Anit-il-23 antibiodies
An IL-23, antibody technology, applied in the direction of antibody, anti-animal/human immunoglobulin, anti-cytokine/lymphokine/interferon immunoglobulin, etc., can solve the high-affinity neutralizing human IL-23 antibody Issues not made public
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Embodiment 1
[0125] IL-23 inhibition assay
[0126] Based on the ability of IL-23 to stimulate mouse splenocytes to secrete IL-17, inhibition of IL-23 activity was detected using a mouse splenocyte assay. Antibodies of the invention were compared with MAb3A8 and the commercially available mouse monoclonal antibody MAB1290 (R&D Systems).
[0127] The basic protocol is as follows: spleens are removed from 1BALB / c or C57BL / 6 mice, splenocytes are harvested, and single-cell suspensions are prepared. Splenocytes were killed with complete RPMI (containing 1% non-essential amino acids, 1% sodium pyruvate, 2.5mM HEPES, 1% L-glutamic acid, 0.00035% 2-mercaptoethanol, 1% penicillin / streptomycin, 10% heat Live serum FCS and 50ng / mL human IL-2 (Andy Biotechnology)) were washed and resuspended. Then splenocytes were seeded in a 96-well culture plate at 500,000 cells / well, with a volume of 100 μl.
[0128]Human IL-23 (Andy Biotechnology) at a concentration of 10 pM was pre-incubated with 3-fold seria...
Embodiment 2
[0134] Neutralization of human IL-23
[0135] Injection of human IL-23 into mice together with IL-2 stimulates splenocytes to produce mouse IL-17. Neutralizing antibodies directed against the IL-23p19 subunit blocked this IL-17 stimulation, as demonstrated by ex vivo IL-17 production readout in an in vivo mouse model as follows.
[0136] C57BL / 6 mice were sensitized by injection of mIL-2 22 hours before stimulation with mouse IL-2 (mIL-2) plus human IL-23 (hIL-23). Mice were injected with a high affinity humanized antibody of the invention or an isotype-matched (IgG4) control antibody 2 hours before stimulation with mIL-2 and hIL-23.
[0137] Then set the following groups:
[0138] At time point 0, mIL-2 (5 μg) alone or mIL-2 (5 μg) plus human IL-23 (10 μg) was injected intraperitoneally (i.p.)
[0139] At 7 hours, mIL-2 (10 μg) alone or mIL-2 (10 μg) plus human IL-23 (10 μg) was injected intraperitoneally
[0140] At 23 hours, mIL-2 (5 μg) alone or mIL-2 (5 μg) plus human...
Embodiment 3
[0146] binding affinity
[0147] The affinity between MAb3A8 and the antibody of the present invention was detected by BIAcore measurement system (Table 3). BIAcore TM is an automated biosensor system that measures molecular interactions (Karlsson et al. (1991) J. Immunol. Methods 145:229-240). In these experiments, antibodies were captured to BIAcore at low density TM on the surface of the chip. Coupling of protein A to carboxymethyl (CM5) BIAcore using active amino groups of ethyldimethylaminopropylcarbodiimide (EDC) TM Flow cell for the sensor chip. Protein A was diluted in sodium acetate buffer, pH 4.5, and immobilized to the flow cell of a CM5 chip using EDC to generate 1000 response units. Unreacted sites were blocked with ethanolamine. A flow rate of 60 μl / min was used. Multiple conjugations were performed by injecting 10 μl of 2 μg / mL antibody solution of the invention per cycle, followed by injections of decreasing concentrations of human IL-23 (e.g., 1500, 750...
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