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,1,¶,1,2,¶
,
,¶
* Department of Oncology, Biology, and Genetics,
Center of Excellence for Biomedical Research, and
Department of Experimental Medicine, University of Genoa, Genoa, Italy;
Laboratory of Experimental Oncology and
|| Laboratory of Immunology, National Cancer Institute (IST), Genoa, Italy;
¶ Istituto Superiore di Oncologia (ISO), Genoa, Italy;
# Institute Giannina Gaslini, Genoa, Italy;
** Institute of Pharmaceutical Science, Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, Zurich, Switzerland; and
Laboratory of Innovative Therapies, Advanced Biotechnology Centre, Genoa, Italy
2Correspondence: Department of Oncology, Biology and Genetics, University of Genoa, L. go R. Benzi 10, Genoa 16132, Italy. E-mail: luca.bagnasco{at}unige.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10–8 M. Internalization of a fluoresceinated Fl-G11-scFv-Int(+) construct was observed in intact human cultured cells with confocal microscopy. After 5 h of incubation in medium containing 1 µM Fl-G11-scFv-Int(+) or Fl-G11-scFv-Int(–), fluorescence intensity was determined in individual cells, both for cytoplasmic and nuclear compartments: concentration levels of Fl-G11-scFv-Int(+), relative to the extracellular culture medium concentration, were 4–5 times higher in the cytoplasm, 7–8 times higher in the nucleus, and 10 times higher in the nucleoli. In the same experimental conditions, the Fl-G11-scFv-Int(–) construct was 3–4 times more concentrated outside of the cells than inside. Cell membranes kept their integrity after 5 h of incubation. The antiproliferative activity of our miniantibody was studied on HCT116 cells. Incubation with 4 µM G11-scFv-Int(+) for 4 days induced very significant statistical and biological growth inhibition, whereas Int alone was completely inactive. Miniantibodies capable of penetrating cell membranes dramatically broaden the potential for innovative therapeutic agents and attack of new targets.—Avignolo, C., Bagnasco, L., Biasotti, B., Melchiori, A., Tomati, V., Bauer, I., Salis, A., Chiossone, L., Mingari, M. C., Orecchia, P., Carnemolla, B., Neri, D., Zardi, L., Parodi S. Internalization via Antennapedia protein transduction domain of an scFv antibody toward c-Myc protein.
Key Words: intracellular delivery • PTD • miniantibody • fluorescence labeling • oncoprotein targeting
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. Antisense oligonucleotides and more recently small interfering RNAs have also been used (4
, 5)
. In particular, but not exclusively, for intracellular signaling proteins/oncoproteins bearing an ATP pocket, medicinal chemists have been able to finding selective and potent small drug-like molecules (6)
. Short basic peptides, derived mainly from transcription factor motifs, such as Int of Drosophila Antennapedia or TAT of HIV-1, can promote cell internalization of linked peptides, even small proteins, and peptidomimetic molecules (7
, 8)
. Non-natural basic peptides endowed with cell-penetrating properties have also been synthesized (9
, 10)
.
These peptides are collectively called protein transduction domains (PTDs) (11
, 12)
. Peptides, antisense oligonucleotides, and proteins conjugated to PTDs have been noted to internalize effectively, and their biological actions have been detected in several cell and animal models (13)
. This noninvasive approach for intracellular delivery of biologically active macromolecules is potentially a very powerful strategy, because intracellular protein targets can be attacked directly. The high specificity and long active half-life of antibodies and their recombinant fragments make them excellent candidates for selective targeting agents. Single-chain fragment variable (scFv) monoclonal antibodies (mAbs) are capable of adopting a functional three-dimensional conformation joining together a VH and VL domain (14)
. The molecular mass of a standard IgG antibody is 150,000 Da and that of an scFv antibody is 30,000 Da (15)
; therefore, it is potentially much more feasible to internalize a smaller scFv molecule. Single-chain mAb expression within the cell can be effectively obtained using recombinant DNA transfection techniques (16)
; however, low general accessibility of target cells to DNA constructs together with lack of pharmacological modulation of antibody levels poses severe limitations from the perspective of therapeutic applications. Fusing PTDs to scFv antibodies allows us to create a "cell-permeable" antibody, potentially capable of effectively inhibiting the function of intracellular targets. These features make these molecules potentially much more appropriate and very interesting from a therapeutic perspective (17)
. In this work we report the internalization of an Int/scFv antibody construct in living cells. Recent reports have shown that cell fixation, even in mild conditions, leads to the artifactual uptake of peptides (18)
and should be avoided in the study of internalization. We have therefore assessed our results directly on unfixed cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Briefly, a cDNA sequence encoding the portion of interest of the protein was subcloned in a pETM11 modified vector (kind gift of G. Stier, EMBL Heidelberg, Heidelberg, Germany) and transformed in the BL21 strain of E. coli. Bacterial pellets were harvested and lysed; proteins were purified on an Ni-NTA column (Qiagen, Valencia, CA). The pETM11 vector contains a tobacco etch virus (TEV) protease recognition site between the His tag and the recombinant protein start codon. After purification the tag could be cut out using a recombinant His-tagged TEV protease; when needed, a second Ni-NTA column was used to further purify the cleaved protein from its His tag and the protease itself.
