Intradiabodies, Bispecific, Tetravalent Antibodies for the Simultaneous Functional Knockout of Two Cell Surface Receptors
2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês
10.1074/jbc.m307002200
ISSN1083-351X
AutoresNina Jendreyko, Mikhail Popkov, Roger R. Beerli, Junho Chung, Dorian B. McGavern, Christoph Rader, Carlos F. Barbas,
Tópico(s)Cell Adhesion Molecules Research
ResumoThe specific and high affinity binding properties of intracellular antibodies (intrabodies), combined with their ability to be stably expressed in defined organelles, provides powerful tools with a wide range of applications in the field of functional genomics and gene therapy. Intrabodies have been used to specifically target intracellular proteins, manipulate biological processes, and contribute to the understanding of their functions as well as for the generation of phenotypic knockouts in vivo by surface depletion of extracellular or transmembrane proteins. In order to study the biological consequences of knocking down two receptor-tyrosine kinases, we developed a novel intrabody-based strategy. Here we describe the design, engineering, and characterization of a bispecific, tetravalent endoplasmic reticulum (ER)-targeted intradiabody for simultaneous surface depletion of two endothelial transmembrane receptors, Tie-2 and vascular endothelial growth factor receptor 2 (VEGF-R2). Comparison of the ER-targeted intradiabody with the corresponding conventional ER-targeted single-chain antibody fragment (scFv) intrabodies demonstrated that the intradiabody is significantly more efficient with respect to efficiency and duration of surface depletion of Tie-2 and VEGF-R2. In vitro endothelial cell tube formation assays suggest that the bispecific intradiabody exhibits strong antiangiogenic activity, whereas the effect of the monospecific scFv intrabodies was weaker. These findings suggest that simultaneous interference with the VEGF and the Tie-2 receptor pathways results in at least additive antiangiogenic effects, which may have implications for future drug developments. In conclusion, we have identified a highly effective ER-targeted intrabody format for the simultaneous functional knockout of two cell surface receptors. The specific and high affinity binding properties of intracellular antibodies (intrabodies), combined with their ability to be stably expressed in defined organelles, provides powerful tools with a wide range of applications in the field of functional genomics and gene therapy. Intrabodies have been used to specifically target intracellular proteins, manipulate biological processes, and contribute to the understanding of their functions as well as for the generation of phenotypic knockouts in vivo by surface depletion of extracellular or transmembrane proteins. In order to study the biological consequences of knocking down two receptor-tyrosine kinases, we developed a novel intrabody-based strategy. Here we describe the design, engineering, and characterization of a bispecific, tetravalent endoplasmic reticulum (ER)-targeted intradiabody for simultaneous surface depletion of two endothelial transmembrane receptors, Tie-2 and vascular endothelial growth factor receptor 2 (VEGF-R2). Comparison of the ER-targeted intradiabody with the corresponding conventional ER-targeted single-chain antibody fragment (scFv) intrabodies demonstrated that the intradiabody is significantly more efficient with respect to efficiency and duration of surface depletion of Tie-2 and VEGF-R2. In vitro endothelial cell tube formation assays suggest that the bispecific intradiabody exhibits strong antiangiogenic activity, whereas the effect of the monospecific scFv intrabodies was weaker. These findings suggest that simultaneous interference with the VEGF and the Tie-2 receptor pathways results in at least additive antiangiogenic effects, which may have implications for future drug developments. In conclusion, we have identified a highly effective ER-targeted intrabody format for the simultaneous functional knockout of two cell surface receptors. Antibodies can bind almost any molecule with high specificity and