Anti-b7s1 polypeptides and their use
An antibody and antigen technology, applied in the direction of peptides, applications, antibodies, etc., can solve the problems that the receptor of B7S1 has not been identified, the molecular mechanism of functional significance has not been established, and the expression pattern of tumors has not been established.
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Embodiment 1
[0205] Example 1. Production of Anti-B7S1 Mouse Antibodies
[0206] BALB / c mice (Vital River, 6 weeks old) were immunized by subcutaneous injection of human B7S1-Fc fusion protein by fusing human B7S1 (also known as B7-H4 and B7X) extracellular domain (Leu25-258, NCBI reference sequence: NP_078902.2) and mIgG2a Fc (Pro222-Lys453 GenBank: AGH20709.1) to construct. For one animal, 10 μg of B7S1-Fc fusion protein in 50 μL of PBS was mixed with 50 μL of complete Freund's adjuvant (CFA, Sigma-Aldrich, Cat# F6881). Immunization was repeated 5 times at 3-day intervals. Three days after the last booster immunization, the lymph nodes close to the injection site were carefully dissected. Lymphocytes were mixed with Ag8.653 myeloma cells (Sigma-Aldrich, cat. no. 85011420) and cloned using HAT selection (Sigma catalog number: H0262) and HFCS (Hybridoma Fusion and Cloning Supplement (Hybridoma Fusion and Cloning Supplement), 50×, Roche catalog number: 11-363-735-001). Hybridoma superna...
Embodiment 2
[0228] Example 2. Analysis of the affinity of humanized anti-B7S1 antibodies
[0229] To test the binding affinity of humanized anti-B7S1 antibodies, ELISA analysis was performed. 2 μg / ml of human B7S1-his (Sinobiologic, catalog number 10738-H08H-100), mouse B7S1-his (R&D system, catalog number 4206-B7-100) and cynomolgus B7S1 extracellular domain (Leu25-Ser259, NCBI reference sequence: XP_005542249.1) was coated on MaxiSorp 96-well plate (NUNC, 449824) in 0.5M carbonate / bicarbonate buffer (pH9.6) and incubated overnight at 4°C. Coated plates were blocked with skim milk (5% in PBST) for 1 hour. Humanized anti-B7S1 antibody was diluted and added to the plate and incubated for 2 hours at room temperature. Wash three times with PBST (1x PBS with 0.05% tween-20) to remove unbound antibody. Goat anti-human IgG-HRP (Easybio, BE0122, 1:5000 in PBS) was added and incubated for 1 hour. Wash three times with PBST (1x PBS with 0.05% tween-20) to remove unbound antibody. Add TMB (eBi...
Embodiment 3
[0236] Example 3. Humanized anti-B7S1 antibodies that bind to B7S1 transfected cell lines
[0237] To evaluate the binding affinity of anti-B7S1 antibodies on the surface of cells overexpressing B7S1, human B7S1 (NCBI reference sequence: NP_078902.2) was constructed on the BaF3 cell line. Cells were washed with PBS and aliquoted into 96-well U-bottom plates at a density of 5x10^5 cells / well. For live / dead / dyed, put Fixable Aqua dead cell staining kit (Life Technologies, L34957) was applied (1:1000 in PBS, 100ul / well) to the cells and incubated at 4°C for 10 minutes. FACS buffer (2% FBS in PBS, 100ul / well) was added to each well to stop staining. Centrifuge (1200 rpm, 5 min) to discard the supernatant and wash once with FACS buffer. The humanized anti-B7S1 antibody was diluted to the corresponding concentration in FACS buffer, and then added to each well (100ul / well). Cells were incubated at 4°C for 30 min and then washed twice with FACS buffer. Add Alexa Labeled goat a...
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