Etk, the 70-kDa member of the Tec family of nonreceptor protein tyrosine kinases, is expressed in a variety of hematopoietic, epithelial, and endothelial cells and was shown to be involved in several cellular processes, including proliferation, differentiation, and motility. In this study, we describe a novel approach using a human single-domain antibody phage display library for the generation of intrabodies directed against Etk. These single-domain antibodies bind specifically to recombinant Etk and efficiently block its kinase activity. When expressed in transformed cells, these antibodies associated tightly with Etk, leading to significant blockade of Etk enzymatic activity and inhibition of clonogenic cell growth in soft agar. Our results indicate that Etk may play a role in Src-induced cellular transformation and thus may represent a good target for cancer intervention. Furthermore, our single-domain antibody-based intrabody system proves to be an excellent tool for future intracellular targeting of other signaling molecules.

Advances in understanding the molecular basis for diseases sets the stage for a wider perception of potential targets that can be used for therapeutic intervention. For cancer in particular, therapy seeks to functionally modify those specific differences between host and tumor that confer on a cancer cell the ability to divide, metastasize, induce new blood vessels formation, and avert cell death. Toward this, various therapeutic approaches have been developed. Since the dawn of cancer therapy, a major concern was the ability to deliver curative agents specifically to tumor cells. Antibodies have become attractive therapeutic means due to their specificity and high affinity toward tumor antigens. Traditionally, antibodies function outside of the cell via several mechanisms of action, including blocking growth factor/receptor interaction, inducing apoptotic processes and mediating immune effector activity. On the other hand, many oncogenic proteins (for instance, fusion proteins resulting from chromosomal translocations and overexpression of nonreceptor protein tyrosine kinases, small GTPases, etc.) are intracellular and therefore may not be amenable to conventional antibody-based therapy. In recent years, the ability to express antibody fragments inside of mammalian cells has led to a gene therapy strategy in which the intracellularly expressed antibodies, commonly known as “intrabodies,” bind to and neutralize or modify an oncogenic protein's function, thereby affecting the malignant phenotype (14). The successful use of intrabodies, to a growing list of cancer-associated targets, in preclinical and clinical studies is providing proof of the value and promise of this new approach in the treatment of cancer as well as other diseases (17).

Etk, the endothelial and epithelial tyrosine kinase, is a member of the Tec family of nonreceptor tyrosine kinases, which also includes Btk, Itk, and Tec (8, 9). These kinases share a high degree of homology and typically contain a NH2-terminal pleckstrin homology domain, a Tec homology domain, SH3 and SH2 domains, and a COOH-terminal kinase catalytic domain (10). The expression of Tec family kinases has been primarily identified in hematopoietic cells (1114). Etk, on the other hand, possesses a much broader expression profile than its counterparts. It was initially identified in bone marrow and subsequently in epithelial cells, fibroblasts, and endothelial cells (1518). Little is known about the biological function of Etk and the signaling pathways in which Etk is involved. It was shown to be involved in various cellular processes, including proliferation, differentiation, adhesion, motility, and survival (10, 1822). The expression and activity of Etk is induced by growth factors, cytokines, G-protein-coupled receptors, the extracellular matrix, antigen receptors, and, possibly, hormones (10, 23, 24), suggesting that its activity may be regulated by physiologic needs dictated by these stimuli. Elevated expression of Etk was reported in several aggressive metastatic carcinoma cell lines (19, 21, 23, 25, 26); however, the role of Etk in such malignancies is yet to be define.

It is well established that Src is a potent oncogene that mediates cellular transformation by engaging several signaling pathways (27, 28). Growing body of evidence suggests that Src activates Etk in vivo through phosphorylation on Tyr566 (18, 25, 29). The direct mechanism by which Etk is involved in Src signaling and Src-induced cellular transformation is, however, yet to be experimentally investigated. In this study, we isolated several antibody fragments that not only bind specifically to the Etk kinase domain but also directly inhibit the enzymatic activity of Etk. When expressed intracellularly in Src-transformed cells, these antibodies interacted with endogenous Etk in the cytoplasm and efficiently blocked its autophosphorylation and activity in phosphorylating substrate, leading to partial inhibition of cellular transformation. Our results support previous data indicating that Etk is directly involved in Src-induced cell transformation process. Further, intrabody technology may provide a useful and specific tool in studying and elucidating the function of various oncogene products in cancer cells.

