/L isopropyl-L-thio-B-D-galactopyranoside (IPTG). The bacterial lysates were resolved on SDS-PAGE and stained with Coomassie blue. Lanes 1 to 4, empty vector encoding GST-MTS; lanes 5 and 6, clones transformed with GST–anti-Akt1–MTS.

 
Human embryonic 293T fibroblasts, BT-474 human breast cancer cells, and mouse cancer cells expressing the PyVmT (middle T antigen) oncogene were used to determine if the recombinant fusion protein could penetrate the membranes of living cells. After treatment with purified GST–anti-Akt1–MTS or GST-MTS, the cells were assayed by immunofluorescence microscopy using a monoclonal antibody specific for the E-tag present in the scFv. Whereas GST-MTS–treated cells did not show any E-tag fluorescent signal, GST–anti-Akt1–MTS–treated cells exhibited a punctate staining pattern (Fig. 3A). In 293T and BT-474 cells, GST–anti-Akt1–MTS was found mainly in the cytosol whereas in PyVmT-expressing cells the fluorescent signal was more diffuse. Staining with a GST antibody showed fluorescent signals in both GST-MTS– and GST–anti-Akt1–MTS–treated cells (data not shown). To confirm that GST–anti-Akt1–MTS was localized intracellularly, we used confocal laser scanning microscopy. Z-axis optical sectioning from bottom (0 µmol/L) to top (11 µmol/L) of cells indicated that the GST–anti-Akt1–MTS fusion protein localized in the midsections of the cells, supporting that intracellular translocation had occurred (Fig. 3B and C).



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  		Figure 3. Intracellular localization of GST–anti-Akt1–MTS. A, 293T, BT-474, and PyVmT cells on cover slips were treated with 10 µmol/L GST–anti-Akt1–MTS or control (GST-MTS) for 24 hours. Cells were then permeabilized, blocked with 5% bovine serum albumin, and stained with an E-tag monoclonal antibody (1:2,000) for 1 hour. After washing thrice with PBS, cells were incubated with anti-mouse IgG-Oregon green (1:1,000) for 1 hour. The samples were washed with PBS, mounted on glass slides, and observed with a confocal microscope. B and C, the same samples were subjected to serial Z-coordinate optical sections by using the Z-sectioning function of a Zeiss laser-scanning confocal microscope: 293T (B) and BT-474 (C).

 
We next determined whether the recombinant scFv inhibited Akt signaling in intact cells. 293T cells were treated with GST–anti-Akt1–MTS for 3 to 6 hours. Intracellular accumulation of the fusion protein was time-dependent as measured by GST immunoblot of lysates from treated cells (Fig. 4A, top). By 6 hours, levels of p-Ser473 Akt and p-GSK-3{alpha}/ß, a cellular substrate of Akt (11), were significantly reduced. Levels of total Akt, p-Thr308 Akt, and total GSK-3{alpha}/ß remained the same. Active MAPK and active p38 levels, as measured by immunoblot analyses using phosphospecific antibodies, were not modified by the anti-Akt1 scFv, suggesting that its effects were Akt specific. This inhibition was dose dependent with partial reduction of p-Ser473 Akt and p-GSK-3{alpha}/ß at a concentration of 1 µmol/L GST–anti-Akt1–MTS (Fig. 4B). The GST-scFv-MTS control fusion also transferred efficiently into 293T cells (10 µmol/L, 6 hours) as indicated by GST immunoblot. However, it did not inhibit p-Ser473 Akt and GSK-3{alpha}/ß phosphorylation (Fig. 4B, lane 5). To confirm binding of the GST–anti-Akt1–MTS to its target (Akt) intracellularly, we precipitated Akt from lysates of cells treated with anti-Akt or control scFv. The anti-Akt scFv, as measured by E-tag immunoblot, but not the control antibody, was present in the Akt precipitates (Fig. 4C), thus providing direct evidence of association of GST–anti-Akt–MTS with its molecular target in intact cells.