affinity, providing powerful biotechnological tools for diagnostic and therapeutic applications. Advances in recombinant DNA technology have facilitated the manipulation of the antibody genes, so that design, cloning, expression, and use of single-chain antibodies have become routine procedures in protein engineering. The potential of single-chain antibody fragments (scFv) 1The abbreviations used are: scFvsingle-chain antibody fragmentERendoplasmatic reticulumHUVEChuman umbilical vein endothelial cellsAng-1 or -2angiopoietin-1 or -2VHheavy chain variable domainVLlight chain variable domainVEGFvascular endothelial growth factorELISAenzyme-linked immunosorbent assayPBSphosphate-buffered salineHAhemagglutininMOImultiplicity of infectionFACSfluorescence-activated cell sortingGFPgreen fluorescent protein. for intracellular applications, termed "intrabodies," has been exploited in a number of laboratories (1Marasco W.A. Haseltine W.A. Chen S.Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7889-7893Crossref PubMed Scopus (235) Google Scholar, 2Beerli R.R. Wels W. Hynes N.E. J. Biol. Chem. 1994; 269: 23931-23936Abstract Full Text PDF PubMed Google Scholar, 3Deshane J. Siegal G.P. Alvarez R.D. Wang M.H. Feng M. Cabrera G. Liu T. Kay M. Curiel D.T. J. Clin. 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To date, intrabodies have been utilized for targeting proteins in a singular fashion. Bispecific and tetravalent antibody fragments could improve and expand the inhibitory potential of intrabodies by exhibiting increased apparent affinity for their antigen and by being more efficient at inhibiting protein function or intracellular trafficking (9Fitzgerald K. Hooliger P. Winter G. Protein Eng. 1997; 10: 1221-1225Crossref PubMed Scopus (84) Google Scholar). Intrabodies present a potent alternative to methods of gene inactivation that target at the level of DNA or mRNA, such as antisense (10Wagner R.W. Flanagan W.M. Mol. Med. Today. 1997; 3: 31-38Abstract Full Text PDF PubMed Scopus (87) Google Scholar), zinc finger proteins (11Beerli R.R. Barbas III, C.F. Nat. Biotechnol. 2002; 20: 135-141Crossref PubMed Scopus (405) Google Scholar), targeted gene disruption, or the relatively new RNA interference (12Hannon G.J. Nature. 2002; 418: 244-251Crossref PubMed Scopus (3543) Google Scholar). Operating at the posttranslational level, intrabodies can be directed to relevant subcellular compartments and precise epitopes on target proteins (2Beerli R.R. Wels W. Hynes N.E. J. Biol. Chem. 1994; 269: 23931-23936Abstract Full Text PDF PubMed Google Scholar, 13Marasco W.A. Gene Ther. 1997; 4: 11-15Crossref PubMed Scopus (86) Google Scholar, 14Bai J. Sui J. Zhu R.Y. St. Clair Tallarico A. Gennari F. Zhang D. Marasco W.A. J. Biol. Chem. 2003; 278: 1433-1442Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), potentially blocking only one out of several functions of an expressed protein. Numerous studies have reported the development of engineering antibodies that are both multispecific and multivalent (15Todorovska A. Roovers R.C. Dolezal O. Kortt A.A. Hoogenboom H.R. Hudson P. J. Immunol. Methods. 2001; 248: 47-66Crossref PubMed Scopus (246) Google Scholar, 16Hudson P. Souriau C. Nat. Med. 2003; 9: 129-134Crossref PubMed Scopus (404) Google Scholar). Applying this knowledge, the goal of our study was to develop an ER-targeted intrabody format for the simultaneous down-regulation of two independent cell surface receptors, in order to investigate the biological consequences of knocking down two receptor-tyrosine kinases. To accomplish this, an ER-targeted tetravalent antibody construct, with dual specificity, was generated. Using a recombinant adenovirus as gene delivery system, we could show that this intrabody construct, termed here an "intradiabody," was expressed in the ER and able to trap both targeted proteins in the same compartment. The intradiabody targets the endothelial transmembrane receptors Tie-2 and VEGF-R2, which are essential for angiogenesis. The interplay of VEGF, VEGF-R2, Tie-2, and Ang-1 and -2 has been suggested as a key modulator in the onset of tumor angiogenesis (17Holash J. Maisonpierre P.C. Compton D. Boland P. Alexander C.R. Zagzag D. Yancopoulos G.D. Wiegand S.J. Science. 