Library, Cells, and Reagents

Human domain antibody phage display library was obtained from Domantis (Cambridge, United Kingdom). NSR cells (mouse NIH3T3 cells overexpressing v-Src) were a gift from Dr. J.E. Darnell (Rockefeller University, New York, NY). ATP, protease inhibitor cocktail, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, glutathione beads, tetracycline, trypsin, and polyEY were purchased from Sigma Chemical Co. (St. Louis, MO). Isopropyl-l-thio-β-d-galactopyranoside, monoclonal anti-Myc-horseradish peroxidase conjugate, low-melting agar, geneticin (G418), NuPage polyacrylamide gel and transfer system, LipofectAMINE 2000, and Opti-MEM were from Invitrogen (Carlsbad, CA). Anti-M13-horseradish peroxidase antibodies and [γ-33P]ATP were from Amersham (Piscataway, NJ). Monoclonal pY20 antibodies were obtained from Oncogene-EMD Biosciences (San Diego, CA). Polyclonal Etk antibodies were from Cell Signaling (Beverly, MA). Glutathione S-transferase (GST) microbeads and μMacs column were from Miltenyi Biotec (Auburn, CA). AffinityPak immobilized protein L and protein A were purchased from Pierce (Rockford, IL). TMB peroxidase substrate was from KPL (Gaithersburg, MD).

Generation of GST-Etk Kinase Domain

The gene fragment encoding Etk kinase domain (amino acids 417–697) was amplified and subcloned into pBac vector (PharMingen, San Diego). Plasmid was used to infect SF21 cells using the baculovirus system, and a soluble fusion protein, GST-Etk kinase domain, was expressed (30). The kinase was purified using glutathione beads column (31), and enzymatic activity of recombinant protein was confirmed by in vitro kinase assay (see details below). GST peptide was generated using the same procedure.

Selection of Anti-Etk Domain Antibodies from Phage Display Library

A large domain phage display library, derived from a single human framework of light chain, containing 1.7 × 1010 clones was used for the selection. Library stock containing 1011 phage units was resuspended in 1 mL PBS containing 3% fat-free milk and mixed with 5 μg GST for 1 hour at 37°C to capture phage displaying anti-GST antibodies and to block other nonspecific binding and followed by incubation with 100 μL anti-GST magnetic beads for additional 30 minutes at room temperature. The mixture was loaded on a μMacs column and the flow-through was collected. Phage preparation, derived from flow-through, was then mixed with 5 μg GST-Etk for 1 hour at room temperature followed by incubation with 100 μL anti-GST magnetic beads for additional 30 minutes at room temperature. The beads-GST-Etk-phage mix was loaded on a μMacs column followed by 5× washes with 1 mL PBS-0.1% Tween 20 and 5× with 1 mL PBS and elution of the bound phages by 500 μL freshly prepared solution of 1 mg/mL trypsin. The eluted phage was incubated with 4 mL mid-log-phase Escherichia coli TG1 cells for 30 minutes at 37°C. TG1 cells were spun down, resuspended, and plated onto several 90-mm TYE plates containing 15 μg/mL tetracycline and incubated overnight at 30°C. All the colonies grown on the plates were scraped into 2 mL 2YT medium, mixed with glycerol to a final concentration of 15%, aliquoted, and stored at −70°C. For further selection rounds, phage was amplified as described previously (32) and 1011 phage units were used for selection using the procedure described above, with decreasing amount of GST-Etk (2 and 1 μg for the second and third rounds, respectively) along with more extensive washes (10–15 times) of the GST-Etk-phage–loaded μMacs columns.

Phage-Binding Assay

Individual E. coli TG1 clones were picked and grown overnight at 37°C in 2TY medium, supplemented with 15 μg/mL tetracycline, in a 96-well plate format. Bacteria culture was spun down, and supernatant containing phage was mixed with fat-free milk to a final concentration of 3% and incubated 1 hour at room temperature. Phage was transferred to Maxi-sorp 96-well microtiter plates (Nunc, Roskilde, Denmark) coated with 100 μL/well of 1 μg/mL GST or GST-Etk and incubated for 1 hour at room temperature. The plates were washed thrice with 1 mL PBS-0.1% Tween 20 and incubated with M13-horseradish peroxidase antibodies for additional 1 hour at room temperature. The plate was washed 5 times, TMB peroxidase substrate was added, and the absorbance at 450 nm was measured using a microplate reader (Molecular Devices, Sunnyvale, CA).