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  		Figure 4. GST–anti-Akt1–MTS binds to and inhibits Akt. A, 293T cells were treated with 10 µmol/L GST–anti-Akt1–MTS for 3 or 6 hours and then washed. Intracellular transfer of the fusion protein (59 kDa) was confirmed by immunoblot with GST antibody. The effects of GST–anti-Akt1–MTS on Akt, MAPK, and p38 MAPK signaling were monitored by immunoblot analyses. B, 293T cells were treated with various concentrations of GST–anti-Akt1–MTS for 6 hours. The GST–control scFv–MTS was only tested at 10 µmol/L for 6 hours. Cell lysates were then subjected to immunoblot analyses with the indicated antibodies. C, 293T cells were treated with 10 µmol/L anti-Akt or control scFv for 6 hours and then lysed. Cell lysates were precipitated with an Akt antibody and immune complexes resolved by SDS-PAGE followed by immunoblot analysis using an E-tag antibody. To control for presence of the control scFv, total lysates (before precipitation with Akt antibody) were tested simultaneously. D, 293T, BT-474, and PyVmT cells treated with 10 µmol/L GST–anti-Akt1–MTS for 6 hours. Fifty micrograms of total cell lysates were separated by SDS-PAGE and then subjected to immunoblot analyses with the indicated antibodies. E, effect of GST–anti-Akt1–MTS on HER2 phosphorylation. BT-474 cells were treated with either 10 µmol/L GST–anti-Akt1–MTS or GST–control scFv–MTS for 6 hours, or 3 µmol/L ZD1839 for 24 hours. Whole cell lysates (0.5 mg) were precipitated with a HER2 antibody; the resulting precipitates were subjected to p-Tyr immunoblot analysis (bottom); 50 µg total cell lysates were tested directly for HER2 content by immunoblot (top).

 
Similar findings were observed in BT-474– and PyVmT-expressing cells. Both cells exhibit constitutive activation of Akt. In BT-474 cells, it has been suggested that this is the result of HER2 (erbB2) overexpression and heterodimerization with erbB3 (26). In PyVmT transgenic cells, middle T antigen binds to and potently activates PI3K and Akt (28). In all three cell lines, GST–anti-Akt1–MTS effectively blocked Ser473 Akt and GSK-3{alpha}/ß phosphorylation (Fig. 4D). However, phosphorylation of Akt on Thr308, the PDK1 phosphorylation site (10), and autophosphorylation of PDK1 on Ser241, which is necessary for PDK1 activation (29), were not affected. Again, active MAPK and active p38 were not inhibited by the anti-Akt1 fusion protein. Because activation of HER2-containing receptor heterodimers activates PI3K/Akt signaling in HER2 overexpressing cells, such as BT-474 (26), we controlled for the effect of GST–anti-Akt1–MTS on HER2 phosphorylation. GST–anti-Akt1–MTS did not inhibit HER2 levels or its phosphorylation as measured by p-Tyr immunoblot of HER2 precipitates from treated BT-474 cells. The small-molecule EGF receptor tyrosine kinase inhibitor ZD1839, which has been shown to inhibit HER2 phosphorylation in BT-474 cells (30), was used as a positive control (Fig. 4E).

Anti-Akt1 single-chain antibodies inhibit Akt2 and Akt3 and induce apoptosis in vivo. To determine the effect of GST–anti-Akt1–MTS on other Akt isoforms, hemagglutinin-tagged, constitutively active versions of Akt2 and Akt3 were expressed in 293T cells. A phosphomimetic mutant of Akt1, where Thr308 and Ser473 are replaced with Asp (Akt1DD), has been shown to exhibit constitutive Akt catalytic activity (31). In this study, we used an Akt2DD mutant, where Asp has replaced Thr309 and Ser474, corresponding to Thr308 and Ser473 in Akt1 (32). Addition of a c-Src–derived myristoylation (myr) signal (MGSSKSKPK) to the amino terminus of Akt renders its kinase constitutively active by tethering Akt to the plasma membrane (33). Expression of Akt1DD, Akt2DD, myr-Akt1, myr-Akt2, and myr-Akt3 in transfected 293T cells was confirmed by hemagglutinin immunoblot analysis (Fig. 5A). Hemagglutinin precipitates from 293T cells transfected with the Akt isoform vectors were preincubated with GST–anti-Akt1–MTS or its control and then tested in the Akt in vitro kinase assay using GSK-3ß fusion protein as enzyme substrate. In all cases, preincubation with the anti-Akt1 fusion protein but not with GST–control scFv–MTS resulted in inhibition of GSK-3ß phosphorylation (Fig. 5B), suggesting that GST–anti-Akt1–MTS can recognize all three Akt isoforms.



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  		Figure 5. GST–anti-Akt1–MTS inhibits other Akt isoforms. A, the indicated hemagglutinin (HA)-tagged isoforms of Akt were transiently expressed in 293T cells. After 48 hours, cells were lysed and the lysates subjected to hemagglutinin immunoblot analysis. B, 293T cells were transfected with various constructs of Akt isoforms as in A. Transfected Akt was precipitated from 293T cell lysates with a hemagglutinin antibody or a control IgG. Antibody pull downs were then tested in an Akt in vitro kinase reaction using 200 µmol/L cold ATP and 1 µg GSK-3ß fusion protein. Before addition to the kinase reaction, the hemagglutinin precipitates were incubated for 1 hour at room temperature with 1 µg GST–anti-Akt1–MTS or GST–control scFv–MTS. Kinase reaction products were resolved by SDS-PAGE followed by immunoblot analyses with p-GSK-3ß and total GSK-3ß antibodies.