1999; 284: 1994-1998Crossref PubMed Scopus (1915) Google Scholar). According to this model, Tie-2 is constitutively engaged with Ang-1 in quiescent blood vessels. The Tie-2/Ang-1 complex stabilizes quiescent blood vessels by promoting their interaction with surrounding perivascular cells, smooth muscle cells, and the extracellular matrix. The constitutive Tie-2/ Ang-1 complex is antagonized by Ang-2, which is up-regulated in endothelial cells that are proximal to the tumor. By competing with Ang-1 for Tie-2 binding, Ang-2 destabilizes the interaction of endothelial cells and their microenvironment. This is thought to sensitize the endothelial cells to VEGF signaling. Thus, Ang-2 produced by endothelial cells promotes tumor angiogenesis in concert with VEGF produced by the tumor. Comparison of the effect of the intradiabody (targeting Tie-2 and VEGF-R2) with the corresponding conventional scFv intrabodies (targeting Tie-2 or VEGF-R2 alone) revealed a remarkable superiority of the intradiabody, as represented by a complete and extended surface depletion of both Tie-2 and VEGF-R2. This finding can be attributed to the extended half-life of our intradiabody, as determined by pulse-chase studies. In addition, we show that the intradiabody strongly inhibits endothelial tube formation beyond that seen with inhibitors of either receptor-tyrosine kinase alone, thus confirming its antiangiogenic properties. Our results confirm that the inhibition of the VEGF receptor pathway cannot be compensated by the Tie-2 pathway, nor vice versa (18Siemeister G. Schirner M. Weindel K. Reusch P. Menrad A. Marme D. Martiny-Baron G. Cancer Res. 1999; 59: 3185-3191PubMed Google Scholar), but also demonstrates that targeting both pathways simultaneously results in additive antiangiogenic effects in vitro. Combining the specific and high affinity binding properties of our intradiabody with its ability to be stably expressed in the ER, we identified a very effective intrabody format for the simultaneous functional knockout of two cell surface receptors. single-chain antibody fragment endoplasmatic reticulum human umbilical vein endothelial cells angiopoietin-1 or -2 heavy chain variable domain light chain variable domain vascular endothelial growth factor enzyme-linked immunosorbent assay phosphate-buffered saline hemagglutinin multiplicity of infection fluorescence-activated cell sorting green fluorescent protein. Cell Culture—Human umbilical vein endothelial cells (HUVEC) (BioWhittaker, Walkersville, MD) were cultured at 37 °C in 5% CO2 in EGM medium (BioWhittaker) supplemented with 2% bovine brain extract (BioWhittaker). 293 cells (human embryonic kidney) (ATCC) cells were cultured at 37 °C in 5% CO2, in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and 1% antibiotics. Library Generation and Selection—The generation and selection of rabbit/human chimeric Fab libraries, as well as the characterization of positive Fabs, were done essentially as described (19Barbas III, C.F. Burton D.R. Scott J.K. Silverman G.J. Phage Display: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001: 9.1-9.113Google Scholar, 20Popkov M. Mage R.G. Alexander C.B. Thundivalappil S. Barbas III, C.F. Rader C. J. Mol. Biol. 2003; 325: 325-335Crossref PubMed Scopus (86) Google Scholar). In brief, rabbit Vκ-, Vλ-, and VH-encoding sequences were amplified from first strand cDNA and fused to human Cκ- and CH1-encoding sequences, respectively, followed by assembly of chimeric rabbit/human light chain- and Fd fragment-encoding sequences and by asymmetric SfiI cloning into phagemid vector pComb3X. Libraries were panned against VEGF-R2/VEGF complex immobilized on Costar 3690 96-well ELISA plates (Corning, Acton, PA). Four rounds of panning (19Barbas III, C.F. Burton D.R. Scott J.K. Silverman G.J. Phage Display: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001: 9.1-9.113Google Scholar, 21Rader C. Ritter G. Nathan S. Elia M. Gout I. Jungbluth A.A. Cohen L.S. Welt S. Old L.J. Barbas III, C.F. J. Biol. Chem. 2000; 275: 