Methods of treating graves' ophthalmopathy using Anti-fcrn antibodies
A technology for antibodies and eye diseases, applied in the field of treatment or prevention of Graves' ophthalmopathy, pharmaceutical composition for treatment or prevention of Graves' ophthalmopathy
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Embodiment 1
[0195] Example 1: Construction of anti-FcRn expression library using transgenic rats
[0196] A total of six transgenic rats ( OMT) for immunization. As the immunogen, human FcRn was used. Both footpads of rats were immunized with 0.0075 mg human FcRn and adjuvant eight times in 24 days at 3-day intervals. On day 28, rats were immunized with 5-10 μg of immunogen diluted in PBS buffer. On day 28, rat sera were collected and used to measure antibody titers. On day 31, rats were euthanized and popliteal and inguinal lymph nodes were recovered for fusion with P3X63 / AG8.653 myeloma cells.
[0197] ELISA assays were performed to measure antibody titers in rat sera. Specifically, human FcRn was diluted in PBS (pH 6.0 or pH 7.4) buffer to make a 2 μg / mL solution, and then 100 μL of the solution was coated in each well of a 96-well plate, and then in Incubate at 4°C for at least 18 hours. Each well was washed 3 times with 300 μL of washing buffer (0.05% Tween20 in PBS) to remov...
Embodiment 2
[0199] Example 2: Evaluation of Antigen Binding Affinity and IgG Binding Blocking Ability of Anti-hFcRn Antibodies of Hybridoma Libraries
[0200] To analyze the binding of antibodies to hFcRn, the same ELISA assays as above (pH 6.0 and pH 7.4) were performed.
[0201] The hFcRn binding affinity was evaluated by FACS at pH 6.0 and pH 7.4 at 5 ng / mL and 25 ng / mL using the culture supernatants of the three hybridoma libraries. HEK293 cells stably expressing human FcRn were isolated from the flasks and suspended in reaction buffer (0.05% BSA in PBS, pH 6.0 or pH 7.4). Dilute the suspension to 2 x 10 6 cells / mL and add 50 μL of the dilution to each well of a 96-well plate. Then, 50 μL of hybridoma bank culture supernatants diluted to 10 ng / mL and 50 ng / mL, respectively, were added to each well and suspended to allow antibody binding. Dilute A488 rabbit anti-IgG goat antibody at a ratio of 1:200 in reaction buffer, add 100 μL of the dilution to each well, and mix with the cell p...
Embodiment 3
[0203] Example 3: Separation of Hybridoma Clones and Selection of Human Antibodies by FACS
[0204] Using hybridoma library A showing the highest human FcRn-binding affinity and blocking effect, clones were isolated by FACS (flow cytometry) to obtain a total of 442 single clones. Isolated single clones were cultured in HT medium, and the supernatant was collected. Antibody-expressing hybridoma clones that bound to hFcRn in the supernatant were selected by FACS.
[0205] RNA was isolated from 100 single clones selected by FACS analysis, and the isolated RNA was sequenced. In the first step of sequencing, 88 out of 100 single clones were sequenced and classified into a total of 35 groups (G1 to G38) based on amino acid sequence. Culture supernatants of representative clones of 33 groups after excluding two clones (G33 and G35) for which medium was not available were diluted at a concentration of 100 ng / mL, and the binding affinity to hFcRn was evaluated by ELISA.
[0206] In ...
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