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Anti-VEGFR-2 scFvs for Cell Isolation. Single-Chain Antibodies Recognizing the Human Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2/flk-1) on the Surface of Primary Endothelial Cells and Preselected CD34⁺ Cells from Cord Blood

^a German Research Centre for Biotechnology, Department of Applied Genetics;
^b Division of Microbiology;
^c Department of Biochemical Engineering;
^d Department of Gene Regulation and Differentiation, Braunschweig, Germany;
^e DKFZ, Institute of Cellular and Molecular Pathology, Heidelberg, Germany;
^f Ulm University Medical Center, Department of Internal Medicine II, Ulm, Germany; Weizmann Institute of Science,
^g Department of Immunology and
^h Department of Molecular Cell Biology, Rehovot, Israel

Key Words. Immune V-gene phage display library • Single-chain antibody • Human VEGF receptor 2 • FACS analysis • Hematopoitic stem cell

Thomas Böldicke, Ph.D., German Research Centre for Biotechnology, Department of Applied Genetics, Mascheroder Weg 1, D-38124 Braunschweig, Germany. Telephone: 49-(0)531-6181-209; Fax: 49-(0)531-6181-202; e-mail: tbo{at}gbf.de

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Immunofluorescence and... Discussion Conclusion References

 
Five specific single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) were selected from a V-gene phage display library constructed from mice immunized with the extracellular domain of VEGFR-2 (Ig-like domain 1-7). All five scFv antibodies (A2, A7, B11, G3, and H1) bound to the purified native antigen in enzyme-linked immunosorbent assay and Dot Blot, and showed no crossreactivity to the human VEGF-receptor 1 (VEGFR-1). The selected antibodies recognize a conformation-dependent epitope of the native receptor and do not recognize denatured antigen in Western blots, as well as linear overlapping peptides comprising the sequence of the human VEGFR-2. The five scFv antibodies bind to the surface of endothelial cells overexpressing human VEGFR-2 c-DNA (PAE/VEGFR-2 cells) as detected by surface immunofluorescence using confocal microscopy. In addition scFv A7 specifically detected VEGFR-2 expressing endothelial cells in the glomerulus of frozen human kidney tissue sections. Therefore, A7 has potential clinical application as a marker for angiogenesis in cryosections of different human tissues. Additionally, two recombinant scFvs (A2 and A7) very efficiently recognize VEGFR-2 on PAE/VEGFR-2 cells and freshly prepared human umbilical vein endothelial cells by fluorescence-activated cell sorter (FACS) analysis. The scFv fragment A7, which was the most sensitive antibody in FACS analysis, recognizes human CD34⁺VEGFR-2⁺ hematopoietic immature cells within the population of enriched CD34⁺ cells isolated from human cord blood. The dissociation constant of A7 was determined to be K_d = 3.8 x 10^–9 M by BIAcore analysis. In conclusion, scFv fragment A7 seems to be an important tool for FACS analysis and cell sorting of vascular endothelial cells, progenitor cells and hematopoitic stem cells, which are positive for VEGFR-2 gene expression.

	INTRODUCTION

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Angiogenesis, the formation of new blood vessels, is crucial both during embryonic development and in the maintenance of normal physiological processes combined with the development of new blood vessels like wound repair, tissue regeneration and during the menstrual cycle [1]. There are a variety of pathophysiological disorders like retinopathies, rheumatoid arthritis and tumor growth, in which angiogenesis is a prerequisite for the progression of the disease. Vascular endothelial growth factor (VEGF or VEGF-A) is a key growth factor and vascular permeability factor for endothelial cells [2]. VEGF-A is not only a specific growth factor for vascular endothelial cells, but also a potent inducer and prime regulator of blood vessel formation for in vivo experiments or in physiologically occurring new vessel formation [3, 4]. Very recently, several new members of the VEGF family have been described and were consequently named VEGF-B to VEGF-E. All these growth factors together with placenta growth factor mediate their biological activity and signals through three receptor tyrosine kinases called VEGFR-1 (flt-1), VEGFR-2 (KDR/flk-1), and VEGFR-3 (flt-4) [5]. From the three receptors, VEGFR-2 is expressed exclusively on vascular endothelial cells and hematopoietic cells [6-10]. During formation of new blood vessels or in tumor angiogenesis, the VEGFR is upregulated, but downregulated in normal vessels [11]. An exception is the human kidney where upregulation of VEGFR-2 gene expression is connected with vascular permeability in the kidney glomerulus [12].

Previously, the production of a rat anti-VEGFR-2 monoclonal antibody (mAb) [13] with neutralizing activity has been reported together with several other mAbs against the human VEGFR-2 suitable for Western blotting and enzyme-linked immunosorbent assay (ELISA) [14]. More recently, several single-chain antibodies against VEGFR-2 have been generated that were able to block binding of VEGF-A to its receptor [15]. These antibodies were able to block VEGF-A-induced DNA synthesis and phosphorylation of the receptors. Very recently it was demonstrated for the first time that pluripotent-hematopoietic stem cells (HSCs) can be purified from bone marrow and cord blood by using a combination of CD34⁺ antibodies followed by VEGFR-2 antibodies for cell sorting of native cells [9]. The VEGFR-2 antibody used by the authors Ziegler et al. was a high affinity hybridoma antibody (mAb clone 260.4) which has been described from our group prior to the start of this work and was generated against the soluble, extracellular domain of VEGFR-2 [16].