Cloning, Expression, and Purification of Soluble Domain Antibody

DNA coding for domain antibodies were amplified and subcloned into the SalI and NotI sites of an expression vector, pDOM5 (Domantis). E. coli BL21 cells transformed with individual expression vector were grown at 37°C in 100 mL Luria-Bertani medium supplemented with 100 μg/mL ampicillin. When A600 nm reached ∼0.5, isopropyl-l-thio-β-d-galactopyranoside was added to a final concentration of 1 mmol/L, and incubation was continued overnight at 30°C. Cells were spun down and the pellet was resuspended in 2 mL binding buffer composed of 100 mmol/L Na2HPO4 and 150 mmol/L NaCl (pH 7.2) supplemented with 1:100 ratio of protease inhibitor cocktail. The cell suspension was sonicated and centrifuged, and the supernatant was collected and incubated with 100 μL protein L beads. The beads were washed with 20 mL binding buffer followed by the elution of the single-domain antibodies using 450 μL of 0.1 mol/L sodium citrate (pH 3.1). The eluant was immediately mixed with 50 μL neutralizing buffer containing 1 mol/L Tris-HCl (pH 8.0).

Quantitative Etk-Binding Assay

Various concentrations of purified domain antibodies were added into a Maxi-sorp 96-well microtiter plate coated with 100 μL/well of 1 μg/mL GST-Etk and incubated for 1 hour at room temperature. The plate was washed thrice with 1 mL PBS-0.1% Tween 20 and then incubated with 100 μL/well of anti-Myc-horseradish peroxidase conjugate (1:5,000 dilution) for 1 hour at room temperature. The plate was washed 5 times, TMB peroxidase substrate was added, and the absorbance at 450 nm was measured.

Construction and Expression of Intrabodies

DNA coding for domain antibodies were amplified and subcloned into the HindIII and EcoRI sites of pcDNA3.1 vector (Invitrogen). NSR cells were transfected with one of the six constructs coding for different intrabodies using the LipofectAMINE 2000 method (33). The cells were transferred into a medium containing 10% FCS and supplemented with 1 μg/mL geneticin (G418) at 24 hours after transfection. Survivor colonies were pooled and gradually scaled up.

Western Immunoblotting and Immunoprecipitation

Cells from a 150-mm dish were harvested in 1 mL lysis buffer containing 25 mmol/L Tris-HCl (pH 7.4), 2 mmol/L sodium orthovanadate, 0.5 mmol/L EDTA, 10 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 25 mmol/L NaCl, and 1% Triton X-100 supplemented with 1:1,000 dilution of protease inhibitor cocktail. Cells were frozen and thawed twice and centrifuged, and supernatant was collected. Samples of protein extracts (50 μg) were resolved by SDS-PAGE under reducing conditions and transferred onto nitrocellulose membrane for Western immunoblotting with the indicated antibodies. For immunoprecipitation, protein extracts (0.5 mg) were incubated with either protein L beads or anti-Etk antibodies coupled to protein A bead overnight at 4°C. The immunocomplex was washed 4 times with 1 mL PBS-0.1% Tween 20 and twice with 50 mmol/L HEPES (pH 7.5), electrophoresed on an SDS-polyacrylamide gel under reducing conditions, and transferred onto nitrocellulose membrane for Western immunoblotting with the indicated antibodies. In addition, the immunocomplex was also used as the source for enzyme (Etk kinase) in the in vitro kinase activity and autophosphorylation assays described below.

In vitro Kinase Assay

Various concentrations of purified domain antibodies were added into a Maxi-sorp 96-well microtiter plate coated with 100 μL/well of 50 μg/mL polyEY, a universal tyrosine kinase substrate. Aliquots of GST-Etk (30 ng) were added to each well to a final volume of 75 μL/well. After incubation for 1 hour at room temperature, 4× phosphorylation buffer (25 μL) containing 200 mmol/L HEPES (pH 7.5), 20 mmol/L MgCl2, 20 mmol/L MnCl2, 2 mmol/L DTT, and 8 μmol/L ATP was added to each well for 15-minute incubation at room temperature. Plate was washed thrice with 1 mL PBS-0.1% Tween 20 and then incubated with 100 μL/well of 1:250 dilution of pY-horseradish peroxidase antibodies for 1 hour at room temperature. The plate was washed 5 times, TMB peroxidase substrate was added, and the absorbance at 450 nm was measured. In another assay, Etk/intrabody immunocomplex derived from NSR cells expressing the intrabody (obtained by immunoprecipitation; see above) was incubated with the polyEY, and the amount of substrate phosphorylation was determined.

