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[Selection and characterization of human antibodies against vascular endothelial growth factor from human phage
1School of Oncology, Beijing Medical University, Beijing Institute for Cancer Research, Beijing 100034, China.
Objective:
To select anti-VEGF (vascular endothelial growth factor) antibodies (Fab fragments) from human phage display antibody library and to identify their specification and activities.
Methods:
Human immunoglobulin heavy chain and light chain genes were separately amplified by RT-PCR from human peripheral lymphocytes using family specific primers and signal sequences of immunoglobulin. Human antibody library was constructed by phage display technology, and phage Fab antibodies to VEGF were screened from this library. ELISA, Western blot and (3)H-thymidine incorporation assays were used for the specification and neutralization activities of these Fab antibodies. Sequencing analysis was carried out for further identification of the antibodies.
Results:
The repertoire of human phage display Fab library was 1.5 x 10(8). After 4 round panning with VEGF(121), 280 clones were checked for their binding activities with ELISA and 12 clones could bind to VEGF(121) specifically. Western blot demonstrated that bacterially expressed soluble Fab could specifically recognize VEGF. Results of (3)H- thymidine incorporation showed that one clone of soluble Fab could neutralize the mitogenic activity of VEGF(165) on HUVEC. Sequencing analysis showed that the obtained V(H) gene belonged to human VH6 subgroup and the light chain was VJC rearranged human VL4 gene.
Conclusions:
Human anti-VEGF antibodies can be obtained from human phage display library, which provides a basis for preparation of high affinity human anti-VEGF monoclonal antibodies through antibody engineer technique.