Basic Information
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Details
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mRNA expression analysis
Strain specific analysis of IL6 and IL6R gene expression in B-hIL6/hIL6R mice by RT-PCR. Mouse Il6 and Il6r mRNA were detectable in spleen of wild-type mice (+/+). Human IL6 was detectable only in homozygous B-hIL6/hIL6R mice (H/H) after stimulated with LPS for 3h. Human IL6R mRNA was detectable in homozygous B-hIL6/hIL6R mice but not in wild-type mice with or without LPS stimulation.
Protein expression analysis
Strain specific IL6 expression analysis in homozygous B-hIL6/hIL6R mice by ELISA. Serum was collected from WT (+/+) and homozygous B-hIL6/hIL6R (H/H) mice stimulated with LPS in vivo, and analyzed by ELISA with species-specific IL6 ELISA kit. Mouse IL6 was detectable in WT mice. Human IL6 was exclusively detectable in homozygous B-hIL6/hIL6R mice but not in WT mice. ND: Not detectable.
Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hIL6/hIL6R mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes were performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. (B) Results of FACS analysis. Percent of T cell, B cell, NK cell, monocyte, dendritic cell and macrophage in homozygous B-hIL6/hIL6R mice were similar to those in the C57BL/6 mice, demonstrating that IL6 and IL6R humanized does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.
Analysis of spleen T cell subpopulations by FACS.
Splenocytes were isolated from female C57BL/6 and B-hIL6/hIL6R mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes were performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live CD45+ cells were gated for CD3+ T cell population and used for further analysis as indicated here. (B) Results of FACS analysis. Percent of CD8+ T cell, CD4+ T cell, and Treg in homozygous B-hIL6/hIL6R mice were similar to those in the C57BL/6 mice, demonstrating that IL6 and IL6R humanized does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.In vivo efficacy of anti-human IL6 antibody
Efficacy of anti-human IL6 antibodies in B-hIL6/hIL6R mice with collagen induced arthritis (CIA) model. Mice in each group were treated with sirukumab (in house). Body weight change(A) and clinical score (B) were evaluated during treatment twice a week. There was no significant change in body weight, while total clinical score increased in the groups except control during days 21 to 28. It indicated that the arthritis mouse model had been constructed successfully. Clinical scores decreased in the two groups treated with sirukumab and dose-dependent. The results indicated the B-hIL6/hIL6R mice provide a powerful preclinical model for in vivo evaluation of anti-human IL6 antibodies.
Efficacy of anti-human IL6 antibody in B-hIL6/hIL6R mice with collagen induced arthritis (CIA) model. Histopathological examination was performed on the joints of the extremities at endpoint. (A) Hematoxylin and eosin (H&E) staining. (B) Score of arthritis histology. G1 showed no significant abnormal changes. G2 showed bone structure damage(d), articular cavity or periarticular space disappeared (e), and pannus were observed (a), suggesting that the CIA model was successfully established. Compared with the G2 group, the low-dose group (G3) showed slight inflammatory cell infiltration (b) and pannus (a) and synovial hyperplasia (c). However, in the high dose group (G4), there was only partial pannus, the arthrosis disappeared and the articular cavity was obvious.