In vitro Autophosphorylation Assay

Etk/intrabody immunocomplexes derived from NSR cells, expressing the intrabody, was used as the source of enzyme (Etk). The immunocomplex, in a final volume of 30 μL, was mixed with 10 μL of 4× phosphorylation buffer containing 200 mmol/L HEPES (pH 7.5), 20 mmol/L MgCl2, 20 mmol/L MnCl2, 2 mmol/L DTT, and 1 μmol/L [γ-33P]ATP. Reaction was allowed to proceed for 15 minutes at room temperature and was terminated by adding 10 μL of 4× SDS-PAGE sample buffer. Samples were resolved by SDS-PAGE under reducing conditions and subjected to autoradiography.

Colony Formation in Soft Agar

Soft-agar assay was done as described previously (34). Briefly, lower layer (in 35-mm diameter dishes) of 0.7% low-melting agar solution in DMEM supplemented with 10% FCS was covered by a second layer of 0.35% agar solution in DMEM in which 105 cells were resuspended. Following solidification, growth medium (DMEM supplemented with 10% FCS) was added to the dishes. The dishes were incubated at 37°C for 10 to 14 days, with growth medium changes every 3 days. Colonies were stained with 1 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in PBS and scored using AlphaEaseFC program.

Selection of Human Domain Antibodies to Etk

A large phage display library (∼1.7 × 1010 diversity), derived from a single human framework of a κ light chain variable region, was used to select for high-affinity, neutralizing domain antibodies against Etk kinase. The library was generated by using side-chain diversification incorporated at positions in the antigen-binding regions, known to be highly diverse in the mature antibody repertoire, with complete randomization at 13 residues. In displaying the domain antibodies on filamentous phage surface, the PCR-amplified variable light chain domain gene was preceded by a signal sequence of GAS leader at the 5′ end. An 11–amino acid c-Myc tag was inserted between the COOH-terminal of the light chain variable region and gene III for purification and detection purposes.

A total of three rounds of selection were done using recombinant GST-Etk, with alternating protein concentrations and number of washes after the antigen/antibody-binding event. Following the third selection round, 540 clones were randomly picked and tested by phage ELISA for binding to both GST and GST-Etk. Over 90% (491 of 540) of the tested clones were positive and specific for Etk binding, suggesting a high efficiency of the selection process. DNA segments encoding the single-domain antibodies, derived from third selection, were then pooled, amplified, and subcloned into pDOM5 vector for the expression of soluble proteins. Following transformation, individual E. coli BL21 clones were grown in 96-well plates and induced for expression of domain antibodies with isopropyl-l-thio-β-d-galactopyranoside. Of 180 randomly picked colonies, 64 (35%) were positive for Etk binding as determined by a soluble antibody ELISA. Thirty-four binders (Fig. 1A) were further assayed for their capability in inhibiting Etk kinase using polyEY as the substrate; 13 showed moderate to strong enzyme-blocking activity (Fig. 1B). Sequence analysis of the 20 best binders revealed 20 different patterns, indicating an excellent diversity of the isolated anti-Etk domain antibodies.

Figure 1.

Selection of human single-domain antibodies to Etk. Thirty-four individual single-domain antibodies were expressed in E. coli BL21 cells. A, crude extracts containing the antibody were subjected to qualitative, ELISA-based binding assay with immobilized recombinant Etk. B, crude extracts containing the antibody were subjected to qualitative, ELISA-based in vitro Etk kinase inhibition assay. Columns, mean of two independent experiments; bars, SD.

Figure 1.

Selection of human single-domain antibodies to Etk. Thirty-four individual single-domain antibodies were expressed in E. coli BL21 cells. A, crude extracts containing the antibody were subjected to qualitative, ELISA-based binding assay with immobilized recombinant Etk. B, crude extracts containing the antibody were subjected to qualitative, ELISA-based in vitro Etk kinase inhibition assay. Columns, mean of two independent experiments; bars, SD.

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Quantitative Etk Binding and Kinase Blocking by Purified Soluble Domain Antibodies

To further study the binding and blocking characteristics of the domain antibodies toward Etk, six clones (L5, L7, L9, L11, L12, and L15) were grown in a large-scale culture and induced using isopropyl-l-thio-β-d-galactopyranoside to express soluble domain antibodies. The domain antibodies were purified from bacterial extract using protein L affinity chromatography. The yield of purified antibodies ranged from 50 to 500 μg per 100-mL culture. SDS-PAGE analysis of each purified antibody preparation showed a single protein band corresponding to the expected molecular size of 15 kDa (Fig. 2A).

Figure 2.

