Basic Information
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Targeting strategy
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Gene targeting strategy for B-hIL5/hIL5RA mice. The exons 1-4 of mouse Il5 gene were replaced by human IL5 exons 1-4 in B-hIL5/hIL5RA mice. The coding sequence of human IL5RA extracellular domain, mouse IL5RA transmembrane and cytoplasmic domain were inserted to replace part of murine exon 5.
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Protein expression analysis
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Strain specific IL5 expression analysis in homozygous B-hIL5/hIL5RA mice by ELISA. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hIL5/hIL5RA mice (H/H; H/H) stimulated with anti-CD3ε and anti-CD28 in vitro, and cell culture supernatant was analyzed by ELISA with species-specific IL5 ELISA kit. Mouse IL5 was detectable in wild-type mice. Human IL5 was exclusively detectable in homozygous B-hIL5/hIL5RA but not wild-type mice.
Strain specific IL5RA expression analysis in homozygous B-hIL5/hIL5RA mice by FACS. Eosinophils of bone marrow were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hIL5/hIL5RA mice (H/H; H/H), and analyzed by flow cytometry with species-specific anti-IL5RA antibody. Mouse Il5ra was detectable in wild-type mice. Human IL5RA was exclusively detectable in homozygous B-hIL5/hIL5RA mice but not wild-type mice.
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In vivo efficacy of anti-human IL5 and anti-human IL5RA antibodies
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The number of BALF immune cells in mouse asthma model. BALF immune cells were isolated from B-hIL5/hIL5RA mice (n=8). The number of eosinophils was analyzed by flow cytometry under the treatment of PBS, mepolizumab or benralizumab (in house). After treatment of mepolizumab or benralizumab (in house), the number of inflammatory cells was much lower than the positive control in homozygous B-hIL5/hIL5RA mice. Data was shown as Mean±SEM, and analyzed using One way ANOVA followed Dunnett compared G1/G3/G4 with G2. (**p<0.01, ***p<0.001, ****p<0.0001) Intraperitoneal (i.p.).
Analysis of eosinophils and basophils in blood by blood routine. Blood was collected at the study endpoint. The results show that the levels of eosinophils in mice treated with mepolizumab or benralizumab (in house) is lower than that in untreated mice.Data was shown as Mean±SEM, and analyzed using One way ANOVA followed Dunnett compared G1/G3/G4 with G2. (**p<0.01, ***p<0.001, ****p<0.0001) Intraperitoneal (i.p.).
H&E staining of asthma-like model in B-hIL5/hIL5RA mice. Lung tissues were collected at the study endpoint. H&E staining results showed that the lung tissues from B-hIL5/hIL5RA mice exposed to PBS aerosols did not show any inflammation. OVA exposure resulted in a significant increase in peribronchial and perivascular inflammation in B-hIL5/hIL5RA mice. A significantly reduction in eosinophils infiltration was observed in mice treated with mepolizumab or benralizumab (in house). Data was shown as Mean±SEM, and analyzed using One way ANOVA followed Dunnett compared G1/G3/G4 with G2. (**p<0.01, ***p<0.001, ****p<0.0001) Intraperitoneal (i.p.); (a) Mucus (b) inflammatory.
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Functional analysis
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Functional analysis of eosinophils expression in homozygous B-hIL5/hIL5RA mice by flow cytometry. Human IL5 was injected daily for 4 days, and blood samples were collected one day after the final injection. The number and proportion of eosinophils was then analyzed using flow cytometry. After stimulation with human IL5 recombinant protein, the proportion of eosinophils in the blood of B-hIL5/hIL5RA mice increased.
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Summary
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Protein expression analysis:
Mouse Il5 and Il5ra were detectable in wild-type mice. Human IL5 and IL5RA were exclusively detectable in homozygous B-hIL5/hIL5RA mice but not wild-type mice.
In vivo efficacy：
Anti-human IL5 antibody and Anti-human IL5RA antibody were efficacious in reducing eosinophils induced by OVA in B-hIL5/hIL5RA mice .
Functional analysis
After injecting human IL5 protein into B-hIL5/hIL5RA mice, an increase in eosinophils can be observed, indicating that the IL5/IL5RA pathway is functioning.
