Novel anti-DR5 antibody
A technology of antibodies and antigens, applied in the direction of antibodies, antibody medical components, antibody mimics/stents, etc., can solve problems such as no correlation
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Embodiment 1
[0393] [Example 1] Generation of mouse antibody B273
[0394] 1) Production of -1 human DR5 protein (human DR5 extracellular domain / human Fc fusion protein)
[0395] 1) Production of-1-1 human DR5 extracellular domain expression vector
[0396] A vector expressing human DR5 protein (isoform 2: NP_671716) was constructed by inserting a gene in which the extracellular domain of human DR5 was fused to the human IgG1 / Fc region downstream of the CMV promoter.
[0397] 1)-1-2 Production of human DR5 protein
[0398] Introduction of the expression vector into 293FreeStyle cells and collection of the culture supernatant were performed by Invitrogen Corporation (now Life Technologies Japan Ltd.).
[0399] 1)-1-3 Purification of human DR5 protein
[0400] The culture supernatant obtained in b) above was purified using protein A affinity column chromatography. 5 L of the culture supernatant was applied to "HiTrap Protein AFF" (GE Healthcare Bio-Sciences Co., Ltd., catalog number 17-5...
Embodiment 2
[0427] [Example 2] Cloning of mouse antibody B273 gene and production of human chimeric antibody gene
[0428] 2)-1 Cloning and sequence determination of mouse antibody B273 cDNA
[0429] 2) Determination of N-terminal amino acid sequences of heavy chain and light chain of -1-1 mouse antibody B273
[0430] In order to determine the N-terminal amino acid sequences of the heavy and light chains of the mouse antibody B273, the mouse antibody B273 purified in Examples 1-8 was separated by SDS-PAGE. After separation, the protein in the gel was transferred from the gel to a PVDF membrane (pore size: 0.45 μm, manufactured by Invitrogen Corporation). Membranes were washed with wash buffer (25 mM NaCl, 10 mM sodium borate buffer pH 8.0) and thereafter stained by immersion in dye solution (50% methanol, 20% acetic acid, 0.05% Coomassie Brilliant Blue) for 5 minutes, followed by destaining with 90% methanol . The band corresponding to the heavy chain (band with smaller mobility) and t...
Embodiment 3
[0471] [Example 3] Measurement of human chimeric B273 (cB273) antibody activity (in vitro)
[0472] 3) Selective binding properties of -1cB273 antibody to human DR5 extracellular domain
[0473] The binding properties of cB273 to human TRAIL R1-R4 and mouse TRAIL R2 extracellular domain proteins (manufactured by R&D Systems, Inc.) were investigated by the direct ELISA method described below. First, each of the ectodomain proteins of TRAIL R was diluted to 1 μg / ml with PBS, the diluted solution was dispensed in an immunoplate (manufactured by Nunc, Inc., #442404) at 50 μl / well, and the plate was kept at 4°C By standing overnight, the protein is thus adsorbed to the plate. The next day, the liquid in each well was removed, and each well was washed once with PBS. Thereafter, in order to suppress nonspecific adsorption of proteins, 200 µl / well of PBS containing 3% fetal bovine serum was dispensed, and the plate was allowed to stand at room temperature for 1.5 hours. The liquid ...
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