Toxicity was determined by determining the body weight of the mice once a week. growth and demonstrated the potential of simultaneous targeting of multiple pathways as a therapeutic strategy. angiogenesis, leading to the hypothesis that simultaneous interference with both pathways should result in additive effects on tumor growth (14, 17). To date, no studies have reported the effect of simultaneous VEGF-R2/Tie-2 antagonism characterization of a human reactive tetravalent endoplasmic reticulum (ER)-targeted intradiabody for the simultaneous surface depletion of two endothelial transmembrane receptors, VEGF-R2 and Tie-2 (17). Because tumor progression is a process involving multiple stages and pathways with built-in redundancies, a potent antitumor strategy should inhibit multiple independent targets. This Mouse monoclonal to TGF beta1 study was performed to test whether a simultaneous phenotypic knockout of VEGF-R2 and Tie-2 is more powerful in antitumor and antiangiogenic therapy than a phenotypic knockout of VEGF-R2 alone. Because VEGF-R2 has attracted so much attention as a target for antiangiogenic therapy, we choose to compare the knockout of VEGF-R2 to the double Ac-Gly-BoroPro knockout of VEGF-R2 and Tie-2. To accomplish this comparison, ER-targeted bispecific (VEGF-R2/Tie-2) and monospecific (VEGF-R2) tetravalent intradiabodies Ac-Gly-BoroPro were generated, and a replication-deficient adenoviral vector was used for local delivery in a melanoma xenograft mouse model. Our results indicate that the simultaneous targeting of the VEGF receptor pathway and the Tie-2 pathway results in a greater antitumor and antiangiogenic effect than targeting VEGF-R2 alone and that the intradiabody approach is an efficient means of blocking two receptors. Materials and Methods Cell Culture. Human umbilical vein-derived endothelial cells (HUVEC) were maintained in endothelium growth medium supplemented with 2% bovine brain extract (BioWhittaker). Human embryonic kidney 293 cells (American Type Culture Collection) were cultured in DMEM supplemented with 10% FBS (HyClone) and 1% antibiotics. Human melanoma cell line M21 (obtained from Donald Morton, John Wayne Cancer Center, Santa Monica, CA) was maintained in RPMI medium 1640, containing 10% FCS and 1% antibiotics. Mouse endothelial cell line MS1 (American Type Culture Collection) was maintained in DMEM supplemented with 4 mM l-glutamine/1.5 g/liter sodium bicarbonate/4.5 g/liter glucose/1 mM sodium pyruvate/10% FCS/1% antibiotics. Immortalized human microvasculuture endothelial cells (HMEC-1) were obtained from Centers for Diseases Control/Emory University (25). Cells were maintained in endothelial basal medium (BioWhittaker), supplemented with 10 ng/ml epidermal growth factor/1 g/ml hydrocortisone/10% FCS/1% antibiotics. All cells were cultured at 37C in 5% CO2. Antibodies and Other Proteins. Recombinant human VEGF-R2 (KDR)/Fc chimeric protein, recombinant mouse VEGF-R2 (Flk-1)/Fc chimeric protein, recombinant human Tie-2/Fc chimeric protein, mouse Tie-2/Fc chimeric protein, biotinylated goat anti-human VEGF-R2 and Tie-2 polyclonal antibodies, and Ac-Gly-BoroPro biotinylated normal goat IgG were purchased from R & D Systems. Rat anti-mouse CD31 (PECAM1) mAb, APC-conjugated streptavidin, and biotinylated goat anti-rat IgG polyclonal antibodies were purchased from Pharmingen. Horseradish peroxidase-conjugated goat anti-human kappa antibody was from Southern Biotechnology Associates. Chimeric rabbit/human Fabs VR05 that binds to the extracellular domain of VEGF-R2 and 2SO8b that recognizes the extracellular domain of Tie-2 were generated as described in refs 26 and 27. Analysis of VEGF-R2 and Tie-2 Binding in ELISA. Binding studies by ELISA for Fab VR05 and 2S08b were performed as described in refs. 26C28. Conversion of a VEGF-R2 and Tie-2-Specific Fab into a scFv. Specific oligonucleotide primers (26, 29, 30) were used to amplify heavy chain variable domain and light chain variable domain gene segments from purified phagemid DNA of Fab VR05 and Fab 2S08b. Light chain variable domain segments of VR05 and 2S08b were amplified with ompseqgtg and RKB9Jo-BL. Heavy chain variable domain regions of VR05 and 2S08b were amplified with RSCVH1 or RSCVH4, respectively, and HSCG1234-B. Overlap extension PCR was done by using primers ompseq and RSC-B. The resulting overlap-PCR products encode a scFv in which the C-terminal light chain variable domain region is linked.
