Gene targeting strategy for B-hIL4/hIL4RA/hIL13/hIL13RA1 mice. The exons 1-4 of mouse Il4 gene that encode the full length coding sequence were replaced by human IL4 exons 1-4 in B-hIL4/hIL4RA/hIL13/hIL13RA1 mice. The exons 4-7 of mouse Il4ra gene that encode the extracellular region coding sequences were replaced by human IL4RA exons 4-7 in B-hIL4/hIL4RA/hIL13/hIL13RA1 mice. The exons 1-4 of mouse Il13 gene that encode the full length coding sequence were replaced by human IL13 exons 1-4 in B-hIL4/hIL4RA/hIL13/hIL13RA1 mice. The exons 2-9 of mouse Il13ra1 gene that encode the extracellular protein were replaced by human IL13RA1 exons 2-9 in B-hIL4/hIL4RA/hIL13/hIL13RA1 mice. B-hIL4/hIL4RA/hIL13/hIL13RA1 mice were derived from crossing B-hIL4/hIL4RA/hIL13 mice and B-hIL13RA1 mice together.

Strain specific IL4RA expression analysis in homozygous B-hIL4/hIL4RA/hIL13/hIL13RA1 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hIL4/hIL4RA/hIL13/hIL13RA1 mice (H/H; H/H; H/H; H/H), and analyzed by flow cytometry with species-specific anti-IL4RA antibody. Mouse IL4RA was detectable in wild-type mice. Human IL4RA was exclusively detectable in homozygous B-hIL4/hIL4RA/hIL13/hIL13RA1 mice, but not in wild-type mice.

Strain specific IL13 expression analysis in homozygous B-hIL4//hIL4RA/hIL13/hIL13RA1 mice by ELISA. CD4+ T cells were isolated from wild-type mice (+/+) and homozygous B-hIL4/hIL4RA/hIL13/hIL13RA1 mice (H/H; H/H; H/H; H/H). The differentiation of CD4+ T cells into Th2 cells was induced, and the supernatants from the Th2 cell cultures were analyzed using a species-specific IL-13 ELISA kit. Mouse IL13 was detectable in wild-type mice. Human IL13 was exclusively detectable in homozygous B-hIL4/hIL4RA/hIL13/hIL13RA1 mice, but not in wild-type mice.

Assessment of IL-13RA1-mediated responses in a model of IL13 induced airway inflammation. Two doses (10 μg per mouse) of mouse IL13 or human IL13 were administered intratracheally every other day for 4 days to wild-type C57BL/6 mice, homozygous B-hIL4/hIL4RA/hIL13 mice and homozygous B-hIL4/hIL4RA/hIL13/hIL13RA1 mice. 24 hours after the final IL13 challenge, the mice were assessed for BALF chemokine. Mouse IL13 and human IL13 strongly induced CCL11 and CCL24 expression in wild-type mice, homozygous B-hIL4/hIL4RA/hIL13 mice and homozygous B-hIL4/hIL4RA/hIL13/hIL13RA1 mice.

Analysis of immune cells in BALF. B-hIL4/hIL4R/hIL13/hIL13RA1 mice (female, 10-week-old, n=8) were immunized with hIL13 to induce asthma. Anti-human  IL4RA antibody (Dupilumab analog, synthesized in-house) and anti-human IL13 antiboday (Lebrikizumab analog, synthesized in-house) were intraperitoneally injected from Day -1 and Day 5. (A&B) The number of CD45+ cells and eosinophils of BALF in the Dupilumab and Lebrikizumab treated groups decreased significantly compared with the hIL13-induced PBS treated group. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***P < 0.001.

Analysis of cytokines in BALF and mouse total IgE in serum. B-hIL4/hIL4R/hIL13/hIL13RA1 mice (female, 10-week-old, n=8) were immunized with hIL13 to induce asthma. Anti-human  IL4RA antibody (Dupilumab analog, synthesized in-house) and anti-human IL13 antiboday (Lebrikizumab analog, synthesized in-house) were intraperitoneally injected from Day -1 and Day 5. The mosue CCL11 and CCL24 of BALF in the Dupilumab and Lebrikizumab treated groups decreased significantly compared with the hIL13-induced PBS treated group. The human IL13 of BALF in the Lebrikizumab treated groups increased compared with the hIL13-induced isotype treated group.  Serum was collected at the study endpoint. IgE level was analyzed by ELISA. The results showed that the levels of total IgE in mice treated with Dupilumab and Lebrikizumab showed a significant reduction compared with untreated mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***P < 0.001.

H&E staining of asthma-like model in B-hIL4/hIL4R/hIL13/hIL13RA1 mice . Lung tissues were collected at the study endpoint and analyzed with H&E staining. The results showed that the group of mice treated with Dupilumab and Lebrikizumab (in-house) in inflammatory infiltration and mucus secretion in lung tissue was lower than that in untreated mice, indicating that B-hIL4/hIL4R/hIL13/hIL13RA1 mice provide a powerful preclinical model for in vivo evaluation of anti-human IL4RA antibodies and anti-human IL13 antibodies. Values are expressed as mean ± SEM. Significance was determined by unpaired t-test.  *P < 0.05, **P < 0.01, ***P < 0.001.