13668-13676Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) were carried out using 500 ng of VEGF-R2/VEGF complex in the first round, 250 ng in the second round, and 100 ng of VEGF-R2/VEGF complex in the third and fourth rounds. To eliminate the selection of clones that bind to the human IgG1 Fc part of the recombinant human VEGF-R2/Fc fusion protein, 2.5 mg/ml human IgG (Pierce) was added to the phage preparations during selection. After the final round of panning, 10 isopropyl-1-thio-β-d-galactopyranoside-induced clones from each library were analyzed for binding to 100 ng of immobilized VEGF-R2, human IgG, and bovine serum albumin by ELISA using a rat anti-HA monoclonal antibody conjugated to horseradish peroxidase for detection. Positive clones were analyzed by DNA fingerprinting and sequencing as described before (20Popkov M. Mage R.G. Alexander C.B. Thundivalappil S. Barbas III, C.F. Rader C. J. Mol. Biol. 2003; 325: 325-335Crossref PubMed Scopus (86) Google Scholar). Soluble Fab were expressed from gene III fragment-depleted phagemid vector pComb3X and purified using goat anti-human F(ab′)2N-hydroxysuccinimide resin columns as described (19Barbas III, C.F. Burton D.R. Scott J.K. Silverman G.J. Phage Display: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001: 9.1-9.113Google Scholar). Library generation and selection for Tie-2 clones is described elsewhere (20Popkov M. Mage R.G. Alexander C.B. Thundivalappil S. Barbas III, C.F. Rader C. J. Mol. Biol. 2003; 325: 325-335Crossref PubMed Scopus (86) Google Scholar). For a control construct, designated T2V2, one clone was selected from each generated library (VEGF-R2 and Tie-2) prior to selection. ELISA was done the same way as for detection of binders after panning, determining no binding to VEGF-R2 or Tie-2. Surface Plasmon Resonance—Surface plasmon resonance for the determination of association (kon) and dissociation (koff) rate constants for the interaction of chimeric rabbit/human Fab with Tie-2 was performed on a Biacore instrument (Biacore AB, Uppsala, Sweden). A CM5 sensor chip (Biacore AB) was activated for immobilization with N-hydroxysuccinimide and N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide according to the methods outlined by the supplier. Recombinant human VEGF-R2/Fc fusion protein was coupled at a low density (500–1000 resonance units) to the surface by injection of 10–20 μl of a 10 ng/μl sample in 20 mm sodium acetate (pH 3.5). Subsequently, the sensor chip was deactivated with 1 m ethanolamine hydrochloride (pH 8.5). Binding of chimeric rabbit/human Fab to immobilized human VEGF-R2 was studied by injection of Fab at five different concentrations ranging from 50 to 150 nm. PBS was used as the running buffer. The sensor chip was regenerated with 20 mm HCl and remained active for at least 20 measurements. The kon and koff values were calculated using Biacore AB evaluation software. The equilibrium dissociation constant Kd was calculated from koff/kon. Conversion of a VEGF-R2 and a Tie-2-specific Fab into a Single-chain Antibody Fragment (scFv)—Specific oligonucleotide primers (19Barbas III, C.F. Burton D.R. Scott J.K. Silverman G.J. Phage Display: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001: 9.1-9.113Google Scholar, 22Rader C. Popkov M. Neves J.A. Barbas III, C.F. FASEB J. 2002; 16: 2000-2002Crossref PubMed Scopus (48) Google Scholar) were used to amplify VH and VL gene segments from purified phagemid DNA of Fab VC06 and Fab 1S05. VL of VC06 and 1S05 were amplified with extompseq (5′-GCG GAG GAG CTT GCT AGC TGC GAG AAG ACA GCT ATC GCG ATT GCA TGT) and RJλO-BL. VH of VC06 and 1S05 were amplified with RSCVH4 or RSCVH3, respectively, and HSCG1234-B. Overlap extension PCR was done using primers ext and RSC-B. The resulting overlap-PCR product encodes an scFv in which the C-terminal VL region is linked to the N-terminal VH region through a peptide linker (SSGGGGSGGGGGGSSRSS). Control construct JC7U (against integrin αvβ3) was constructed as described (22Rader C. Popkov M. Neves J.A. Barbas III, C.F. FASEB J. 2002; 16: 2000-2002Crossref PubMed Scopus (48) Google Scholar). The scFv encoding sequences were cloned into