Antibody fragment isolation
A phage display library of human antibodies in the scFv format (ETH-2) was used for the selection of recombinant antibodies. We used the NH2-terminal [1–152 amino acids (aa)] of the human c-Myc protein as antigen and performed three rounds of panning for the selection, as described previously (20)
. Ninety ampicillin-resistant independent colonies were screened to identify those producing antigen-binding scFv fragments by ELISA, making use of the c-Myc fragment to coat the immunotiter plates. One of these anti-c-Myc clones (G11-scFv) was chosen, sequenced on both strands, and fused to the Int peptide.
Construction and purification of anti-Myc-Int (G11-Int) fusion protein
With use of the cDNA of G11-scFv as template and the primers LMB3 (21)
(which contains the SfiI restriction enzyme sequence), BC-803 (sequence: gcg ccg att ctg gaa cca aat ctt tat ctg gcg tag gac ggt cag ctt ggt ccc tcc; part of the Int sequence is underscored), and BC-804 (sequence: ctc cgc ggc cgc tta tca ctt ctt cca ctt cat gcg ccg att ctg gaa cca aat ctt, which contains the NotI restriction enzyme sequence; part of the Int sequence is underscored), high-fidelity polymerase chain reactions (PCRs) were performed to obtain the G11-Int insert. The construct was cloned in the Sfi1/NotI sites of a pDN5 expression vector (21)
, expressed in HB2151 E. coli cells and sequenced on both strands. G11-Int fusion protein purification was performed by immunoaffinity using the recombinant c-Myc fragment (1–152 aa) conjugated to Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden) as described previously (20)
.
Labeling reaction
Each antibody sample, suspended in 0.1 M phosphate buffer, pH 7.3, was reacted with fluorescein-5-EX succinimidyl ester (Molecular Probes Inc., Eugene, OR) under mild stirring and dark conditions at 10°C for 1 h (final protein concentration 1 x 10–4 M). Different molar ratios of reacting dye/protein, ranging from 1:1 to 10:1, were used.
To stop the labeling reaction, hydroxylamide hydrochloride (Fluka Chemie AG, Buchs, Switzerland), pH 8.5, was added (final concentration 0.3 M), and the mixture was incubated at 20°C for 1 h. The sample was then dialyzed against 0.1 M phosphate buffer, pH 7.3, using dialysis cassettes (10K MWCO; Pierce Biotechnology, Rockford, IL). The relative efficiency of a labeling reaction was determined by measuring the absorbance of the protein-dye conjugate at 280 nm (A280) and at the 
max for the dye (Amax=491 nm) and correcting for the contribution of the dye to the absorbance at A280. Protein concentration was calculated, assuming 1.4 Aprotein units 
1 mg/ml.
The degree of labeling (DOL) was thus determined:
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dye is the extinction coefficient of the dye at its absorbance maximum, and the protein concentration is in mg/ml (22)
. Fluorescence measurements on serial dilutions of labeled samples were performed with a Polarion fluorescence reader (Tecan Austria GmbH, Grödig, Austria). Linear regression best-fit values for slopes were matched with DOL values obtained by absorption spectroscopy and were thus routinely used as a check for labeling reactions.
