APRIL receptor (BCMA) and uses thereof
A use, tumor cell technology, applied in the direction of cytokine/lymphokine/interferon receptors, receptors/cell surface antigens/cell surface determinants, antibodies, etc. Insufficient types of tumors, etc.
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Embodiment 1
[0181] Embodiment 1: Detect the combination of APRIL and APRIL-R with plate test
[0182] In this example, the binding of BCMA to Apil was tested.
[0183] To test whether BCMA binds April, we performed a co-immunoprecipitation assay. Two soluble proteins, hgCMA-Fc and myc-mApril, were used in this assay.
[0184] HBCMA-Fc and LTbR-Fc and the following different TNF ligands were added to medium containing 10% FBS: myc-mApril; myc-CD40L and myc-RANKL and incubated for 1 / 2 hour at room temperature. Fc protein was combined with protein A beads for 1-2 hours, washed 3 times with 1ml PBS, immunoblot analysis was performed with mouse monoclonal antibody 9E10 (anti-myc), and color development was performed with enhanced chemiluminescence.
[0185] The detection of myc-APRIL in hBCMA-Fc immunoprecipitate showed that the interaction between BCMA and April was specific because other TNF ligands myc-CD40L and myc-RANKL could not bind to BCMA. Myc-April does not bind LTbR-Fc.
[0186]...
Embodiment 2
[0188] This example describes the interaction between hBCMA-Fc and FLAG-hAPRIL.
[0189] ELISA test: Coat the plate with pH 9.6 carbonate solution (1 ug / ml) of receptor-Fc fusion protein (hBCMA-Fc739 or hTNFR2-Fc-492), overnight at 4°C. Block with PBS / 5% skim milk powder / 0.5% Tween-20 for 2 hours at room temperature. Use 100 μl blocking solution to make 2-fold serial dilutions of the ligand (TNFa-197 starts from 1000ng / ml, muAPRIL-657 starts from 1000ng / ml, hApril-507 starts from 2000ng / ml (inactive), hApril-429 starts from 5× concentrated medium to start). After incubation with ligand, the plates were washed with PBS containing 0.5% Tween-20 and then hybridized with anti-FLAG mAb M2 in dilution buffer at a concentration of 0.5 μg / ml. The antibody was then detected with anti-mouse-PO 1 / 2000 and enzymatic color development (OPD).
[0190] Immunoprecipitation assay: 293T cells were transfected with the indicated expression plasmids (Rec-Fc or flag ligand) in a 9cm plate. The...
Embodiment 3
[0192] This example describes the binding of myc-mAPRIL, hKayL-440(hAPRIL) and Flag-mAPRIL to hBCMA-Ig, hLT-R-Ig or hp80TNFR-Ig. The conditions for all experiments were: 25°C, flow rate of 10 μl / ml min.
[0193] All experiments used HBS buffer (10 mM HEPES, 150 mM NaCl, 0.005% P20 surfactant, pH 7.4). This solution is also used as running buffer and sample diluent. .
[0194] First, the surface of a CM5 chip (BIAcore, Inc.) was activated with N-hydroxysuccinimide / N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide hydrochloride (BIAcore). Dilute hBCMA-Ig, hLT-R05-Ig, hp80-TNFR to 30g / ml with 10mM acetic acid, take 2μl, 15μl and 10μl of these three solutions respectively, then block with 30μl and then 15μl ethanolamine-HCl (pH 8.5) . This results in a surface density of 1600-3700 resonance units (RU). Chips were regenerated with 20 μl 1 mM formic acid. These were repeated 5 times to establish a repeatable stable baseline.
[0195] In the experiment, 100 μl each of myc-mApril,...
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