Generation of Monoclonal Antibodies Against Mouse VEGFR2
Monoclonal hybridomas were generated by fusing splenocytes from immunized rats with 653CrmA
cells or 653 cells. The initial screening of supernatants derived from 653CrmA
fusants on immobilized Flk-1 antigen in ELISA yielded 110 wells having supernatants that were highly positive (higher than 2 OD), compared with only two wells in a similarly performed fusion deriving from the 653 cells. The higher fusion efficiency of the CrmA-transfected myeloma cells has been observed in several fusions and is possibly due to stable expression of the antiapoptotic protein, CrmA [24
], by myeloma partner cells. From this extensive primary pool, we selected eight stable clones (RAFL-1 to RAFL-8) secreting high-affinity antibodies with diverse functional properties. All the antibodies were rat IgG2a
. All but RAFL-8 had κ light chain.
Binding of Anti-VEGFR2 Antibodies to Immobilized Soluble Domain of Flk-1 in ELISA
RAFL-1 to RAFL-8 antibodies bound strongly and specifically to sFlk-1 in ELISA. Half-maximal binding was observed at concentrations that ranged between 10 and 67 pM (). RAFL-4 was the antibody having the strongest binding from this panel with a half-maximal binding of 10 pM. All antibodies reached saturation at concentrations of 0.2 to 0.4 nM. None of the antibodies reacted with purified mouse Flt-1 or Flt-4 proteins, which have structural similarity to Flk-1 (data not shown).
Figure 1 Binding of RAFL antibodies to mouse VEGFR2 in ELISA. The extracellular domain of mouse VEGFR2 was immobilized on plastic by incubating 96-well plates with 1 µg/ml purified protein. RAFL antibodies were added at concentrations ranging from 6.7 (more ...)
Recognition of Mouse VEGFR2 Expressed on Surface of Cultured EC
The ability of RAFL antibodies to bind to VEGFR2 on intact fixed or unfixed mouse bEnd.3 endothelial cells was examined. RAFL-1, RAFL-5, and RAFL-8 stained unfixed cells but not cells after glutaraldehyde fixation, indicating preferential recognition of native epitope(s). RAFL-6 stained fixed cells but not unfixed cells, suggesting that it recognizes an epitope in denatured VEGFR2. RAFL-2, RAFL-3, and RAFL-7 bound equally well to native and fixed cells (). RAFL-4 did not stain fixed or unfixed cells.
Functional Properties of Rat Monoclonal Antibodies Against VEGFR2.
RAFL antibodies bound preferentially to cells in mitosis (, A and B, arrows). Strong binding to cells during metaphase () and telophase () was observed. Nondividing cells, having distinct nuclear membranes and nucleoli, were more weakly stained (, arrowheads). In contrast, antimouse CD31 antibody uniformly stained all endothelial cells with strong intensity regardless of stage of division (data not shown). This observation suggests that VEGFR2 on endothelial cells is upregulated during mitotic division.
Figure 2 Binding of RAFL-1 and RAFL-6 antibodies to mouse endothelial cells. Cultures of bEnd.3 endothelial cells, which contained both dividing and nondividing cells, were grown on sterile glass slides. Binding of RAFL antibodies to unfixed cells (A) or fixed (more ...)
Blocking of Binding of [125I]VEGF165 to VEGFR2 on Mouse Endothelial bEnd.3 Cells
The six RAFL antibodies that bound to intact unfixed cells were tested for their ability to block the binding of [125I]VEGF165 to VEGFR2 on bEnd.3 cells. We established in prior experiments that bEnd.3 cells express approximately equal numbers of Flk-1 and Flt-1 receptors. This is based on the observation that both antimouse Flk-1 antibody TO14 and antimouse Flt-1 antibody 10.2, when added at a saturating concentration of 10 µg/ml, were able to compete out only ~50% of [125I]VEGF165 binding (). Thus, antibodies reactive exclusively to the Flk-1 receptor are expected to block the binding of VEGF by a maximum of 50%. Noncompeting anti-Flk-1 antibody, 1A8 (10 µg/ml), reduced the binding of [125I]VEGF165 by 10% under the same conditions, indicating that 90% of [125I]VEGF165 was binding specifically to cell surface receptors.
Figure 3 Inhibition of [125I]VEGF165 binding to the surface of endothelial cells by RAFL antibodies. Log phase or confluent endothelial cell cultures were incubated with [125I]VEGF165 (25 pM) in the presence of various RAFL antibodies; nonblocking anti-VEGFR2 (more ...)
RAFL-1, RAFL-2, RAFL-3, RAFL-5, RAFL-7, and RAFL-8 inhibited the binding of [125I]VEGF165 to dividing, sparsely seeded endothelial cells by 33% to 55%, indicating that RAFL antibodies are efficient blockers of VEGF interaction with R2. RAFL-2 was the most efficient antibody in this assay (). All competing antibodies had little or no effect on [125I]VEGF165 binding to confluent monolayers (). This is in accord with the above observation that dividing endothelial cells have higher surface expression of VEGFR2 than nondividing cells ().
Binding to Vascular Endothelial Cells on Frozen Tumor Sections
The ability of RAFL antibodies to recognize VEGFR2 on tissue sections was of particular interest because of the paucity of available reagents having this ability. Two of the RAFL antibodies (RAFL-1 and RAFL-2) showed strong recognition of vascular endothelial cells on frozen tumor sections, ranked as 4+ (, ). Four additional antibodies (RAFL-3, RAFL-5, RAFL-6, and RAFL-7) had moderate binding, ranked as 2+. RAFL-4 antibody, which had high affinity for immobilized Flk-1 in ELISA, had no recognition of the cell surface receptor and did not stain tumor vessels.