phagemid vector pComb3X using asymmetric SfiI sites and binding activity of the expressed scFv was confirmed by ELISA. Cloning of Diabodies and Corresponding scFv—The scFv 1S05 and VC06 genes were linked through the second and third heavy chain constant domains of human IgG1 to generate a bispecific diabody. ScFv 1S05 was PCR-amplified using extompseq and Tie-2-B (5′-GCC AGA CCC ACC GCC TCT AGA TGA GGA GAC GGT GAC CAG GGT G-3′). CH2-CH3 was amplified using CH2-F (5′-TCT AGA GGC GGT GGG TCT GGC GGG GGC TCG-3′) and CH3-B (5′-CGA CTG AGT CAG CAC GAG CTC GGC CGC CTG TGC CGA GCC ACC CCC AGA ACC-3′). ScFv VC06 was amplified using VEGF-R2-F (5′-TCT CCG GGT GGC GCG CCT GGT GGC GGT TCT GGC GGT GGT TCT GGG GGT GGC TCG GCA CAG GCG GCC GAG CTC GTG CTG ACT CAG TCG CCC TC-3′) and dpseq (19Barbas III, C.F. Burton D.R. Scott J.K. Silverman G.J. Phage Display: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001: 9.1-9.113Google Scholar). ScFv 1S05 and CH2-CH3 were combined by overlap extension PCR using primer ext (22Rader C. Popkov M. Neves J.A. Barbas III, C.F. FASEB J. 2002; 16: 2000-2002Crossref PubMed Scopus (48) Google Scholar) and CH3-B. ScFv VC06 was SacI/SpeI-cloned into phagemid vector pComb3X. Finally, both constructs were combined by SacI cloning and confirmed by DNA sequence analysis. The T2 scFv was amplified using primers extompseq and T2-B (5′-GCC AGA CCC ACC GCC TCT AGA TGA GGA GAC GGT GAC CAG-3′), CH2-CH3 was amplified with primers CH2/T2-F (5′-TCT AGA GGC GGT GGG TCT GGC GGG GGC TCG-3′) and CH3/V2-B (5′-AGT CTG GGT CAT CAC GAG CTC GGC CGC CTG TGC CGA GCC ACC CCC AGA ACC-3′), and the scFv for V2 was amplified using dpseq and primer V2-F (5′-TCT GGG GGT GGC TCG GCA CAG GCG GCC GAG CTC GTG ATG ACC CAG ACT-3′). scFv T2 and CH2-CH3 were combined by overlap extension PCR using ext and CH3/V2-B. ScFv V2 was SacI/SpeI cloned into phagemid vector pComb3X. Both constructs were then combined in the same manner as the Tie-2/VEGF-R2 intrabody. Assembly of Intrabody Constructs in pAdTrackCMV and Generation of Adenoviral Plasmids by Homologous Recombination—Intrabody coding regions were initially assembled in pBabePuro essentially as described (23Steinberger P. Andris-Widhopf J. Buehler B. Torbett B.E. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 97: 805-810Crossref Scopus (110) Google Scholar). In these constructs, the scFv coding regions are flanked by a human κ light chain leader sequence at the 5′-end and a sequence encoding the HA tag (YPYDVPDYA) and the ER retention signal (KDEL) at the 3′-end. The intrabody coding regions were then excised by digestion with BamHI and SalI and ligated into pAdTrackCMV (24He T. Zhou S. Da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3256) Google Scholar) digested with BglII and SalI. This adapter fragment contains also compatible SfiI sites, which were used for cloning the different intrabodies (intradiabody against Tie-2/VEGF-R2, control intradiabody T2V2, scFv intrabody against Tie-2, and scFv intrabody against VEGF-R2) into the adenovirus vector. The generation of recombinant adenoviruses was done essentially as described (24He T. Zhou S. Da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3256) Google Scholar). High titer viral stocks were produced and purified by CsCl banding. All virus preparations were GFP-corrected (25Hitt D.C. Booth J.L. Dandpani V. Pennington L.R. Gimble J.M. Metcalf J. Mol. Biotechnol. 2000; 14: 197-203Crossref PubMed Scopus (26) Google Scholar). Infection of HUVEC with Intrabodies Using Recombinant Adenoviruses—1.5 × 106 HUVEC were infected with 10 MOI (∼80% of the cells were infected) of adenovirus encoding Tie-2/VEGF-R2-specific intradiabody, Tie-2-specific intrabody, VEGF-R2-specific intrabody, control intrabody, or no insert (mock). Tie-2 and VEGF-R2 expression of HUVEC cells was monitored for 15 days by flow cytometry. Flow Cytometry—HUVEC were washed once with Hepes-buffered saline solution, trypsinized, washed once with PBS, and resuspended at 106 cells/ml in FACS buffer (1% (w/v) bovine serum albumin, 0.03% (w/v) NaN3, 25 mm Hepes in PBS, pH 7.4). 