Novel vascular endothelial growth factor humanized monoclonal antibody
A monoclonal antibody and humanized technology, applied in the direction of anti-growth factor immunoglobulin, antibody, biochemical equipment and methods, etc., can solve the problem of inter-individual differences in sequence removal of CDR parts
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specific Embodiment 1
[0020] (1) Antigen preparation
[0021] human vascular endothelial growth factor VEGF 165 (Human Vascular endothelial growth factor, hVEGF 165 ) gene was amplified from the human gene transcriptome by PCR, the upstream primer was: 5'-CGCGGATCCGCACCCATGGCAGAAGGAGG-3'; the downstream primer was: 5'-CTCCTCGAGTCACCGCCTCGGCTTGTCACATC-3'. Using BamH I and Xho I double enzyme digestion, the gene fragment was connected into the prokaryotic expression vector pET32a (+), PCR and double enzyme digestion identified positive clones and then sequenced. After the sequence was determined correctly, the plasmid was extracted. Prokaryotic expression vector pET32a-VEGF 165 Transform Rosetta strain in 100ml LB medium, OD 600 When reaching about 0.5, 1mM IPTG induced expression for 4h, VEGF165 Proteins form inclusion bodies. After sonication, the inclusion bodies were dissolved with 1×SDS loading buffer, and the expression level of the target protein was measured. Prepared VEGF 165 The prote...
specific Embodiment 2
[0032] (1) Humanization of mouse monoclonal antibodies
[0033] Compare the mouse antibody amino acid sequence with the human antibody amino acid sequence to obtain the framework of the human subclass consensus sequence of the highest homologous sequence, that is, the human light chain V L Kappa subgroup II (V L κII) and heavy chain V H Subgroup I (V H I), as a humanized framework. According to the definition of kabat et al., CDR1:24-34, CDR2:50-56, CDR3:89-97 of the L chain; CDR1:26-35, CDR2:50-65, CDR3:95-102 of the H chain (sequences of proteins of immunological Interest, (5 th ), Public Health Service, National Institutes of Health, Bethesda, MD (1991). Utilize computer to construct the three-dimensional map model of mouse monoclonal antibody VL-VH region (Carter et al., Proc.Natl.Acad.Sci.USA 89:4285-4289 (1992) and Eigenbrot et al., J.Mol.Biol.) 229:969- 995 (1993)), identifying those murine antibody framework region residues to introduce into humanized antibodies...
specific Embodiment 3
[0039] (1) VEGF affinity analysis
[0040] The affinity of monoclonal antibody 18-5, h18-5 and Bevacizumab was determined by Scatchard method. Table 1 shows the results of the assay, indicating that the two monoclonal antibodies have strong binding affinity to VEGF.
[0041]
[0042] (2) Analysis of inhibition of endothelial cell proliferation in vitro
[0043] 2% fetal calf serum and growth factors (BulletKit, Lonza) were added to the basic endothelial cell medium (EGM-2, Lonza) to prepare endothelial cell growth medium. When human umbilical vein endothelial cells (HUVECs) (Lonza, Walkersville, MD) grew to a dense monolayer, 4 × 10 3 The density of cells / well was seeded in a 96-well plate, and no growth factors were added to the analysis medium EGM-2 at this time. The positive control antibody was Bevacizumab (Genetech), and the humanized VEGF antibody was added in a certain dilution gradient. After incubation for 1 hour, human VEGF was added 165 Make the final concent...
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