Our goal was to establish a novel generation of single-chain antibodies for a broad spectrum of analytical assays, and their employment as high affinity antibodies for the isolation and characterization of primary human endothelial cells and other progenitor cell types, known to be positive for VEGFR-2 gene expression.

	MATERIALS AND METHODS

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Cell Lines and Proteins
The soluble VEGFR-2 protein was expressed in insect cells and purified as described previously in detail. It consists of the first seven Ig-like domains of the extracellular receptor part [16]. Human VEGFR-1 and VEGFR-2-transfected porcine aortic endothelial cells (PAE/VEGFR-1/ VEGFR-2 cells) were described earlier [17] and cultivated in Ham's F12 medium containing 10% fetal calf serum (FCS) and 0.4 mg/ml G418 sulfate (Seromed; Berlin, Germany). Human umbilical vein endothelial cells (HUVECs) were prepared according to Quadros et al. [18] and cultivated in endothelial cell Basal Medium-2 (Clonetics; San Diego, CA; http://www.clonetics.com).

Human cord blood mononuclear cells from full-term deliveries were isolated by standard separation on Ficoll-Paque (Amersham Pharmacia Biotech; Freiburg, Germany; http://www.apbiotech.com). Enrichment of CD34⁺ cells was performed using a MACS separation kit (Miltenyi Biotech; Bergisch Gladbach, Germany; http://www.miltenyibiotec.com). Adherent HUVECs were removed for fluorescence-activated cell sorter (FACS) staining using Trypsin/EDTA. All procedures performed with the human cord blood mononuclear cells were performed at the Weizmann Institute of Science and approved by the human experimentation and ethics committees.

All cells were grown under normoxic conditions with 5% CO₂ in a humidified cell culture incubator (Heraeus; Hanau, Germany; http://www.heraeus.com). All chemicals, if not otherwise indicated, were obtained from Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany; http://www.sigma-aldrich.com).

Construction of the Immune V-Gene Phage Display Library and Selection of Specific scFv Fragments by Biopanning
Three BALB/c mice (Zentralinstitut für Versuchstierzucht; Hannover, Germany) were immunized subcutaneously, each mouse with 10 µg/injection of the extracellular domain of VEGFR-2 in 50 µl phosphate-buffered saline (PBS) (145 mM NACl, 7.5 mM Na₂HPO₄, 2.5 mM NaH₂PO₄x2H₂O, pH 7.1) mixed with 50 µl complete Freund's adjuvant. Immunizations were repeated at days 7, 15, and 23 with incomplete adjuvant. At day 28 lymphocytes were harvested from popliteal lymph nodes. After extraction of total RNA [19], mRNA was isolated using an mRNA isolation kit (Quiagen; Hilden, Germany; http://www.quiagen.com) and the cDNA synthesized using a "First-strand cDNA synthesis kit" (Amersham Pharmacia Biotech). Then the V_H and V_L domains were amplified using primers (GIBCO BRL; Eggenstein, Germany) as outlined by Clackson et al. and McCafferty et al. [20, 21]. Amplification reactions were performed in five different polymerase chain reaction (PCR) tubes (one tube for amplification of the heavy chains using the V_H1Back and the V_H1FOR-2 primer; the other four tubes were used for amplification of the VK light chains employing the VK2Back primer and one of the 4 J-region primers [MJK1FONX, MJK2FONX, MJK4FONX, MJK5FONX]), [21].

The scFv DNA was generated by fusion of V_H- and V_L-PCR fragments via a short DNA fragment encoding for 15 amino acids ("Recombinant Phage Antibody System," Amersham Pharmacia Biotech). Another PCR reaction was used for the addition of vector-compatible restriction sites (SfiI/NotI) to both ends of the single-chain-encoding DNA fragment. Thus assembled scFv DNA was then pooled, unincorporated desoxyribonucleotides and primers removed by agarose gel electrophoresis, and cleaved by restriction endonucleotides SfiI and NotI. The fragment was gel-purified and ligated into the pCANTAB 5E vector (Amersham Pharmacia Biotech) and used to transform electrocompetent E. coli XL-1-Blue cells. After electroporation E. coli XL-1-Blue cells were plated onto SOB-A-G-T plates (20 g Bacto-tryptone, 5 g Bacto-yeast extract, 0.5 g NaCl per liter medium, plus 2% glucose, 100 µg/ml ampicillin, and 10 µg/ml tetracyclin) and incubated overnight at 30°C. On the next day cells were scraped into 25 ml 2YT-G-A-T medium (17 g Bacto-tryptone, 10 g Bacto-yeast extract, 5 g NaCl, plus 2% glucose, 100 µg/ml ampicillin, and 10 µg/ml tetracyclin) and diluted until OD₆₀₀ of 0.4 was reached. Bacteria were grown to OD₆₀₀ of 0.6 followed by infection with helper phage M13K07 for phagemid rescue. Amplification was performed in 2YT-A-T medium at 30°C overnight. The packaged phagemids were precipitated in 4% PEG/0.5 M NaCl and finally resuspended in 1ml PBS containing 0.02% Na-acid.