 
Akt function is causally associated with cell survival (10, 11) . Thus, we next examined if GST–anti-Akt1–MTS exhibited proapoptotic activity in cell lines with constitutive Akt activity. U87-MG human glioma cells contain a homozygous deletion of the phosphatase PTEN resulting in high PI3K and Akt activities (34). Subconfluent 293T, BT-474, PyVmT, and U87-MG cells were treated with GST–anti-Akt1–MTS or GST–control scFv–MTS. All four cell lines exhibited markedly enhanced cleavage of caspase-3, a hallmark of apoptosis (35), when treated for 72 hours with the anti-Akt1 scFv but not with the control fusion protein (Fig. 6A). Furthermore, quantification of cell death by TUNEL analysis of all four cell lines showed a 2-fold to >4-fold increase in the proportion of TUNEL+ cells as a function of treatment with the anti-Akt1 fusion protein (Fig. 6B).



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  		Figure 6. GST–anti-Akt1–MTS induces apoptosis of cells in culture. A, 293T, BT-474, PyVmT, and U87-MG cells were treated with 10 µmol/L GST–anti-Akt1–MTS or GST–control scFv–MTS in serum-free medium for 72 hours. Controls were maintained in 10% fetal bovine serum. Cell lysates were subjected to an immunoblot procedure using an antibody specific to cleaved caspase-3. B, cells were treated as in A and fixed in 1% paraformaldehyde. The proportion of apoptotic cells was quantitated by Apo-BrdUrd analysis and flow cytometry as described in Materials and Methods. The proportion of FITC+ (TUNEL+) cells ± SD (n = 3 wells) is indicated in the gated area in each panel.

 
Finally, we determined the inhibitory activity of GST–anti-Akt1–MTS against MMTV/PyVmT transgenic tumor transplants in vivo. Tumorigenicity and metastases in this transgenic mouse model of mammary cancer depend on the ability of middle T to activate PI3K, the major kinase upstream Akt (28). In addition, inhibition of PI3K and Akt in these cells has been shown to reduce cell motility, survival, and metastases (25, 36). Mice bearing established and vascularized MMTV/PyVmT transgenic transplants in dorsal mouse chambers were treated systemically with GST–anti-Akt1–MTS or the control scFv. Both tumor volume and tumor neovascularization, as inferred from the vascular length density, were markedly reduced in tumors present in mice treated with the anti-Akt scFv treated for 7 days compared with controls (Fig. 7A). In addition, there was an 8-fold increase in the average proportion of TUNEL+ cells in the former tumors compared with those treated with the control scFv (Fig. 7B).



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  		Figure 7. Anti-Akt1 scFv induces apoptosis in vivo. A, ~0.5 mm3 fragments from a MMTV/PyVmT mammary tumor were transplanted into individual dorsal window chambers that had been implanted in FVB mice. Seven days later, mice were started on treatment with GST–anti-Akt1–MTS or GST–control scFv–MTS every other day (n = 3 per group). Tumor volume and neovascularization, as measured by the tumor vessel length density, were assessed serially on days 3 and 7 after the start of treatment as described in Materials and Methods. Day 7 data are shown (n = 3 per group; P < 0.05). B, on day 7 after treatment initiation, the tumors were harvested, paraffin embedded, and processed for both H&E staining and TUNEL analysis. Shown is the average of TUNEL+ cells ± SD of three randomly chosen fields (n = 3 per group; P < 0.012).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The serine-threonine kinase Akt promotes tumor cell proliferation and survival both in vitro and in vivo. Several components of the PI3K/Akt pathway are dysregulated in a variety of human cancers. These include inactivation and mutation of the tumor suppressor phosphatase PTEN (34, 37, 38), PI3K gene amplification and/or mutation (39–42), and Akt gene amplification or Akt protein overexpression (16, 17). Studying the specific role of Akt in cancer biology has been somewhat limited by the lack of specific inhibitors of Akt function. We report herein that phage-displayed scFvs screened for inhibitory activity in vitro against the Akt kinase can be genetically engineered to acquire cell membrane permeability and inhibit Akt function in intact cells. GST–anti-Akt1–MTS specifically inhibited Akt but had no detectable activity against PDK-1, MAPK, p38, and HER2 kinases (Fig. 4). This effect cannot be subscribed to GST action in that a similar fusion protein containing an irrelevant scFv had no effect on Akt or cell survival (Figs. 6 and 7) both in cultured cells and a PI3K/Akt-dependent tumor model in vivo. GST–anti-Akt1–MTS also inhibited constitutive active versions of Akt2 and Akt3 in vitro (Fig. 5). Because the anti-Akt1 scFv acts as a competitive substrate inhibitor for rAkt1 (Fig. 1D), the anti-pan Akt activity of GST–anti-Akt1–MTS (Fig. 5B) suggests that the fusion protein binds the catalytic domain of the enzyme, which is highly conserved in all three Akt isoforms (10).

