Vascular endothelial cell growth factor variants and use thereof
A variant, cell technology, applied in the field of VEGF variants, which can solve problems such as decreased affinity of FLT-1
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Embodiment 1
[0174] Screening of KDR-specific VEGF variants
[0175] To generate KDR-specific variants, two phage libraries were constructed in which VEGF(1-109) residues important for Flt-1 binding but not KDR binding were randomly mutated.
[0176] Phagemid construction
[0177] To construct the phage library, phagemid vectors with cDNA encoding VEGF residues 1-109 were first generated. Phagemid vector pB2105 was generated by PCR amplification of cDNA encoding VEGF residues 1-109 using primers allowing subsequent ligation of Nsi / Xba I restriction fragments into phagemid vector phGHam-g3 (Genentech, Inc.) (Genentech, Inc.). This allowed the introduction of an amber codon immediately after residue 109 and the fusion of the VEGF1-109 cDNA to the C-terminal half of the gIII site containing residues 249 to 406.
[0178] In one library, positions 18, 21, 22 and 25 of VEGF1-109 allow all possible combinations of residues (with the aid of oligonucleotides that change the target codons to NNS ...
Embodiment 2
[0215] Binding of VEGF variants to KDR receptors
[0216] The ability of VEGF(1-109) variants and VEGF165 variants (described in Example 1) to inhibit binding of biotin-labeled native VEGF(8-109) to the KDR receptor was evaluated for the binding of the variants to the KDR receptor. combined. The VEGF variants evaluated contained the mutations shown in Table 2.
[0217] Receptor binding assays were performed in 96-well immunoplates (Maxisorp, Nunc-Immunoplate, Nalge Nunc International, Rochester, New York). Each well was coated with 100 μl of a solution containing 8 μg / ml anti-KDR monoclonal antibody called MAKD5 (Genentech, South San Francisco, Caliromia) in 50 mM carbonate buffer, pH 9.6 and incubated overnight at 4°C. Discard the supernatant, wash each well three times in washing buffer (PBS containing 0.05% Tween 20), block the plate with blocking buffer (PBS containing 0.5% BSA, 0.01% thimerosal) at room temperature (150 μl per well) 1 hour. Discard the supernatant and...
Embodiment 3
[0222] Binding of VEGF variants to the Flt-1 receptor
[0223] The ability of VEGF(1-109) variants and VEGF165 variants (described in Example 1) to inhibit the binding of biotinylated native VEGF(8-109) to the Flt-1 receptor was assessed by measuring the binding of the Flt-1 receptor. Binding of receptors. The VEGF variants evaluated contained the mutations shown in Table 2.
[0224] Receptor binding assays were performed in 96-well immunoplates (Maxisorp, Nunc-Immunoplate, Nalge Nunc International, Rochester, New York). Each well was coated with 100 μl of a solution containing 2 μg / ml anti-human IgG Fc rabbit F(ab')2 (Jackson Immuno Research, West Grove, Pennsylvania) in 50 mM carbonate buffer at pH 9.6 and incubated at 4° C. Incubate overnight. Then the supernatant was discarded, each well was washed three times in washing buffer (PBS containing 0.05% Tween 20), and the plate was blocked at room temperature with blocking buffer (PBS containing 0.5% BSA, 0.01% thimerosal) ...
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