For panning on microtiter plates (MaxiSorb^TM polystyrene assay plates, Nunc; Wiesbaden, Germany; http://www.nalgenunc.com) each well was coated with 10 µg/ml of the extracellular domain of VEGFR-2 overnight at 4°C, followed by blocking with 5% skimmed milk for 30 min at room temperature. Then 2.5 x 10⁹ colony-forming units of packaged phagemids were blocked in 100 µl PBS supplemented with 5% skimmed milk for 30 min at room temperature and added to each antigen-coated well for 2 h at room temperature. In order to remove unbound phages, the wells were washed 12x with PBS after the first panning, whereas after the second and third pannings, wells were washed 12x in PBS containing 0.05% Tween 20 (PBST) followed by additional short washing steps (12x) in PBS. Bound phages were eluted with 200 µl 100 mM glycin-HCL (pH 2.2) by incubation for 15 min at room temperature. After neutralization with saturated Tris-base solution (~pH 11), 400 µl eluted packaged phagemids (from two different wells) were used to infect 5 ml log-phase E. coli XL-1 Blue. Cells were plated onto SOB-A-G-T plates, incubated overnight at 30°C and titers determined in parallel.

Phage ELISA
Individual clones obtained after the third panning were grown overnight at 30°C in 96-well microtiter plates in 2YT-G-A-T medium and phagemid rescue performed as described above. Phage antibodies containing supernatants were blocked in 2.5% skimmed milk for 1 h at room temperature and transferred into the respective wells of other 96-well plates (MaxiSorb^TM polystyrene plates, Nunc) which were coated with 100 µl/well antigen at a concentration of 1 µg/ml. Incubation was carried out for 2 h at room temperature. Plates were washed 10x with PBST and bound phages were detected by incubation with a horseradish peroxidase (HRP)-labeled mouse anti-M13 antibody (Amersham Pharmacia Biotech) for 1 h at room temperature. The ELISA was developed for 10-20 min with an ortho-phenylene-diamine (OPD)-solution according to the supplier's instructions (Sigma). The reaction was stopped by adding 100 µl/well 10% H₂SO₄ and absorption red at 490 nm. The DNA of positive clones was purified and sequenced using the pCANTAB 5 sequencing primer set (Amersham Pharmacia Biotech) and an ABI PRISMTM 310 Genetic Analyzer (Perkin-Elmer; Weiterstadt, Germany; http://www.instruments.perkinelmer.com/index.asp).

Preparation of Soluble scFv
The nonsuppressor E. coli host HB2151 was infected with individual positive phages and selected on SOB-A-G plates at 30°C overnight. Next-day cells were cultured in 2YT-A-G (0.1% glucose) medium to OD₆₀₀ of 1.0. Expression of scFv fragments was induced with 1mM isopropyl-ß-D-thiogalactopyranoside cells grown overnight at 30°C and antibodies harvested from the supernatant after centrifugation. Expression of the scFvs was analyzed in an ELISA with the extracellular domain of VEGFR-2 as antigen using an HRP-labeled mouse anti-E tag antibody (Pharmacia Amersham Biotech). Soluble antibodies were purified from bacterial supernatants using a HiTrap anti-E Tag column from Amersham Pharmacia Biotech (RPAS Purification Module).

Immunoblot Analysis of the scFvs with Native and Denatured VEGFR-2 Antigen
The antigen was spotted onto a cellulose membrane in its native, untreated state or denaturated. The membrane was blocked with 5% skimmed milk in PBS for 1 h and incubated with the purified scFv antibodies (2 µg/ml 2% skimmed milk) for 2 h. Bound antibodies were detected with the HRP-labeled mouse anti-E tag antibody (diluted 1:2,500 in 2% skimmed milk). The binding of a polyclonal serum obtained from one of the three immunized mice was detected with an HRP-labeled goat antimouse IgG-Fc antibody (diluted 1:1,000 in 2% skimmed milk, Dianova; Hamburg, Germany). All incubation steps with the secondary antibodies were performed at room temperature for 1 h. For all washes in between each incubation step, PBS supplemented with 0.05% Tween 20 was used. Blots were developed with an OPD solution according to the instructions from Sigma for 5-10 min, and the reaction stopped by two washes with PBS.