Peptide synthesis
All peptides were chemically synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis on a Rink amide AM resin (Advanced Biotechnologies, Ltd., Epsom, Surrey, UK). After deprotection of the Fmoc group with 20% piperidine in dimethylformamide, the resin was treated with a coupling reaction mixture containing 4 Eq of the appropriate Fmoc amino acid (Advanced Biotechnologies, Inc.), 3.8 Eq of o-(7-azabenzotriazol-1-yl)-1,1,3,3 tetramethyluroniumhexafluorophosphate (Advanced Biotechnologies, Inc.), 4 Eq of N,N-di-isopropylethyl amine (Fluka Chemie AG), and 6 Eq of sim-collidine (Fluka) at a 0.2 M amino acid final concentration in anhydrous N-methylpirrolidone (Merck, Darmstadt, Germany). The analysis on the crude powders was performed by HPLC-electrospray ionization-mass spectrometry (MS) using an Agilent 1100 series LC/MSD trap instrument. All synthesized compounds were purified by reverse-phase HPLC on a Shimadzu (Kyoto, Japan) LC-9A preparative high-performance liquid chromatograph equipped with a Waters C18 Bondapack column (19–300 mm). The diode array detector, during analysis, was set at 220 nm and a linear gradient from 100% of water [0.1% trifluoroacetic acid (TFA)] to 100% of acetonitrile (0.1% TFA) was applied. The molecular weights of the products were finally confirmed by electrospray ion-trap MS.
Cell culture and internalization experiments
Internalization of fusion protein constructs or peptides was performed in the HCT-116 human colon cancer cell line, which overexpresses c-Myc, or in MRC-5 human normal lung fibroblasts. MRC-5 cells (catalog no. CCL-171; American Type Culture Collection, Manassas, VA) were grown in 10% FBS Eagle’s minimal essential medium (ATCC) and 10 mM Hepes, pH 7.3, with ampicillin and streptomycin (EuroClone, Pero, Italy). HCT-116 cells were grown in 10% FBS RPMI 1640 (EuroClone). Cells grown on cover slides (5 cm2) were incubated, with treatment, at 37°C in a humidified incubator using a 0.1-mm deep chamber: 50 µl of complete medium containing the fluorescent peptide (10 µM) or protein (1 µM) was added over the intact cells. The cover slide was inverted and placed over a glass slide, and its margins were sealed with a tiny amount of nail polish (19)
. Cells were subjected to confocal microscopy observation without prior fixation.
Quantification of fluorescence in internalized cells
Confocal image acquisitions were performed using an Olympus confocal laser scanning biological microscope (Fluoview 500 software version 4.3). The light source was a 488 argon ion laser with a dichroic mirror and barrier filter combination (excitation DM 351/488, barrier filter BA 505 IF). Gain and offset parameters were set, as for quantification experiments, respectively, to 1 and 0 values. Image size was set to 800 x 600 pixels, with each acquired image having 12 bits of brightness data per pixel. Acquisitions were overlaid with a network of rectangles (n=50–60, average area 2.0 µm2) covering internal cell districts as well as external adjacent regions (see Results,
Fig. 4
). Approximately 10 cells were used for each quantification experiment. Mean fluorescence intensity was obtained as arbitrary units/pixel using the Image Analysis option of Fluoview 500
(Fig. 2)
.
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Fluorescence anisotropy
The formation of antigen-antibody complexes was measured in terms of increased anisotropy using a Polarion fluorescence reader (Tecan Austria GmbH). Excitation wavelength was 485 nm and emission wavelength was 535 nm.
All experiments were performed in 0.1 M phosphate buffer (pH 7.4) and in 96-well black nonbinding plates (Corning Glassworks, Corning, NY); the experimental volume was always 200 µl/well. The reagents were mixed at room temperature and maintained at constant temperature (30°C). Anisotropy measurements were obtained after 60 min of incubation.
Antiproliferative experiments in cell culture
To assess the antiproliferative activity of our anti-c-Myc miniantibody, HCT116 cells (a human colon cancer line) were used. This cell line had been used by our group previously to study the antiproliferative activity of retro-inverso peptidomimetic inhibitors of c-Myc (9)
and also for internalization studies in this work.
We performed two independent duplicate experiments, using slightly different growth conditions. In the first experiment cells were cultured in 60% RPMI 1640 + 40% PBS, supplemented with 10% (v/v) FCS, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37°C and 5% CO2. In a second experiment we used 90% RPMI 1640 + 10% PBS, supplemented with 10% (v/v) FCS, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37°C and 5% CO2. During exponential growth, cells showed a slower growth rate in the first experiment (doubling time
35.4 h) than in the second (doubling time24.0 h), probably as a consequence of a difference in nutrient concentration, that was decidedly lower in the first experiment than in the second.
