Anti-il-25 antibodies and use thereof
One-IL-25, IL-25 technology, applied in the direction of antibodies, antibody medical components, applications, etc., can solve the problem of little understanding of the significance of white blood cell anti-tumor immune function
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Embodiment 1
[0121] Example 1. Preparation of Anti-IL-25 Antibody
[0122] IL-25KO mice (16-18g, 6 weeks old, n=3) were immunized by subcutaneous injection of 10 μg human IL-25 protein (NCBI Gene ID: 64806) with complete Freund's adjuvant (Sigma-Aldrich, Cat#F6881) , provided by Professor Chen Dong's laboratory at Tsinghua University), the human IL-25 protein was prepared in-house according to general standard methods for protein expression. Immunization was repeated 5 times at 3-day intervals. 3 days after the last booster, carefully dissect the lymph nodes close to the injection site. Lymphocytes were mixed with Ag8.653 myeloma cells (Sigma-Aldrich , Cat#85011420) fusion and cloned with HAT selection (Sigma cat#: H0262) and HFCS (Hybridoma Fusion and Cloning Supplement, 50X, Roche cat#: 11-363-735-001). The hybridoma supernatants were screened by ELISA to prepare antibodies that can bind to human IL-25. In addition, antagonistic anti-IL-25 mAbs were selected based on cell-based funct...
Embodiment 2
[0150] Example 2. Expression and Purification of Anti-IL-25 Antibodies
[0151] DNA sequences encoding humanized IgG heavy chain (variable domain amino acid sequence of SEQ ID NO: 7, 8 or 9) and light chain (variable amino acid sequence of SEQ ID NO: 10 or 11) were synthesized and synthesized. The pCDNA3.1 vector (commercially available from LifeTechnology) was inserted to construct an expression plasmid for full-length IgG. Expression of the parental antibody was performed in 100 ml of HEK293 cell culture (HEK293 cells are commercially available from ThermoFisher Scientific), and the supernatant was purified with a protein A affinity column (commercially available from GE Healthcare life Science). Purified antibodies were buffer exchanged into PBS using PD-10 desalting columns (commercially available from Thermofisher Scientific). The concentration and purity of the purified protein were determined by OD280 and SDS-PAGE, respectively. Humanized antibodies were expressed in ...
Embodiment 3
[0152] Example 3. SPR analysis of the binding affinity of anti-IL-25 antibody to human IL-25
[0153] An anti-human Fcγ-specific antibody (Jackson Immuno Research, Lot #124448, code 109-008-098) was immobilized on a Biacore T200 sensor chip using the amine coupling method. The four antibodies secreted into the culture medium were injected along with the parental antibody and captured by the anti-human Fc antibody alone by the Fc (capture phase). After equilibration, IL-25 was injected for 300 seconds (association period), followed by running buffer for 1200 seconds (dissociation period). The response of the reference flow cell (flow cell 1 ) was subtracted from those of the humanized antibody flow cell during each cycle. The surface was regenerated prior to injection of another humanized antibody. This process was repeated until all antibodies were analyzed. The off-rates of the humanized antibodies were obtained by locally fitting the experimental data to a 1:1 interaction...
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