Cell Surface Immunofluorescence
Immunofluorescence with adherent cells: PAE/VEGFR-2 cells were grown for 48 h on sterile coverslips coated with 0.1% gelatin in HAM's F-12 medium containing 10% FCS (v/v) and 0.4 mg/ml G418. After discarding the medium, cells were directly incubated for 1 h with 0.5 µg/ml (diluted in PBS/2% FCS) of purified antibody, followed by a 1-h incubation with the anti-E tag antibody (diluted 1:2,500 in PBS/2% FCS) and a 1-h incubation step with a TRITC-labeled goat antimouse IgG (H+L) antibody (diluted 1:160 in PBS/2% FCS, Dianova). All incubation steps were performed at 4°C and in between these steps washings were performed four times in PBS/1% bovine serum albumin. Finally, cells were fixed in 1% paraformaldehyde in PBS for 30 min on ice, washed four times, embedded in moviol (Hoechst; Frankfurt/Main, Germany) and analyzed with the confocal microscope. For intracellular staining, cells were fixed with acetone/methanol for 30 sec at room temperature and processed as described above.

Immunofluorescence with PAE/VEGFR-2 cells in suspension: adherently growing PAE/VEGFR-2 cells were detached by incubation with cold Trypsin/EDTA solution at room temperature. The cells were centrifuged at 1,000 rpm for 5 min at 4°C, washed with PBS/2%FCS and aliquots of 5 x 10⁴ cells were incubated with 0.5 µg/ml scFv in 100 µl PBS/2% FCS in a 96-well microtiter plate for 1 h at 4°C. The following incubation steps with the secondary antibodies were the same as described for the adherent cells. After each incubation step, washing was performed once in PBS/2% FCS. Cells were fixed with 1% paraformaldehyde, followed by two washing steps, resuspended in 50 µl PBS, and absorbed to polylysin-coated cover slips for 10 min at room temperature. Cells were embedded in moviol and analyzed with the confocal microscope.

For control images to test the cross-reactivity of scFv A2 and scFv A7 with VEGFR-1, living PAE/VEGFR-1 cells in suspension were stained with fluorescein isothiocyanate (FITC)-labeled mitochondrion-selective dye MitoTracker^TM Green FM (Mo Bi Tec; Göttingen, Germany; http://www mobitec.de.) prior to labeling with the antibodies. VEGFR-1 cells in suspension were incubated with serum-free growth medium containing 1,000 nM MitoTracker dye for 45 min under growth conditions. After pelleting, cells were suspended in fresh serum-free medium and incubated for another period of 15 min to allow the dye to accumulate in the mitochondria. Then cells were pelleted once more and labeled with the scFv fragments as described before.

Immunohistochemistry and Western Blotting
5 µm frozen tissue sections were prepared from adult kidneys that were received immediately after surgical removal from patients suffering from renal carcinoma. Tissue sections, fixed in acetone at –10°C for 10 min, were incubated with 10 µg/ml scFv A7 for 2 h at 22°C. Then sections were incubated with the HRP-labeled mouse anti-E tag antibody (1:2,500) at 22°C for 1 h, followed by a 1-h incubation with a rabbit antimouse antibody (Z 259, DAKO; Hamburg, Germany; http://www.dako.dk) and a 1-h incubation with alkaline-phosphatase-labeled mouse mAb (1:40). All dilutions were performed in PBS pH 7.6. Bound antibodies were visualized with a solution of sodium nitrite (28 mM), new fuchsin (basic fuchsin, 21 mM), naphthol-AS-BI-phosphate (0.5 mM), dimethylformamide (64 mM), and levamisole (5 mM) in 50 mM Tris/HCL buffer, pH 8.4, containing 146 mM NaCl (incubation time: 15 min). Nuclei were stained with hämatoxilin. Washing was performed with H₂O and PBS.

Immunoprecipitation and Western blotting for VEGFR-2 detection were done very similarly as described before by Siemeister et al. [22]. Briefly, unstimulated HUVECs were lysed and the polyclonal rabbit anti-VEGFR-2 antibody r212 [12] was used for immunoprecipitation of 1 x 10⁶ cells. Then Western blotting was done with the C-terminal polyclonal rabbit anti-VEGFR-2 antibody 504 from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

FACS Analysis
FACS analysis was performed with scFv A7 using A) recombinant PAE/VEGFR-1 and PAE/VEGFR-2 cells; B) HUVECs, and C) CD34⁺ cells isolated from cord blood.

1. Adherently growing PAE/VEGFR-2 and PAE/VEGFR-1 cells were detached from the bottom of a flask by incubation with a cold Trypsin/EDTA solution (1x, Sigma). After washing cells with PBS/2% FCS they were used directly for FACS analysis. All incubation steps with antibodies were performed for 30 min at 4°C in a 96-well microtitre plate (Nunclon^TM Surface plate, Nunc) as described in the section on immunofluorescence with cells in suspension. Binding of the scFvs was detected with the HRP-labeled mouse anti-E tag antibody and an FITC-conjugated sheep antimouse IgG antibody (diluted 1:125 in PBS/2% FCS, Sigma). Cells were resuspended in 500 µl PBS/2% FCS containing 10 µg/ml Propidiumjodid and measured with a FACSCalibur^TM (Becton Dickinson; San Jose, CA; http://www.bd.com).
   