Treatment with GST–anti-Akt1–MTS resulted in inhibition of Ser473 but not Thr308 phosphorylation in Akt1 (Fig. 4). Although PDK1 has been well characterized as the kinase that phosphorylates Thr308 of Akt1 (11), the kinase responsible for Ser473 phosphorylation is a matter of debate (reviewed in ref. 43). In the presence of PDK1-interacting fragment from protein kinase C–related kinase-2, PDK1 phosphorylates both Thr308 and Ser473 of Akt1 in a phosphatidylinositol 3,4,5-triphosphate (PIP3)-dependent manner (44). Integrin-linked kinase has also been shown to regulate phosphorylation of Ser473 in Akt1 (45). For example, knocking-out integrin-linked kinase by double-stranded RNA interference inhibits Ser473 phosphorylation of Akt without affecting Thr308 phosphorylation (46). However, another report suggested that kinase-dead integrin-linked kinase may contribute to Ser473 phosphorylation by providing an adaptor function and, thus, recruiting other kinases to Akt (47). Akt Ser473 kinase activity was also found in detergent-insoluble plasma membrane rafts, which did not contain integrin-linked kinase (48). Protein kinase C ßII has also been shown to phosphorylate Akt on Ser473 in a cell-type-specific fashion (49). Another study with kinase-dead and heat-inactivated Akt suggested that Ser473 is autophosphorylated by Akt itself (50). Our results support this last possibility in that the recombinant scFv exhibited kinetics consistent with competition with the kinase substrate, whereas blocking phosphorylation of Akt at Ser473 and downstream GSK-3{alpha}/ß phosphorylation. However, we could not rule out the possibility that binding of GST–anti-Akt1–MTS to Akt may sterically hinder access of other kinase(s) capable of phosphorylating Ser473.

Recent reports have claimed the development of Akt inhibitors. Derivatives of dichlorotriazine and dichloropyrimidine were reported as Akt3 inhibitors with an IC50 of <1 µmol/L. However, these compounds were also effective against EGF and insulin-like growth factor receptor tyrosine kinases (51). A series of phosphatidylinositol phosphate lipid analogues with modified 3-OH group have been developed. These analogues inhibit Akt indirectly via the suppression of PI3K-mediated formation of PIP3 (52). One of these derivatives was shown to act directly on Akt by binding to the pleckstrin homology domain of the serine-threonine kinase, thus inhibiting membrane translocation and/or dimerization of Akt (53). The alkyl-lysophospholipid perifosine has also been reported to perturb membrane translocation of the pleckstrin homology domain of Akt and, in turn, inhibit its activation by PDK1 (54). Because these compounds can, in principle, affect translocation of all pleckstrin homology–containing signal transducers, their Akt specificity is unclear. Finally, a small-molecule Akt pathway inhibitor (API-2), from the National Cancer Institute Diversity Set, was recently discovered (55). In this study, API-2, previously known as tricyclic nucleoside, exhibited predominant activity against tumor cells with aberrant Akt signaling. However, the ability of this compound to also inhibit DNA synthesis and viral activity clearly suggests that API-2 may have a broad spectrum of molecular targets in addition to Akt.

The penetration of MTS-containing fusion peptides into the cell membrane is thought to occur through interactions between the amino acid residues in the MTS and membrane phospholipids (56). Cell-penetrating peptides derived from either cell-permeable proteins or their signal sequence have been used for the delivery of macromolecules into cells and may have important therapeutic applications (reviewed in ref. 57). Dendritic cells loaded with a tyrosinase-related protein 2 peptide covalently linked to MTS showed prolonged presentation of class I MHC and protected immunized mice from B16 tumor formation and lung metastases (58). A fusion protein containing a growth factor receptor binding protein 2 (Grb2) SH2 domain and MTS entered cells and, in dominant-negative fashion, inhibited the association of the EGF receptor with Grb2 (23). An MTS peptide that was chemically attached to antibodies was shown to enter living cells in culture (59). To our knowledge, this is the first report of an scFv that is genetically engineered to have inherent cell membrane-translocating activity, whereas retaining biochemical inhibitory function against its molecular target as well as cellular activity in vivo. These results have important clinical implications as they suggest that this approach can be applied to the generation of compounds that target tumor cells dependent on aberrant Akt signaling for their survival.


   Acknowledgments
 
Grant support: U.S. Army Breast Cancer Program grant DAMD17-01-10660 (C.L. Arteaga), R01 grants NS45888 (P.C. Lin) and CA80195 (C.L. Arteaga), Breast Cancer Specialized Program of Research Excellence grant P50 CA98131, and Vanderbilt-Ingram Comprehensive Cancer Center Support grant P30 CA68485.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 8/10/04. Revised 1/ 7/05. Accepted 1/14/05.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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