Cells were seeded at day 0 (30,000 cells in the first experiment and 15,000 cells in the second experiment) and treated with 4 µM G11-scFv-Int(+) or 4 µM Int control peptide at days 2 and 4. Untreated cells received only fresh medium at the same time. Notice that 4 µM G11-scFv-Int(+) is close to the solubility limit in our experimental conditions.
Growth curves of treated and control cells were obtained by means of a hemocytometer count and the trypan blue dye exclusion test. Cells were counted at days 2, 4, and 6.
Doubling time (T) was calculated using the formula:
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A clone we named G11-scFv was chosen, and its cDNA was sequenced on both strands. Sequencing of the V segments of the selected G11-scFv anti-c-Myc clone, identified a VH segment DP47 and a V segment DPL-16, containing a VH-CDR3 sequence LTPHKWFDY and a V-CDR3 sequence NSSAGPRRRVV (23)
.
Figure 1
A shows the chimeric construct, having the cDNA corresponding to the Int peptide appended to the 3' of the cDNA of the G11-scFv clone. Chimeric cDNA was cloned in an E. coli expression vector, and the fusion protein was purified from the bacteria culture medium by an affinity chromatography column (see Material and Methods). Figure 1B
shows SDS-PAGE of the purified protein, which has an apparent molecular mass of 30 kDa.
Labeling of the constructs for internalization purposes
Nonradioactive labeling of polypeptides/proteins commonly requires procedures that use fluorochromes, such as fluorescein or rhodamine succinimidyl esters, capable of reacting with nonprotonated aliphatic amino groups. The difference between pKa values for terminal 
-amino groups (ranging from 6.8 to 8.0) and those for -amino groups of lysine or arginine (>10.4) in proteins (24)
favors labeling at the level of the -amino terminus (22
, 25)
. The aim of the labeling procedure we adopted was to minimize the involvement of these internal basic amino acids in the labeling of the construct. A minimum involvement of internal basic amino acids is especially important when the reaction is quantitatively organized to obtain, on average, slightly less than one bound fluorochrome per polypeptide chain. The results of a typical experiment performed using a scFv anti-lysozyme antibody are reported in Fig. 2
, which shows the dependence of the DOL on the [dye]/[protein] ratio used. Macromolecules having a minimum degree of spurious DOL are more convenient for binding experiments such as those using fluorescence anisotropy techniques. These were performed to ascertain the fact that labeled antibodies maintained their binding ability (data not shown).
Internalization experiments by confocal microscopy
Confocal microscopy techniques were used in internalization experiments, as they offer several advantages over conventional wide-field optical microscopy. Measurements of fluorescence intensity were taken in a central confocal section among those collected starting from the cell adherence plane (Fig. 3
). Acquisitions taken using high-gain values were not considered for quantification of internalization. Initial experiments were performed with fluoresceinated L-Int peptide, the 16-aa internalizing sequence from Antennapedia (FlRQIKIWFQNRRMKWKK), used at 1 or 10 µM concentrations, with BH3 of Bak peptide (FLGQVGRQLAIIGDAINR) as a negative control. Serial confocal sections of HCT-116 cells treated with fluoresceinated L-Int peptide were acquired after 2.5 or 5 h of incubation. The frequency distributions of green fluorescence intensity at 5 h were obtained for different cell districts, and the ratios of fluorescence values for these districts to the external environment were reported. These are shown in Fig. 4
and Table 1
, which summarizes for comparison results obtained with our reference peptides and with miniantibodies. Subsequent experiments with miniantibodies were performed using an anti-Myc-Int(+) scFv antibody and, as a negative control, either anti-lysozyme or anti-Myc antibodies, both lacking the internalization sequence. At 5 h a 3- to 5-fold overall increase of fluorescence values over the external environment was measured in both the HCT-116 human cancer cell line and MRC-5 human normal fibroblasts treated with anti-Myc-Int(+) scFv antibody (Fig. 5
; Table 1
). Results of experiments performed with treatments at 2.5 h are shown in Fig. 5B
. To test whether the high fluorescence content of cells treated with the anti-Myc-Int(+) scFv construct should be ascribed to cell membrane damage, experiments were performed with a mixture of unlabeled anti-Myc-Int(+) and fluoresceinated anti-Myc lacking the internalization sequence (Int) (Fig. 6
A) and, as a negative control, only fluoresceinated anti-Myc lacking the Int sequence (Fig. 6B
). As shown in Fig. 6A, B
, results were very similar.