2. For analyzing the expression of VEGFR-2 on the surface of cultivated HUVECs, they were freshly prepared out of the umbilical cord. One-half of cells were analyzed directly by flow cytometry for surface expression of CD34 and VEGFR-2. The rest of the cells were cultivated using endothelial cell Basal Medium-2 and analyzed after different passages. Expression of VEGFR-2 was detected with scFv A7 and expression of CD34 was directly detected with a phycoerythrin (PE)-labeled mouse anti-CD34 antibody (clone No. 581, diluted 1:10 in PBS/2% FCS, Becton Dickinson) and analyzed as in A.
   
3. VEGFR-2 expression on the cell surface of human cord blood CD34⁺ cells was detected using the following antibodies: two steps staining with 2 µg/ml biotinylated anti-VEGFR-2 mAb (clone 260.4, Sigma) followed by staining with streptavidin-PE (Pharmingen; San Diego, CA; http://www.pharmingen.com). Three steps staining with 0.5 µg/ml scFv-A7 followed by staining with the HRP-labeled mouse anti-E tag antibody and donkey antimouse PE (Jackson; West Grove, PA). FACS analysis was performed by FACsort (Becton Dickinson).
   

BIAcore Analysis of the Soluble scFv
The binding kinetics of the soluble scFv A7 were measured using BIAcore biosensor (Amersham Pharmacia Biotech; http://www.apbiotech.com). ScFv A7 was immobilized onto a sensor chip, and VEGFR-2 was injected at concentrations ranging from 10 to 100 nM. The sensorgrams were evaluated using the BIA Evaluation 2.0 program, and the rate constants k_on and k_off and the dissociation constant (K_d) were determined.

	RESULTS

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Selection of Recombinant Anti-VEGFR-2 scFv Antibodies Using the Immune V-Gene Phage Display Library
An immune V-gene phage display library was constructed starting from the mRNA isolated from mice immunized with the extracellular domain of VEGFR-2. For cloning the antibody V_H- and V_L-gene repertoire the "Recombinant Phage Antibody System" from Amersham Pharmacia Biotech was applied, except that the primer sequences for the amplification of V_H and V_L domain encoding cDNA sequences were taken from the publications by Clackson et al. [20] and McCafferty et al. [21]. All transformations gave rise to 2 x 10⁶ primary transformants, which were used to generate an antibody-phage library. After three rounds of panning on the extracellular domain of VEGFR-2, a primary phage ELISA screening was performed. Fifty (26%) out of 192 antibody phage clones were found to react specifically with the antigen. Twenty-three clones which gave a strong signal (OD₅₉₅ > 0.5, color development after 30 min) were sequenced and five different clones were found (A2, A7, B11, G3, and H1 [Fig. 1A and Fig. 1B]). In total scFv clone A7 was represented 17 times (74%), clones A2, 11B, and G3 once (4%), and clone H1 3 times (13%). As seen from the consensus sequences, all CDR domains of the variable light chains show a high homology. A similar observation was made for CDR1 and CDR2 of the variable domain of all heavy chains. On the other hand, the CDR3 region of the variable heavy chain shows the highest variability. A consensus sequence of only three amino acids (GDY) could be deduced.

View larger version (33K): [in this window] [in a new window]   Figure 1. Deduced amino acid sequences of the VH and VL domains of the scFv fragments A2, A7, B11, G3, and H1 isolated from the phage display library. (A) Amino acid sequences of the VH domains. The complementary-determining regions (CDR1-CDR3) are printed in bold according to Kabat et al. [37]. Sequences were aligned using the Multalin program. Consensus sequences are printed in italics. (B) Amino acid sequences of the VL domains. Linker: Amino acid sequence (Gly4Ser)3 between the VH and VL domains; NotI: restriction site used for ligation of the assembled scFv into the pCANTAB 5E vector.

View larger version (154K): [in this window] [in a new window]   Figure 2. Immunoblot analysis: binding of the anti-VEGFR-2 scFv fragments to native and denatured VEGFR-2. The antigen (0.1 µg/2.5 µl) was spotted onto several scraps of a cellulose membrane which had been placed into the wells of a 48-well microtitre plate. Treatments of the VEGFR-2 were as follows: (A) native; (B) denaturation with PBS containing 2% SDS for 10 min at 37°C; (C) denaturation with the same buffer as in (B) for 5 min at 95°C; (D) denaturation with PBS containing 2% SDS and 1% DTT (Laemmli-buffer). After the blocking step the scraps were treated with the scFv fragments (1) A2, (2) A7, (3) B11, (4) G3, (5) H1, and (6) polyclonal mouse serum (diluted 1:500 in PBS). Columns (7) and (8) are background controls for the secondary antibodies HRP-labeled anti-E tag antibody without scFv fragment (7) and HRP-labeled goat antimouse IgG-Fc antibody without polyclonal mouse serum (8), respectively.