Figure 4 Detection of VEGFR2 on vasculature of various tumors by RAFL-1 antibody. NCI-H358 human nonsmall lung carcinoma and L540 Hodgkin's disease tumors were grown in SCID mice. Meth A mouse fibrosarcoma and 3LL mouse lung carcinomas were grown in BALB/c and (more ...)
Six different tumors growing in mice were tested for staining with RAFL-1, RAFL-2, RAFL-3, and RAFL-5. The tumors were NCI-H358 human NSCLC (), L540 human Hodgkin's disease (), Meth A mouse fibrosarcoma (), 3LL mouse lung tumor (), orthotopic MDA-MB-431 human breast carcinoma (), and orthotopic L3.6pl human pancreatic carcinoma (). The four antibodies gave similar patterns of staining in each individual tumor. They stained moderately to strongly 50% to 80% of vessels expressing CD31, a pan-mouse endothelial marker (). This corresponds to 50 to 160 vessels/mm2
. Rat IgG2a
of irrelevant specificity did not stain tumor sections. TO14, a rabbit polyclonal antibody to mouse VEGFR2 [23
], showed the same intensity and pattern distribution of vascular staining as did RAFL antibodies. However, TO14 cross-reacts with cytosolic components of the human tumor cells, giving a high background. In contrast, RAFL antibodies specifically recognized mouse tumor blood vessels.
Figure 5 Biodistribution of RAFL-1 antibody in mice bearing orthotopic MDA-MB-231 human breast xenografts. Nu/nu mice bearing MDA-MB-231 tumors in their MFPs were injected intravenously with 50 µg of RAFL-1 antibody. One hour later, their blood circulation (more ...)
Figure 6 Expression of VEGFR2 on vessels of normal mouse pancreas and orthotopically implanted human pancreatic tumor, L3.6pl. Human pancreatic tumor, L3.6pl, was orthotopically grown in nude mice. Frozen sections of normal pancreas from nontumor bearing mice (more ...)
Binding to Vascular Endothelial Cells on Sections of Normal Tissues
A limited panel of normal tissues including heart, lung, liver, kidney, pancreas, and brain was examined with RAFL antibodies by direct staining. Vessels in the heart and lungs (), liver, and brain cortex (not shown) were not visibly stained. In the kidney, RAFL antibodies discretely stained glomerular endothelium (). In the pancreas, most of the CD31-positive vessels in the islets were positive for VEGFR2 (). Vascular expression of VEGFR2 in normal kidney glomeruli and pancreas was also confirmed by a rabbit anti-VEGFR2 antibody, TO14.
Localization of Anti-VEGFR2 Antibodies to Tumor Vessels in Orthotopic MDA-MB-231 Breast Tumors in Mice
RAFL-1, RAFL-2, RAFL-3, and a control rat IgG2a antibody were intravenously injected into nude mice with human MDA-MB-231 breast tumors growing in their MFPs. One hour later, the mice were sacrificed and their blood circulation was perfused to remove free antibody. Frozen sections were prepared from the tumor and normal organs and examined immunohistochemically to determine the localization of the RAFL antibodies or control rat IgG (). About 60% of tumor vessels that were positive for CD31 were also positive for localized RAFL-1, RAFL-2, and RAFL-3. Vessels with bound RAFL antibodies were homogeneously distributed within the tumor vasculature. All VEGFR2-positive vessels detected by direct staining of tumor sections also had localized RAFL-1, indicating that all VEGFR2-positive vessels were accessible to intravenously administered antibody. Vessels in normal organs were unstained, with the exception of the kidney glomeruli () and the pancreas (). Control rat IgG did not localize to tumor or normal vessels in any of the mice, indicating specificity of detection of anti-VEGFR2 antibodies.
Inhibition of Tumor Vasculature by RAFL-1 Antibody in MDA-MB-231 Breast Cancer Model
Groups of six tumor-bearing mice were treated with either saline or RAFL-1. Mice were sacrificed when the mean tumor volume of the control and treated groups reached 1250 and 640 mm3, respectively. Treatment with RAFL-1 reduced the tumor volume by an average of 48%. Tumors from all treated and control mice were excised and microvessel density and morphology was assessed as described under Materials and Methods section.
The mean number of vessels per square millimeter was 164±7 and 57±5 in control and RAFL-1-treated groups, respectively. Vessels in control tumors mainly consisted of capillaries that were homogeneously distributed in all regions except necrotic areas. The vessel phenotype, number, and distribution of tumors from RAFL-1-treated mice were strikingly different from those of control tumors (). Microvasculature was almost absent from treated tumors and residual vessels mainly consisted of mid-sized mature vessels coated by smooth muscle cells/pericytes. Necrotic areas occupied 80% to 90% of cross sections, leaving only a rim of viable cells of approximately 130 µm in width (). No effects were observed on the vascularization of normal MFP epithelium adjacent to treated tumors. RAFL-1-treated and control mice had similar numbers and patterns of vessels in normal MFP epithelium as well as in other normal organs. These observations indicate that RAFL-1 treatment specifically inhibits VEGF-dependent angiogenesis in tumors.
Figure 7 RAFL-1 antibody reduces microvascular density in MDA-MB-231 human breast tumors growing in the MFP of mice. Tumors from RAFL-1-treated (A) or rat IgG control antibody-treated (B) mice were removed, frozen sections were cut, and microvessels were detected (more ...)