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Analogous experiments were also performed using, as an alternative miniantibody, fluoresceinated anti-lysozyme lacking the Int sequence. We obtained the same results (data not reported).
Antiproliferative experiments in cell culture
The results obtained in our two independent experiments are reported in Fig. 7
A, B. To convey clearer information on the variability of our results, in both experiments the dots corresponding to each individual result are shown. The two independent dots, corresponding to the same treatment at the same time, are always quite close to one another, suggesting reproducible experimental conditions. In both experiments we never observed a growth crossing-over of the individual dots for G11-scFv-Int(+)-treated cells compared with control or Int-treated cells. This information is also the basis of our nonparametric statistical analysis.
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If only statistical fluctuations were present, considering that cell growth in each well is totally independent from that in other wells and that each well is destroyed at the moment of cell counting, looking at days 4 and 6, when an effect of treatment can be measured, we found that the binomial probability of a total lack of any crossover for each of the four dots concerning treated cells toward the corresponding control dots is 1/256 (P=0.0039, one-tailed). The situation is identical for a comparison of G11-scFv-Int(+)-treated cells against Int-treated cells. In addition, we are confronted with an identical situation in both independent experiments (Fig. 7A, B
). Theoretically, by including Int treatments in the comparisons also, we would be tempted to say that the real probability of being confronted with statistical fluctuations determined by pure chance could be (1/256)4, a probability that is vanishingly small.
In actively dividing cells c-Myc is mostly expressed in the G1 phase of the cell cycle (26)
. Using the growth conditions of the second experiment (richer medium), we seeded six subconfluent wells (1.5x106 cells/well) at day 0. At day 3 we counted (duplicate experiment) 5.8 x 106 cells/well. Two wells were left as controls, and two wells were treated with 4 µM G11-scFv-Int(+). Two days later cell numbers were 10.4 x 106 in control cells (doubling time T57.0 h) and 9.3 x 106 in treated cells; a growth inhibition of roughly 10.6% was observed. With the same richer medium but with cells growing at lower density (Fig. 7B
) a much higher inhibition (47.3% after the first 2 days of treatment) had been observed. At a higher cell density we would expect fewer cells in exponential growth and therefore fewer cells expressing c-Myc in G1, and consequently also a reduced effect of our c-Myc inhibitor (a smaller fraction of potentially sensitive cells).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 28)
.
This work is placed in the broad area of strategies and attempts to inhibit protein-protein interactions. For this purpose, small drug-like molecules are expected to have a narrower spectrum of applications, because in principle they can only "see" a small target size (6)
. On the contrary, miniantibodies could potentially be able to "see" protein motifs more extended in space.
c-Myc was selected as a target because of its established relevance in several solid tumors and hematological malignancies (29)
. In the present study, attention was focused on the amino-terminal transactivation domain (aa 1–152) of c-Myc, containing both Myc Box I and Myc Box II. Myc Box II mediates binding to a large molecular weight nuclear cofactor complex called TRRAP and to TIP48/49 protein, directly correlated with c-Myc transcriptional and oncogenic activities (30)
.
Because the binding surfaces between protein subunits often consist of multiple interactions dispersed over a wide area, molecules that disrupt these interactions may need to be relatively large and complex (such as, for instance, the scFv peptides). These types of molecules have the potential for more selective inhibitory docking at the level of protein-protein interactions (6)
.
Monoclonal antibodies against intracellular targets have been used extensively in basic research to silence gene functions in cells, by means of a number of techniques such as microinjection, red cell ghost fusion, or electroporation (31
32
33)
. This is, however, an approach suitable from the perspective of acquiring new basic biological knowledge, but not from a pharmacological/therapeutic perspective, for which reversible and dosage-modulated effects and the potential capability of entering practically every cell are crucial pharmacological requirements.