	IMMUNOFLUORESCENCE AND IMMUNOHISTOCHEMISTRY

Top Abstract Introduction Materials and Methods Results Immunofluorescence and... Discussion Conclusion References

 
For cell surface immunofluorescence, the adherently growing PAE/VEGFR-2 cells were incubated with the five scFv fragments and binding to the cell surface expressed VEGFR-2 analyzed with a laser scanning confocal microscope (Figs. 3 and 4). In Figure 3A binding of the clone A7 to the surface area of the cell is clearly shown and in Figure 3B, a section 2 µm underneath the surface area of the cell was stained. Binding of scFv A7 was restricted mostly to the cell membrane. In contrast to this observation, areas around the nuclei were stained when fixed cells had been used. A section 4 µm underneath the surface area is shown (Fig. 3C). In Figures 3D and 3E controls are shown with living PAE/VEGFR-1 cells and scFv A7. No unspecific binding of scFv A7 to the cells was seen. This confirms our data that scFv A7 did not cross-react to the homologous soluble extracellular domain of VEGFR-1 in ELISA. The other four scFv fragments behaved similarly and therefore results are not separately shown. The staining of the surface-expressed VEGFR-2 on adherent cells was confirmed with scFv fragments A2 and A7 and PAE/VEGFR-2 cells in suspension. The fluorescent images representing optical sections scanned at 2 µm were collected simultaneously using the laser scanning confocal microscope and are shown in Figure 4 I /A-D (clone A2) and Figure 4 II/A-D (clone A7). Binding of the scFv fragments A2 and A7 to the surface of the cells was clearly evident. The VEGFR-2 seems to be unequally distributed and was observed as focal and localized patches on the cell surface. Figure 5 B and C and Figure 5 E and F show control images using PAE/VEGFR-1 cells in suspension incubated with A7 or A2, respectively. No binding to the cells was detected. Furthermore scFv A2 and A7 recognized VEGFR-2 on the surface of VEGFR-2 cells which had been fixed with 1% paraformaldehyde for 30 min on ice before staining (data not shown).

View larger version (76K): [in this window] [in a new window]   Figure 3. Immunofluorescence of scFv A7 with adherent PAE/ VEGFR-2 cells analyzed by laser scanning confocal microscopy. Confocal imaging, three sections of a Z-Series are depicted: (A) Confocal section on the surface area of the cell. (B) Section 2 µm underneath the surface area of the cell. (C) Cells fixed with acetone/methanol, section 4 µm underneath the surface area of the cell. The bars represent 10 µm in (A) and (B) and 50 µm in (C). (D) and (E) are negative controls with PAE/VEGFR-1 cells: (D) light microscopy, (E) immunofluorescence; in (D) and (E) cells were analyzed with a photomicroscope (Zeiss Axiophot); bar represents 50 µm.

View larger version (37K): [in this window] [in a new window]   Figure 4. Laser-scanning confocal micrographs of PAE/VEGFR-2 cells in suspension incubated with scFv A7 (I) and scFv A2 (II). (I, A-D) A z-axis section series from top to bottom of a representative cell after incubation with scFv A7; section thickness is 2 µm. Bar represents 10 µm. (II, A-D) A z-axis section series as in (I, A-D), representative cell after incubation with scFv A2. Bar represents 5 µm.

View larger version (56K): [in this window] [in a new window]   Figure 5. Control images with PAE/VEGFR-1 in suspension incubated with scFv A7 (A-C) and scFv A2 (D-F). Immunofluorescences were analyzed with an Axiovert 135TV (Zeiss). Figure 5 A and D show the positive control (labeling of PAE/VEGFR-2 cells with scFv A7 (A) and scFv A2 (D). Double immunofluorescence staining of PAE/VEGFR-1 cells with FITC-labeled MitoTracker dye (5B and E) and scFv A7 (5C) and scFv A2 (5F) is shown to the right of it.

View larger version (199K): [in this window] [in a new window]   Figure 6. Immunohistology of frozen human kidney tissue sections with scFv A7. (A) Cryosection of an interlobular artery. A monolayer of cells inside the artery with a red positive label for VEGFR-2 is shown. (B) Negative control with the secondary antibodies: HRP-labeled mouse anti-E tag antibody, rabbit antimouse mAb and alkaline-phosphatase-labeled mouse antibody without scFv A7. (C) Glomerulus with a red positive label for VEGFR-2 in capillaries. (D) Negative control with the secondary antibodies as in (B). Nuclei are stained with hematoxilin.

View larger version (18K): [in this window] [in a new window]   Figure 7. Binding of anti-VEGFR-2 scFv fragments to PAE/VEGFR-2 cells analyzed by flow cytometry. Shown is the binding of the scFv fragments A7 (A), A2 (B), G3 (C), B11(D), and H1(E) to PAE/VEGFR-2 cells. Percentages represent the number of gated, living VEGFR-2+ cells detected with the corresponding antibody. (F) shows the negative control with the secondary antibodies: HRP-labeled mouse anti-E tag antibody and FITC-conjugated sheep antimouse IgG antibody. Binding of the five scFv-fragments to PAE/VEGFR-1 cells was negative. Shown representatively are the clones A7 (G), A2 (H), and G3 (I). (J) shows the negative control with the secondary antibodies without the primary antibodies and (K) shows the positive control with a rabbit polyclonal anti-VEGFR-1 serum. In this case binding was detected with an FITC-labeled goat antirabbit IgG (H+L) antibody.