Expression and characterization of the soluble single-domain antibody. Soluble single-domain (S.D.) antibodies were expressed in E. coli BL21 cells and purified using protein L affinity chromatography. A, purified single-domain antibody preparations were resolved by SDS-PAGE and gels were stained with coomassie blue. B, indicated concentrations of purified single-domain antibodies were subjected to quantitative, ELISA-based binding assay with immobilized recombinant Etk. C, indicated concentrations of purified domain antibodies were subjected to quantitative, ELISA-based in vitro Etk kinase inhibition assay. Points, mean of two independent experiments done in duplicates; bars, SD.

Figure 2.

Expression and characterization of the soluble single-domain antibody. Soluble single-domain (S.D.) antibodies were expressed in E. coli BL21 cells and purified using protein L affinity chromatography. A, purified single-domain antibody preparations were resolved by SDS-PAGE and gels were stained with coomassie blue. B, indicated concentrations of purified single-domain antibodies were subjected to quantitative, ELISA-based binding assay with immobilized recombinant Etk. C, indicated concentrations of purified domain antibodies were subjected to quantitative, ELISA-based in vitro Etk kinase inhibition assay. Points, mean of two independent experiments done in duplicates; bars, SD.

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To measure the binding of the domain antibodies to immobilized Etk, various concentrations of the purified proteins were allowed to bind to 96-well plates coated with GST-Etk. As shown in Fig. 2B, all six domain antibodies bound Etk in a dose-dependent manner, with L9 as the best binder followed by L11, L5, L7, L12, and L15. The antibody concentration required for 50% of maximum binding to Etk was 5 and 8 nmol/L for L9 and L11, respectively, and ∼20 nmol/L for L5, L7, L12, and L15. The control, nonrelevant domain antibodies, IRK3 and ERK3, did not bind to Etk even at high doses (Fig. 2B). Consistent with the binding data, all six domain antibodies blocked Etk enzymatic activity dose-dependently as indicated by the reduction in the ability of recombinant Etk to phosphorylate the substrate, polyEY, in the presence of the domain antibodies (Fig. 2C). The domain antibody concentration required to achieve 50% inhibition of Etk kinase activity was ∼20 nmol/L for L5, L7, L9, and L12, 40 nmol/L for L11, and ∼3 μmol/L for L15 (Fig. 2C). As expected, both control domain antibodies failed to show any kinase-blocking activity in this assay. In addition, 1 μmol/L of all six purified domain antibodies failed to bind or block the kinase activity of epidermal growth factor receptor, KDR, fibroblast growth factor receptor 1, platelet-derived growth factor receptor β, Fyn, Fms, c-Met, insulin receptor, and, most noteworthy, Src as well as Btk and Itk, two Tec family members (data not shown). These findings suggest that these neutralizing anti-Etk domain antibodies possess high specificity and good kinase-blocking activity and therefore may have a potential to serve as intracellular antibodies to block endogenous Etk enzymatic activity and interfere with Etk-related signaling cascades.

Expression of Anti-Etk Domain Antibody as Intrabodies

To study the effect of the intracellularly expressed anti-Etk intrabodies on endogenous kinase activity, we proceeded to introduce the domain antibodies into transformed cells. The DNA segments encoding the domain antibodies were subcloned into pcDNA3.1 vector, fused to a c-Myc tag, and transfected into NSR cells, a NIH3T3 cell line overexpressing v-Src (34). Due to low transfection yield common to NIH3T3 cells (10%), transfected cells were selected using geneticin (G418). Survivor colonies were pooled, gradually up scaled during a period of 4 weeks, and used for various assays. In total, six domain antibodies were expressed as intrabodies in NSR cells. Clones were named K5, K7, K9, K11, K12, and K15 accordingly.

The cells were first assayed for intrabody expression via Western blotting. As shown in Fig. 3A, five of six clones, K5, K7, K9, K11, and K15, highly expressed intrabodies as shown by a single protein band corresponding to the expected molecular size of 12 kDa when detected by an anti-c-Myc antibody. In contrast, cells transfected with K12 failed to produce any detectable intrabody (Fig. 3A). Only four of the five expressed intrabodies (K5, K7, K9, and K11) were capable of interacting with endogenous Etk as shown by immunoprecipitation of the cell lysate with immobilized protein L, which is proficient in binding the κ light chain domain antibodies, followed by blotting with an anti-Etk antibody (Fig. 3B). This was further confirmed when the cell lysate was immunoprecipitated with an anti-Etk antibody followed by blotting with an anti-c-Myc antibodies (Fig. 3C). Although K15 intrabody expressed well in NSR cells, it failed to interact with endogenous Etk (Fig. 3). These results indicate that the intracellularly expressed K5, K7, K9, and K11 intrabodies fold appropriately, thus allowing them to interact with their target in the cytoplasm compartment.

