Fusion protein and application thereof
A fusion protein and targeting antibody technology, applied in the field of biomedicine, to achieve the effect of inhibiting angiogenesis, promoting angiogenesis, and low ADCC effect
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Embodiment 1
[0121] Example 1 Expression of fusion protein VEGFR2 / TGFβRII trap
[0122] Using the extracellular domain (SEQ ID NO: 12) of the TGFβRII receptor (SEQ ID NO: 16 and SEQ ID NO: 17) as the immunomodulatory molecular part in the fusion protein, the Ramucirumab antibody (SEQ ID NO: 10 and SEQ ID NO:11) as the targeting part of the fusion protein, with (Gly 4 Ser) 4 G as linker sequence (SEQ ID NO: 13). The N-terminus of the extracellular domain of TGFβRII and the Ramucirumab antibody were linked to form Anti-VEGFR2-TGFβRII extracellular domain fusion protein (R0469) (SEQ ID NO: 11 and SEQ ID NO: 18). At the fusion junction, the C-terminal lysine residue (K) of the antibody heavy chain is mutated to alanine (A), reducing cleavage hydrolysis of the fusion protein. Using standard protocols for transient or stable transfection for R0469, mammalian cells were transfected with DNA encoding the anti-VEGFR2 light chain and DNA encoding the anti-VEGFR2 / TGFβRII receptor, either in the sa...
Embodiment 2
[0136] Example 2 Expression of fusion protein VEGFR2 / TGFβRII trap
[0137] The expression plasmid was constructed to transiently transfect human embryonic kidney HEK 293 cells, and the anti-VEGFR2-TGFβRII Trap (R0469) produced by the isolated and purified cells had a band molecular weight of about 170kD on SDS-PAGE under non-reducing conditions ( figure 1 B), the molecular weight of the band on SDS-PAGE under reducing conditions is about 80kD. There was a small peak after the 190 kD peak on size exclusion chromatography, which was identified by mass spectrometry as the antibody portion of the anti-VEGFR2-TGFBR2 trap cleaved at a site in the N-terminal portion of TGFβRII.
Embodiment 3
[0138] Example 3 ELISA detection of VEGFR2-TGFβRII trap antibody terminal binding activity
[0139] The antigen used for antibody end binding detection is human VEGFR2-his (10012-H08H, purchased from Sinobiological), and the detection process is as follows:
[0140] 1) Dilute anti-His Tag rabbit monoclonal antibody (31-1048-00, purchased from RevMABBioscience) with 1× phosphate buffered saline (PBS) to 0.5 μg / ml, 100 μl / well coated 96-well microtiter plate, 4°C overnight;
[0141] 2) 250 μl of 1×PBST (PBS+0.5% Tween20) was washed 3 times, and 200 μl of PBS containing 2% bovine serum albumin (BSA) was added to block for 1 hour at room temperature;
[0142] 3) Wash 3 times with 250 μl 1×PBST, add human VEGFR2-his, and incubate at room temperature for 1 hour;
[0143] 4) Wash 3 times with 250 μl 1×PBST, add serially diluted anti-VEGFR2-TGFβRII trap, VEGFR2 antibody (purchased from Lilly) as a positive control, and incubate at room temperature for 2 hours;
[0144] 5) Wash 3 ti...
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