View larger version (20K): [in this window] [in a new window]   Figure 8. The amount of VEGFR-2- and CD34-expressing HUVECs in function of the number of cell passages analyzed with scFv A7 by flow cytometry. HUVECs were freshly prepared from the umbilical cord (day 0), cultivated over different passages and the percentage of gated living VEGFR-2+ cells determined with scFv A7. Bars represent the mean deviation of four different experiments. Inset: Immunoprecipitation (IP) followed by SDS-PAGE immunoblotting under reduced conditions for VEGFR-2 detection using cell lysates from passage no. 1 HUVECs (lane 1) and passage no. 8 HUVECs (lane 2). The VEGFR-2 protein (see arrow) was visualized with an HRP-labeled goat antirabbit antibody (Promega; Mannheim, Germany) using the ECL-system from Amersham Pharmacia Biotech. The amount of gated living CD34+ cells was determined in parallel. Bars represent as in (A) the mean deviation of four and three different experiments, respectively.

View larger version (37K): [in this window] [in a new window]   Figure 9. Flow cytometry analysis of VEGFR-2 expression on human cord blood CD34+ cells with scFv A7. Enriched human cord blood CD34+ cells were stained with streptavidine-PE (A), negative control; biotinilated anti-VEGFR-2 mAb clone 260.4 (B) or scFv A7 (C). Indicated are the amounts (%) of CD34+ cells expressing VEGFR-2. (D) shows staining of HUVECs with (a) irrelevant mouse IgG-PE, (b) biotinilated anti-VEGFR-2 mAb clone 260.4 and (c) scFv A7.

	DISCUSSION

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At first a polyclonal antibody generated in rabbit against the purified soluble receptor was reported [12]. Interestingly, this antibody has a strong neutralizing activity but also has been used for immunoprecipitation and Western blotting [23, 24]. With a similar approach, several mAbs against the soluble receptor protein were described and characterized for different potential uses, including immunostaining techniques and Western blot studies [5, 12, 16]. A different immunization protocol was used recently for the generation of mAbs against the soluble receptor part [14]. Baculo-virus-infected Sf-9 cells expressing the whole receptor protein in combination with a stringent and rapid screening procedure were used. This method was successful to identify two hybridomas highly specific and with high affinity for the human VEGFR-2, whereas mice immunized with isolated receptor protein alone were not successful.

VEGF ligand-receptor interaction and induction of the signaling cascade are necessary for angiogenesis, regardless of whether this process is induced under normal or pathogenic conditions. Early attempts to block this interaction also lead to the development of neutralizing antibodies, binding either to VEGF itself or to its receptor [13, 25]. An efficient receptor blocking mAb for the mouse homologue of VEGFR-2, flk-1, was characterized in detail some years before [13]. These studies were extended recently by the same group, establishing blocking single-chain antibodies against VEGFR-2. The approach chosen was very similar to that described and selected for this study [15]. The described blocking antibodies were generated with a similar antigen but with the difference that a fusion protein of VEGFR-2 with human alkaline phosphatase (VEGFR-2-AP) was used. The screening process was optimized to isolate and characterize two blocking antibodies from a library of 2.7 x 10⁸ clones. From our earlier studies using the same antigen and generation of a polyclonal serum in rabbits, we were also expecting that some of the high affinity binders were blocking, but this observation was not made. Probably higher numbers of phages have to be screened in order to select a blocking single-chain antibody. However, from the single-chain antibodies generated and described from the group of Zhu et al. [15], it was not shown that they may be used for selection of primary progenitor cells under native conditions by flow cytometry.

In general, recombinant antibodies could be successfully selected from immune, naïve or synthetic libraries using phage display [26]. Similar to Zhu et al. [15], we constructed an antigen-biased library because they contain antibodies with high affinity (10⁸ – 10⁹ M^–1 [15, 27]) and are easier to construct than large naive or large synthetic libraries. A library possessing a size of 2 x 10⁶ clones was generated. The complementary regions (CDR1-CDR3) of the heavy and light chains of two (the dominant nonblocker clone p2A6 and the most interesting VEGF blocker p1C11 that inhibit VEGF-induced KDR phophorylation) of the selected and described antibodies from Zhu et al. [15], were compared with the regions of scFv A7. The result showed that the CDR 3 regions of the heavy chains and light chains possessed the lowest homology. The homology was defined in percentage of identical amino acids at the same positions of the aligned sequences. Furthermore, the CDR3 regions of the heavy chains of the scFv fragments selected by Zhu et al. [15], possessed the most variability as detected by our selected clones. From the sequence data of p2A6 and p1C11, and from the fact that p1C11 blocks the binding of VEGF in contrast to A7, we conclude that A7 recognizes other epitopes of VEGFR-2 in addition to p1C11 and p2A6.