Figure 3.

Expression of intrabodies and their interaction with Etk in the cytoplasm of transfected NSR cells. NSR cells were transfected with pcDNA3.1-L5, pcDNA3.1-L7, pcDNA3.1-L9, pcDNA3.1-L11, pcDNA3.1-L12, and L15, and clones were selected and named K5, K7, K9, K11, K12, and K15 accordingly. A, total cell extracts were prepared from parental and transfected cells and samples of protein extracts (50 μg) were resolved on SDS-PAGE and immunoblotted with an anti-Etk or an anti-c-Myc antibody. B, samples of protein extracts (500 μg) were subjected to immunoprecipitation using immobilized protein L. Immunocomplexes were resolved by SDS-PAGE and immunoblotted with an anti-Etk or an anti-c-Myc antibody. C, samples of protein extracts (500 μg) were subjected to immunoprecipitation using an immobilized anti Etk antibody. Immunocomplexes were resolved by SDS-PAGE and immunoblotted with an anti-Etk or an anti-c-Myc antibody. Representative of three independent experiments.

Figure 3.

Expression of intrabodies and their interaction with Etk in the cytoplasm of transfected NSR cells. NSR cells were transfected with pcDNA3.1-L5, pcDNA3.1-L7, pcDNA3.1-L9, pcDNA3.1-L11, pcDNA3.1-L12, and L15, and clones were selected and named K5, K7, K9, K11, K12, and K15 accordingly. A, total cell extracts were prepared from parental and transfected cells and samples of protein extracts (50 μg) were resolved on SDS-PAGE and immunoblotted with an anti-Etk or an anti-c-Myc antibody. B, samples of protein extracts (500 μg) were subjected to immunoprecipitation using immobilized protein L. Immunocomplexes were resolved by SDS-PAGE and immunoblotted with an anti-Etk or an anti-c-Myc antibody. C, samples of protein extracts (500 μg) were subjected to immunoprecipitation using an immobilized anti Etk antibody. Immunocomplexes were resolved by SDS-PAGE and immunoblotted with an anti-Etk or an anti-c-Myc antibody. Representative of three independent experiments.

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Inhibition of Etk Kinase Activity by Intrabodies

We then evaluated the effect of the intrabodies on the intrinsic enzymatic activity of endogenous Etk. Etk from both the intrabody-transfected cells and the parental NSR cells were immunoprecipitated using an anti-Etk antibody and subjected to an in vitro autophosphorylation assay using [γ-33P]ATP. As shown in Fig. 4A, Etk derived from clones K5, K7, K9, and K11 showed a significantly lower rate of tyrosine autophosphorylation, retaining ∼49%, 37%, 38%, and 30% of the wild-type enzyme (from NSR cells) activity, respectively. Consistent with our expression and binding data in Fig. 3, Etk derived from clones K12 (the clone without expression) and K15 (the clone showing no Etk interaction) showed full kinase activity to a level that is comparable with that of Etk derived from NSR cells (Fig. 4A). Similar outcome was observed when the endogenous Etk was used as the source of kinase for a substrate polyEY in a quantitative in vitro kinase assay (Fig. 4B). The enzymatic activity of Etk derived from clones K5, K7, K9, and K11 was reduced to ∼25% of that observed with Etk derived from NSR cells. In contrast, Etk derived from clones K12 and K15 showed similar levels of enzymatic activity to that derived from NSR cells (Fig. 4B).

Figure 4.

In vitro kinase assay of the endogenous Etk purified from the parent and the intrabody-transfected NSR cells. Total cell extracts were prepared from parental and transfected cells and samples of proteins (500 μg) were subjected to immunoprecipitation using an immobilized Etk antibody. A, immunocomplexes were subjected to in vitro autophosphorylation assay and resolved by SDS-PAGE under reducing conditions and followed by autoradiography. The bands corresponding to [γ-33P]ATP-bound Etk were quantitated by densitometry. Columns, mean of two independent experiments; bars, SD. B, immunocomplexes were subjected to ELISA-based in vitro kinase assay as described in the text. Columns, mean of two independent experiments done in duplicates; bars, SD.

Figure 4.