105 cells were stained with biotinylated Tie-2 or VEFG-R2 polyclonal antibodies (R&D Systems, Minneapolis, MN) (1:200) in FACS buffer, followed by streptavidin-APC (1:100). Incubation times were 30 min at room temperature. Flow cytometry was performed using a FACSort instrument from Becton-Dickinson measuring at FL4. For control purposes, cells were also stained with LM609 (αvβ3) and P1F6 (αvβ5) (Chemicon, Temecula, CA) and detected with biotinylated donkey anti-mouse antibody followed by streptavidin-APC. The formula for calculations of surface inhibition, after subtracting the background of normal goat IgG-stained cells was as follows: VEGF-R2GFP or Tie-2GFP minus VEGF-R2intrabody or Tie-2intrabody, divided by VEGF-R2GFP or Tie-2GFP. Immunocytochemical Analysis of Antigen/Intrabody Colocalization— For analysis of Tie-2, VEGF-R2, and intrabodies on GFP-positive HUVEC, cells were seeded on collagen-coated Lab-Tek coverglasses and infected with 50 MOI of adenovirus encoding intradiabody or control intrabody (JC7U). Forty-eight hours postinfection, HUVEC were washed with copious amounts of PBS, followed by incubation in a humidifying chamber at room temperature for 1 h with a primary antibody mixture of rat anti-HA monoclonal antibody (5 μg/ml; Roche Applied Science) and biotinylated goat anti-human Tie-2 or anti-human VEGF-R2 polyclonal antibodies (R&D Systems). The cells were then stained for 1 h at room temperature with the mixture of Cy5-conjugated donkey anti-rat IgG polyclonal antibodies and streptavidin/rhodamine red-X (both from Jackson Immunoresearch, West Grove, PA) diluted to 1:100 in FACS buffer, 0.1% saponin. Finally, the cells were covered with SlowFade Antifade reagent. Three-color (GFP, rhodamine red-X, and Cy5) three-dimensional data sets were collected with a DeltaVision system (Applied Precision, Issaquah, WA); this consisted of an Olympus IX-70 fluorescence microscope, a motorized high precision xyz stage, a 100-watt mercury lamp, and a KAF1400 chip-based cooled chargecoupled device camera. Exposure times were 0.2–0.5 s (2-binning), and images were obtained with a ×60 oil objective. Three-dimensional reconstructions were generated by capturing 150-nm serial sections along the z axis. Images were deconvolved (based on the Agard-Sadat inverse matrix algorithm) and analyzed with softWorX version 2.5. Pulse-chase for Determination of Half-life—HUVEC cells were seeded at a density of 1 × 106 cells in T175 flasks and infected 24 h later with recombinant adenoviruses using an MOI of 10. After infection for 24 h, cells were washed with Hepes-buffered saline solution and trypsinized. Cells were starved for 2 h in 10 ml of serum-free, methionine-free, cysteine-free minimal essential medium at 37 °C and swirled periodically. Samples were then labeled with Tran35S-label medium (50 μCi/ml; ICN, Aurora, OH) for 2 h at 37 °C and subsequently chased with EGM medium, containing a 40-fold excess of methionine and 20-fold excess of cysteine for various time points (0, 4, 8, 16, 24, and 48 h). At each time point, cells were washed once with ice-cold PBS containing 1 mm phenylmethylsulfonyl fluoride and lysed (Promega lysis buffer, containing complete protease inhibitor mixture). Supernatants were collected and stored at –80 °C. Samples were precipitated using protein G and monoclonal anti-HA antibody (Covance) for scFv or protein G alone for the diabody. Immunocomplexes were washed twice with lysis buffer and twice with TBS, boiled in 1.5× SDS loading buffer, and separated by a 4–20% gradient SDS-PAGE gel under reducing conditions. The gels were stained, dried, and exposed to autoradiography as well as quantitatively analyzed with a PhosphorImager. Endothelial Cell Tube Formation Assay—6 × 104 HUVEC cells were infected with recombinant adenovirus using an MOI of 20 in an Eppendorf tube for 45 min and were then transferred to 6-well plates. Three days after infection, cells were washed with Hepes-buffered saline solution, trypsinized, and counted. 