Method for the treatment of fibrotic disease
A technology for fibrotic diseases and renal fibrosis, which can be used in chemical instruments and methods, skin diseases, gene therapy, etc., and can solve problems such as unmet medical needs for improvement and treatment of fibrotic diseases.
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Embodiment 1
[0141] Example 1 Isolation of anti-mouse CSF-1R antibody
[0142] Two rabbits received 5 immunizations with cells transiently expressing residues 1-512 of mouse CSF-1R (Uniprot entry p09581). Antibody responses were monitored by ELISA using NuncMaxisorp plates coated with 2 μg / ml mouse CSF-1R-rabbit Fc. Serum titers exceeding 1:100,000 dilutions were observed in both rabbits. Serum binding to M-NFS-60 cells was also determined by FACS (Metcalf et al., 1970). Median FL1 is plotted against antibody dilution. Binding was observed beyond a dilution of 1:10,000.
[0143] Using the CSF-1-dependent M-NFS-60 cell line (Metcalf et al., 1970), it was shown that sera from both rabbits were able to block the survival of CSF-1-dependent cells at dilutions exceeding 1:100 (data not shown).
[0144] One hundred 96-well plates were seeded with 1000-5000 rabbit PBMCs per well and grown at 37C for 1 week in the presence of EL4-B5 mouse thymoma cells and rabbit T cell conditioned medium (...
Embodiment 2Ab535
[0153] In vitro analysis of embodiment 2Ab535
[0154] Ab535 blocks the binding of mouse CSF-1 to CSF-1 receptor positive cell lines in vitro
[0155] The ability of receptor-bound Ab535 to prevent the binding of CSF-1 to CSF-1R was investigated. The murine CSF-1R positive cell line M-NFS-60 was used in the assay in which cells were pre-incubated with Ab535 or a control antibody prior to exposure to CSF-1. Receptor-bound CSF-1 was then detected using fluorescently labeled antibodies and flow cytometry. The Ab535-dependent decrease in cellular fluorescence intensity was interpreted as indicative of the ability of receptor-bound Ab535 to prevent CSF-1 from binding to CSF-1R.
[0156] M-NFS-60 cells (LGC Promochem, Teddington, UK) were maintained in suspension supplemented with 10% fetal calf serum (PAA), Hepes (Invitrogen, final concentration 10 mM), sodium pyruvate (Invitrogen, final concentration 1 mM) , glucose (Sigma-Aldrich, final concentration 4.5g / l), sodium bicarbon...
Embodiment 3
[0164] Example 3 Effect of anti-CSF-1R antibody in bleomycin-induced pulmonary fibrosis model in vivo
[0165] Bleomycin is an antibiotic first isolated from Streptomyces verticillatus and has been used as a chemotherapeutic agent for various cancers. The bleomycin model of pulmonary fibrosis is a well established model and was used essentially as described in Madtes, DK et al., 1999, AmJ RespirCellMolBiol, 20, 924-34. Detailed protocols can be found in Morschl, E., Molina, J.G., Volmer, J., Mohsenin, A., Pero, R.S., Hong, J.S., Kheradmand, F., Lee, J.J. and Blackburn, M.R. (2008), A3adenosine receptor signaling influences pulse monary inflammation and fibrosis. Am. J. Respir. Cell Mol. Biol. 39:697-705.
[0166] All mice used were wild-type C57Blk6 female mice (20 g) purchased from Harlan Labs. Intratracheal (IT) instillation was used in which mice were anesthetized with avertin and a tracheostomy was performed to instill 3.5 unit doses of bleomycin in 50 μl saline or 50 ...
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