Two of our five selected scFv fragments (A7 and A2) efficiently recognize the membrane-bound VEGFR-2 on the surface of recombinant PAE/VEGFR-2 cells as shown in this study by surface immunofluorescence and FACS analysis. Furthermore, clone A7 detects human endothelial cells freshly isolated out of the umbilical cord expressing VEGFR-2. Interestingly, the expression of VEGFR-2 on the surface of the HUVECs is dependent on the number of cell passages. Maybe the VEGFR-2 is upregulated in vitro by the growth factors VEGF or FGF-2 which are supplements of endothelial cell Basal Medium-2. Recently this was shown for VEGF with bovine adrenal cortex endothelial cells [28]. On the contrary, we could show that the expression of CD34 was downregulated with proliferation in continuous culture, and this confirms the results published by Delia et al. [29]. In the future the scFv fragment A7 will be very useful to study the upregulation of VEGFR-2 expression on the surface of primary endothelial cells in more detail by FACS analysis (for example influence of hormones and growth factors).

Mesoderm-inducing factors, like FGF-2, are involved in the generation of the so-called hemangioblast, which is a bipotential (so far hypothetical) precursor for the formation of angioblasts and HSCs [11, 30, 31]. VEGFR-2 is an early marker of the so-called hemangioblast. After differentiation, VEGFR-2 is downregulated in differentiated hematopoietic but not endothelial cells. The ligand for VEGFR-2, VEGF itself, acts in a paracrine fashion and it is produced by the endoderm. Threshold VEGF seems to be needed to maintain angioblast (endothelial cell precursor) differentiation as demonstrated by VEGF gene knockout experiments [2, 32]. Further evidence was demonstrated by gene disruption studies which indicated that the mouse homologue of VEGFR-2, flk-1, is required for initiation of hematolymphopoiesis and vasculogenesis [33]. Other studies also suggested the existence of embryonic cells positive for VEGFR-2 with hemoangiogenic potential, but did not further allow identification of prenatal repopulating HSCs [34-36]. Furthermore human AC133⁺CD34⁺VEGFR-2⁺ endothelial progenitor cells have been isolated using antibodies against CD34, and it was shown that these cells have the capacity to expand and differentiate in vitro into mature AC133^–VEGFR-2⁺ endothelial cells after incubation with VEGF and (FGF)-2 [10].

Very recently it was found that VEGFR-2/KDR is a major functional marker for postnatal HSCs [9]. The authors purified CD34⁺ cells and separated CD34⁺VEGFR-2⁺ from CD34⁺VEGFR-2^– fractions. It is shown that 0.1% to 0.5% of purified CD34⁺ cells from bone marrow, peripheral blood and cord blood were VEGFR-2⁺, and that only these cells are pluripotent stem cells whereas the CD34⁺VEGFR-2^– cells are lineage-commited progenitor cells. The KDR expression was monitored with the high-affinity hybridoma clone 260.4 that binds like our scFv A7 to the extracellular domain of VEGFR-2 as previously described by our group [12]. The most exciting fact is that scFv A7 also recognizes CD34⁺ cells purified by cell sorting of human cord blood. Comparison of scFv A7 with clone 260.4 shows that scFv A7 detects the CD34⁺VEGFR-2⁺ cells with similar efficiency. Thus scFv A7 offers the possibility of purifying VEGFR-2⁺ HSCs by cell sorting for analyzing the cellular and molecular phenotype and functional properties of these stem cells and subsets. This is very crucial for clinical applications such as stem cell transplantation and in vitro blood cell generation.

	CONCLUSION

Top Abstract Introduction Materials and Methods Results Immunofluorescence and... Discussion Conclusion References

 
The study describes five specific single-chain antibodies selected from an immune V-gene phage display library recognizing the soluble extracellular part of VEGFR-2 in its native form in ELISA. Two of the clones (A2 and A7) bind efficiently to the surface-expressed VEGFR-2 on recombinant PAE/VEGFR-2 cells. It is shown that one scFv fragment (A7) recognizes HUVECs and more interesting human CD34⁺VEGFR-2⁺ hematopoetic immature cells within the population of enriched CD34⁺ cells isolated from human cord blood. Therefore scFv A7 can be used to select vascular endothelial cells, progenitor cells and HSCs, all expressing VEGFR-2 on their surface by cell sorting. The scFv fragment A7 recognizing CD34⁺VEGFR-2⁺ cells may be very important for clinical application such as stem cell transplantation and in vitro blood cell generation where purified VEGFR-2⁺ HSCs are needed urgently.

	ACKNOWLEDGMENT

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We thank Dr. B. Haase for performing the BIAcore Analysis and Dr. W. Röckl for purification of VEGFR-2. This study was supported in part by a grant from Deutsche Forschungsgemeinschaft (SFB451, Project B1) to J.W.

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