Several types of monoclonal antibodies are the result of enormous progress made in recent years in the field of antibody engineering technology (14
, 15)
and are now being used clinically for therapy (against extracellular targets) and also for diagnostic purposes (34)
. Fusing PTDs to scFv antibodies allows us to create a cell-permeable antibody, potentially capable of effectively inhibiting the function of intracellular targets. We investigated the delivery to human cultured cells of a constructed fusion scFv antibody, directed toward the NH2-terminal domain of c-Myc, bearing the Antennapedia 16-aa PTD (Int). A fluorescence-labeled scFv-Int construct should remain unaltered as much as possible to maintain its functions. It was therefore convenient to attach a fluorescent label to our constructs, preferentially at the amino terminus (-NH2 group), rather than in –NH2 groups of basic side chains. A very efficient internalization of the peptide/cargo construct was observed in intact cells kept in culture medium, utilizing confocal microscopy. Fluorescence intensity was determined in multiple spots of each individual cell examined. In a recent study, penetration of a scFv-TAT against Bcl-XL in cultured cells was reported. These authors studied internalization in the presence of fixative agents (35)
. Other authors have shown, however, that cell fixation, even in mild conditions, leads to the artifactual uptake of peptides (18)
. Aware of this artifact, we studied in this work the internalization of an scFv-Int(+) construct (miniantibody against c-Myc) in unfixed living cells, using a sealed microchamber for our fluorescence measurements. Quantification of nonradioactively labeled extracellular and intracellular peptidic/peptidomimetic molecules or miniantibodies (scFv format, molecular mass 30,000 Da) was performed by means of a confocal laser scanning biological microscope, by which local fluorescence intensity of regions of interest can be obtained, with the overt advantage of the reduction of background information away from the focal plane and the possibility of collecting serial sections.
Pharmacologically attainable concentrations in a micromolar range for peptides or antibodies were used. Experiments performed at 2.5 h suggest that internalization is already well under way. Results obtained at 5 h with 1 µM mAb concentrations showed a 5-fold average intracellular/extracellular ratio and an even higher nuclear fluorescence. c-Myc protein should be accessible both at nuclear and cytoplasmic locations. To check maintenance of membrane integrity in our experimental conditions, cells were cotreated with an internalizing nonfluorescent antibody and a fluorescent antibody lacking the internalization sequence. Our results show a negligible internalization increase of the fluorescent antibody lacking the Int sequence, when coincubated with a cold anti c-Myc Int(+) scFv.
Experience with scFv antibodies showed that Kd values toward the target in a 10–8 to 10–10 M range can be often achieved (36)
. In addition, a miniantibody-Int(+) peptide containing 250 aa can be very specific and selective in terms of its interaction with the target. It was therefore especially interesting to have obtained very efficient internalization of a 250-aa peptide.
The results reported in Fig. 7A, B
and the statistical analysis show that G11-scFv-Int(+) is endowed with a clear antiproliferative activity in exponentially growing cells. When cells are close to confluency (third antiproliferative experiment), a smaller fraction of c-Myc-expressing cells and, therefore, sensitive to our c-Myc inhibitor is expected, and this expectation was also verified.
We will try to find a higher affinity, more potent, and perhaps more soluble anti-c-Myc miniantibody, and we will expand our studies to different cells lines and strains in different experimental conditions. These experiments could lead to molecules endowed with a higher intracellular stability and improved pharmacodynamic and pharmacokinetic properties. These developments will be the object of future studies. However, the molecule in this study already showed not only clear capability for efficient cell internalization but also clear antiproliferative activity, not dependent on the accompanying Int internalization sequence and probably dependent on the abundance of the c-Myc target protein.
We also plan to demonstrate in future studies the fact that internalized scFv antibodies lacking a specific intracellular target tend to be inactive. It is evident that our approach of miniantibody internalization worked well in our specific case but could have a much more general application.
Many other intracellular proteins and also intracellular viruses have the potential to be targets for inhibition/neutralization. These prospective, more general applications are the core of the real relevance of our study.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication June 6, 2007. Accepted for publication October 25, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]-ATP. Nat. Biotechnol. 14,485-490[CrossRef][Medline]This article has been cited by other articles:
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  J. Y. Wu, D. J. Yang, L. S. Angelo, S. Kohanim, and R. Kurzrock Molecular imaging of Bcr-Abl phosphokinase in a xenograft model Mol. Cancer Ther., March 1, 2009; 8(3): 703 - 710. [Abstract] [Full Text] [PDF]
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