2 × 104 cells/well (volume 50 μl) in complete EGM medium were seeded in triplicates for each virus in a 96-well plate coated with Matrigel Basement Membrane Matrix (BD Bioscience, Bedford, MA) and incubated for 15.5 h. Cells were then stained and fixed with the Diff-Quik® staining set (DADE BEHRING Inc., Newark, DE). For this, cells were fixed with Diff-Quik Fixative, followed by Diff-Quik Solution I and II, each with 100 μl for 2–3 min. Cells were then washed four times with distilled H2O, and pictures were taken under an inverted light microscope at ×2 magnification. The number of tube branches for each virus was counted in triplicates to calculate the average ± S.D. Cell Proliferation Assay—HUVEC cells were seeded (5 × 104) in 96-well plates in complete EGM medium and infected with different recombinant adenoviral constructs with an MOI of 10 and 50. Three days later, the cells were trypsinized, stained with trypan blue, and counted in triplicate by a hemacytometer. Cell proliferation was also measured by adding [3H]thymidine (ICN Radiochemicals) in a concentration of 0.5 μCi/well (1 Ci = 37 GBq) during the last 24 h of incubation. The cells were frozen at –80 °C overnight and subsequently processed on a multichannel automated cell harvester (Cambridge Technology, Cambridge, MA) and counted in a liquid scintillation β-counter (Beckman Coulter). The background was defined by uninfected cells. The inhibition was calculated according to the following formula: (background – infected cell count)/background × 100%. Selection of Rabbit Fabs Binding to VEGF-R2—Using phage display, several New Zealand White rabbit Fabs against VEGF-R2 were selected by panning (19Barbas III, C.F. Burton D.R. Scott J.K. Silverman G.J. Phage Display: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001: 9.1-9.113Google Scholar) chimeric rabbit/human antibody libraries (20Popkov M. Mage R.G. Alexander C.B. Thundivalappil S. Barbas III, C.F. Rader C. J. Mol. Biol. 2003; 325: 325-335Crossref PubMed Scopus (86) Google Scholar, 21Rader C. Ritter G. Nathan S. Elia M. Gout I. Jungbluth A.A. Cohen L.S. Welt S. Old L.J. Barbas III, C.F. J. Biol. Chem. 2000; 275: 13668-13676Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) on a human VEGF-R2·VEGF complex. For further analysis, chimeric rabbit/human Fab VC06 was selected and produced as soluble Fab in Escherichia coli and purified by affinity chromatography using goat anti-human F(ab′)2N-hydroxysuccinimide resin columns. Fab VC06 demonstrated a strong binding to human VEGF-R2 in ELISA and to HUVEC in flow cytometry (not shown). Thus, the VEGF-R2 epitope recognized by VC06 is displayed by native VEGF-R2 expressed on the cell surface and is an accessible target for antiangiogenic therapy. Surface plasmon resonance studies of VC06 revealed that it possessed a monovalent dissociation constant of ∼1 nm to human VEGF-R2, whereas Fab 1S05, which we described earlier (20Popkov M. Mage R.G. Alexander C.B. Thundivalappil S. Barbas III, C.F. Rader C. J. Mol. Biol. 2003; 325: 325-335Crossref PubMed Scopus (86) Google Scholar), bound with a dissociation constant of 14 nm to human Tie-2 (Table I).Table IBinding parameters of chimeric rabbit/human Fab 1S05 directed to human Tie-2 and Fab VC06 directed to human VEGF-R2Fabkon/104koff/10-4Kdm-1s-1s-1nm1S059.813.513.8VC064.60.531.2 Open table in a new tab Generation of an Intradiabody against Tie-2 and VEGF-R2 and scFv Intrabodies against Tie-2 or VEGF-R2 Alone—Fabs were converted into scFv, in which the VL and VH fragments were covalently linked with a peptide linker consisting of 18 amino acids. Preserved binding to their respective antigens was confirmed for both scFv VC06 (VEGF-R2) and scFv 1S05 (Tie-2) by ELISA (not shown). Next, scFv 1S05 and scFv VC06 were linked through the second and third heavy chain constant domains of human IgG1, resulting in a scFv-CH2-CH3-scFv expression cassette. As a key feature, the scFv-CH2-CH3-scFv expression cassette provides for the production of a bifunctional tetravalent antibody construct from a single polypeptide. Through the homophilic interaction of CH3, two scFv-CH2-CH3-scFv molecules associate to form a 150-kDa dimer, which displays both the N-terminal and C-terminal scFv module bivalently as the intradiabody (Fig. 1). The sc
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