In vitro kinase assay of the endogenous Etk purified from the parent and the intrabody-transfected NSR cells. Total cell extracts were prepared from parental and transfected cells and samples of proteins (500 μg) were subjected to immunoprecipitation using an immobilized Etk antibody. A, immunocomplexes were subjected to in vitro autophosphorylation assay and resolved by SDS-PAGE under reducing conditions and followed by autoradiography. The bands corresponding to [γ-33P]ATP-bound Etk were quantitated by densitometry. Columns, mean of two independent experiments; bars, SD. B, immunocomplexes were subjected to ELISA-based in vitro kinase assay as described in the text. Columns, mean of two independent experiments done in duplicates; bars, SD.

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Partial Inhibition of Cellular Transformation by Anti-Etk Intrabodies

It was shown previously that Etk is phosphorylated by Src on residue 566 (18) and that this phosphorylation activates Etk intrinsic kinase domain and enables it to phosphorylate downstream effectors, such as signal transducers and activators of transcription 3 (STAT3), to further propagate pathways involved in Src-induced cellular transformation (18). Therefore, the effect of the anti-Etk intrabodies on STAT3 phosphorylation was assessed. No alteration in the phosphorylation content of STAT3 was observed as measured by Western immunoblotting with phosphospecific STAT3 antibody directed against Tyr705 (data not shown).

When seeded and grown as an adherent culture, the overall growth rate of the domain antibody-transfected clones was comparable with that of the parental NSR cells. Next, the effect of the anti-Etk intrabodies on Src-induced cellular transformation was studied. Toward this end, parental NSR cells as well as the intrabody-transfected cells were plated and grown in a soft-agar medium for 14 days (Fig. 5). Although colonies of ≥50 cells were observed with both parental NSR cells and cells expressing the intrabodies, the parental cells showed higher colony formation efficiency with 387 ± 25 colonies per plate. Cells transfected with K5, K7, K9, and K11 showed colony formation rate of 302 ± 19 (P = 0.001), 245 ± 6 (P = 0.0002), 262 ± 43 (P = 0.0069), and 234 ± 18 (P = 0.0016), respectively. These numbers are significantly lower than that observed with NSR cells and represent a 22%, 37%, 31%, and 40% reduction in colony formation for clones K5, K7, K9, and K11, respectively. As expected, both clones K12 and K15 showed a similar growth and colony formation rate to NSR cells, with 354 ± 24 and 371 ± 26 colonies per plate, respectively (Fig. 5).

Figure 5.

Colony formation on soft agar by the parent and the intrabody-transfected NSR cells. The parental and the transfected NSR cells were plated (105 per plate) in soft-agar medium and grown for 10 to 14 d. Colonies were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and counted using the AlphaEaseFC program. Columns, mean of three independent experiments done in triplicates; bars, SD.

Figure 5.

Colony formation on soft agar by the parent and the intrabody-transfected NSR cells. The parental and the transfected NSR cells were plated (105 per plate) in soft-agar medium and grown for 10 to 14 d. Colonies were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and counted using the AlphaEaseFC program. Columns, mean of three independent experiments done in triplicates; bars, SD.

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Here, we describe a novel approach, using a human single-domain antibody phage display library, for the generation of intrabodies directed against the nonreceptor protein tyrosine kinase, Etk. These single-domain antibodies bind specifically to recombinant Etk and efficiently block its kinase activity. When expressed in transformed cells, these antibodies associated tightly with Etk in the cytoplasm compartment leading to significant blockade of Etk enzymatic activity and partial inhibition of clonogenic cell growth in a soft-agar assay. Our results support the notion that Etk plays a role in Src-induced cellular transformation. Further, our single-domain antibody-based intrabody system should prove to be an excellent tool for future intracellular targeting of other signaling molecules involved in a wide spectrum of diseases, cancer in particular.

Several observations in this study are noteworthy. First, we showed that a single variable domain of an antibody, the smallest immunoglobulin-based recognition unit (35), could be highly efficacious as a source of intrabodies. Despite some early promising observations both in vitro and in several disease models (6, 7, 36, 37), the main obstacle in the use of intrabodies remains those problems associated with their instability and tendency to aggregate when expressed inside the cell. These problems are a result of the reducing environment intrabodies are exposed to in the cytoplasm of the cell, which prevents the formation of disulfide bonds, leading to protein insolubility, instability, or incorrect folding (1, 38). The formation of disulfide bonds is usually critical for the structural integrity and function of the conventional antibody fragments, especially the single-chain Fv (scFv), the most used antibody format in intrabody technology. Several approaches have been exploited to enhance the production of stable and functional intrabodies in the cellular environment with various success [e.g., generation of cysteine-free scFv fragments (39), grafting of antigen-binding regions onto the scaffold of a reducing environment-resistant scFv (40), engineering highly stable variable domains for intrabody construction (41, 42), and direct selection of intrabodies in the intracellular environment (3941, 4346)]. Compared with scFv, the single-domain antibodies not only process all the essential features of the antigen-binding site but also show excellent intracellular stability profile—they do not need any disulfide bonds for proper folding and function (35). In this study, five of six single-domain antibodies showed good levels of expression inside the cells and four of five expressed antibodies associated tightly with their target, Etk, and efficiently inhibited the enzymatic activity of the kinase (Figs. 3 and 4). These results clearly support the notion that single-domain antibody may represent a novel and perhaps superior class of antibody fragments to the traditional scFv format for intrabody application.

The second noteworthy observation in this report is that we generated several intrabodies that target directly an intracellular kinase and block its enzymatic activity. The intrabodies have been proven very effective in inhibiting the function of culprit proteins both in vitro and in animal models of a wide spectrum of diseases, for example, in HIV infection (6, 47, 48), in neurodegenerative diseases, such as Alzheimer's (46), in diseases caused by mutated proteins, such as Huntington's (5, 4648), and others. In the field of oncology, intrabodies have been used to modulate the expression of proteins up-regulated in tumors, such as ErbB2 and Cyclin E in breast and ovarian cancer (1, 2, 4, 49), interleukin-2 receptor in some sorts of leukemia (3), and epidermal growth factor receptor in glioblastoma and epithelial cancers (50). In most of these cases, intrabodies were used as means to alter the intracellular trafficking and localization of their targets, for example, by trapping oncogene products (e.g., epidermal growth factor receptor and ErbB2) that were destined for cell surface expression in the endoplasmic reticulum or isolating transcription factors (e.g., Cyclin E) in the cytoplasm. Alternatively, intrabodies have been used to block protein-protein or protein-nucleic acid interactions (51), to directly promote cell death by inducing apoptosis-related proteolysis cascade (5254), or, albeit indirectly, to facilitate selective degradation of target proteins by recruitment to the ubiquitin-proteasome pathway (55). In this study, by targeting the enzymatic activity of an intracellular kinase (i.e., Etk) with an intrabody, we showed that we could modulate the role of a kinase plays in cell transformation in an extremely specific manner. Our study with Etk thus provides a model system and should be readily applicable to other intracellular kinases.

Etk, a member of the Tec family of nonreceptor protein tyrosine kinases, represents a wide spectrum of intracellular signaling molecules involved in various cell transformation and tumorigenesis processes. It has been reported previously that activation of Etk enhances the proliferation, anchorage-independent growth, and tumorigenicity of human breast cancer cells (23). High expression levels of Etk were observed in several aggressive malignancy-derived cell lines (19, 21, 23, 25, 26) and were correlated with stimulation of cell proliferation and protection against apoptosis (23, 26). The precise mechanism by which Etk exerts its biological effects on cancer cells remains, however, elusive. Thus, the third noteworthy observation in this report is that we describe, for the first time, an attempt to inhibit Etk kinase by specific intrabodies and to study the consequence of Etk blockade in transformed cells. We showed that the single-domain antibodies directed against the kinase domain of Etk were capable of binding to recombinant Etk and blocking its enzymatic activity in vitro (Fig. 2). When expressed in v-Src-expressing NIH3T3 cells (NSR cells) as intrabodies, these domain antibodies were capable of interacting with endogenous Etk (Fig. 3) and inhibiting its intrinsic kinase activity (Fig. 4). Additionally, although the intrabodies had no significant effect on the overall growth rate of the above v-Src-transfected cells, their expression resulted in a lower transformation potential as shown in the soft-agar colony formation assay (Fig. 5). These findings, taken together with those reported previously (18, 23, 26), suggest that Etk plays a role in governing cell proliferation and survival. The fact that Etk inhibition by intrabodies only led to 22% to 40% inhibition of colony formation of NSR cells indicates that inhibition of Etk alone is not sufficient to abolish Src-induced transformation. It may be, at least partially, due to the oncogenic potential of v-Src is greater than that of c-Src.

In conclusion, we here described a novel approach for generation of anti-Etk intrabodies using the single-domain antibodies as the functional entity. These intrabodies were active both in vitro and in cell cytoplasm in blocking Etk enzymatic activity. Using these specific anti-Etk intrabodies, we showed that Etk functions in the process of Src-induced cellular transformation. Intrabody, similar to knockout methods, such as RNA interference and antisense oligonucleotides, represents an excellent tool in target validation in functional genomics.

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.

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