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Gene targeting strategy for B-hTSLP/hTSLPR plus/hOX40/hOX40L mice.

The exons 1-5 of mouse Tslp gene that encode the whole molecule (ATG to STOP codon) were replaced by human counterparts in B-hTSLP/hTSLPR plus/hOX40/hOX40L mice. The promoter, 5’UTR and 3’UTR region of the mouse gene are retained. The human TSLP expression is driven by endogenous mouse Tslp promoter, while mouse Tslp gene transcription and translation will be disrupted.

A chimeric CDS that encodes human TSLPR extracellular domain, transmembrane and mouse TSLPR cytoplasmic domain, followed by mouse 3’UTR-STOP is inserted right after mouse Tslpr signal peptide sequence to replace part of the exon 2 of mouse Tslpr gene. The chimeric TSLPR protein expression will be driven by endogenous mouse Tslpr promoter, while mouse Tslpr gene transcription and translation will be disrupted.

The exons 1~6 of mouse Il7r gene that encode the extracellular region were replaced by human IL7R exons 1~6 in B-hTSLP/hTSLPR plus/hOX40/hOX40L mice.

The exons 1-5 of mouse Ox40 gene that  encode the extracellular domain were replaced by human OX40 exons 1-5 in B-hTSLP/hTSLPR plus/hOX40/hOX40L mice.

The exons 2-3 of mouse Ox40l gene that  encode the extracellular region were replaced by human OX40L exons 2-3 in B-hTSLP/hTSLPR plus/hOX40/hOX40L mice.

B-hTSLP/hTSLPR plus/hOX40/hOX40L mice were derived from mating B-hTSLP/hTSLPR mice plus (112744) and B-hOX40/hOX40L mice (120543).

Strain specific TSLP expression analysis in homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice by ELISA. Calcipotriol (MC903) was dissolved in ethanol and applied on ears of wild-type C57BL/6JNifdc mice and homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice for 7 days. The ear grinding supernatant collected from the two strains of mouse were analyzed by ELISA (anti-mouse TSLP antibody: Biolegend, 434107; anti-human TSLP antibody: Biolegend, 434207). Mouse TSLP was only detectable in wild-type C57BL/6JNifdc mice. Human TSLP was only detectable in homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice but not in wild-type mice. ND: not detectable.

Strain specific TSLPR expression analysis in homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice by flow cytometry. Bone marrow derived dendritic cells (BMDCs) were produced by culturing the bone marrow from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice (H/H;H/H;H/H;H/H;H/H),  which were induced with FLT3L (acrobiosystems, FLL-H5218) for 6 days and respectively stimulated with 1 μg/mL LPS in vitro for 24 h. Protein expression was analyzed with anti-mouse TSLPR antibody (Biolegend, 151805) and anti-human TSLPR antibody (Biolegend, 322906) by flow cytometry. Mouse TSLPR was detectable in wild-type C57BL/6JNifdc mice. Human TSLPR was detectable in homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice. WT：C57BL/6JNifdc, HO: homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice.

Strain specific IL7R expression analysis in homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice(+/+) and homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice (H/H;H/H;H/H;H/H;H/H), protein expression was analyzed by flow cytometry with species-specific anti-mouse IL7R antibody (Biolegend, 135011) and anti-human IL7R antibody (Biolegend, 351303). Mouse IL7R was detectable on T cells, DCs and NK cells of wild-type C57BL/6JNifdc mice. Human IL7R was detectable on T cells, DCs and NK cells of homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice. WT：C57BL/6JNifdc, HO: homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice.

Strain specific IL7R expression analysis in homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice(+/+) and homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice (H/H;H/H;H/H;H/H;H/H) stimulated with PBS or anti-mouse CD3ε antibody in vivo for 24 h, protein expression was analyzed by flow cytometry with species-specific anti-mouse OX40 antibody (Biolegend, 119414) and anti-human OX40 antibody (Biolegend, 350004). Mouse OX40 was detectable on T cells of wild-type C57BL/6JNifdc mice. Human OX40 was detectable on T cells of homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice. WT：C57BL/6JNifdc, HO: homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice.

Strain specific OX40L expression analysis in homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice by flow cytometry. Bone marrow derived dendritic cells (BMDCs) were produced by culturing the bone marrow from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice  (H/H;H/H;H/H;H/H;H/H), which were stimulated with LPS in vitro for 24 h. Protein expression was analyzed with anti-mouse OX40L antibody (Biolegend, 108814) and anti-human OX40L antibody (BD, 563766) by flow cytometry. Mouse OX40L was detectable in wild-type C57BL/6JNifdc mice. Human OX40L was detectable in homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice. WT：C57BL/6JNifdc, HO: homozygous B-hTSLP/hTSLPR plus/hOX40/hOX40L mice.

Analysis of immune cells in BALF. B-hTSLP/hTSLPR plus/hOX40/hOX40L mice (female, 7-week-old, n=6) were immunized with OVA etc. inducer to induce asthma. Anti-human TSLP antibody (SHR-1905 analog, provide by the client), anti-OX40L antibody (Amlitelimab analog, provide by the client), and anti-human IL13 antibody (Lebrikizumab analog, provide by the client) were intraperitoneally injected to B-hTSLP/hTSLPR plus/hOX40/hOX40L mice. (A&B) The number of CD45+ cells and eosinophils of BALF in the anti-human  IL13 antibody, the combination therapy group of anti-human  TSLP antibody, anti-human IL13 antibody and anti-human OX40L antibody, and the combination therapy group of anti-human OX40L antibody, and anti-human IL13 antibody or anti-human TSLP antibody treated groups decreased significantly compared with the PBS treated group. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ****P < 0.0001, ***P < 0.001, ns，No significance.

Analysis of mouse total IgE in serum. B-hTSLP/hTSLPR plus/hOX40/hOX40L mice (female, 7-week-old, n=6) were immunized with OVA etc. inducer to induce asthma. Anti-human TSLP antibody (SHR-1905 analog, provide by the client), anti-human OX40L antibody (Amlitelimab analog, provide by the client), and anti-human IL13 antibody (Lebrikizumab analog, provide by the client) were intraperitoneally injected to B-hTSLP/hTSLPR plus/hOX40/hOX40L mice. 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 the anti-human  IL13 antibody, the combination therapy group of anti-human  TSLP antibody, anti-human IL13 antibody and anti-OX40L antibody, and the combination therapy group of anti-human OX40L antibody, and anti-human IL13 antibody or anti-human TSLP antibody 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 < 0.0001, ***P < 0.001, ns，No significance.

H&E staining of asthma-like model in B-hTSLP/hTSLPR plus/hOX40/hOX40L mice. Lung tissues were collected at the study endpoint and analyzed with H&E staining. The results showed that the group of mice treated the anti-human IL13 antibody, the combination therapy group of anti-human TSLP antibody, anti-human IL13 antibody and anti-OX40L antibody, and the combination therapy group of anti-human OX40L antibody, and anti-human IL13 antibody or anti-human TSLP antibody in inflammatory infiltration and mucus secretion in lung tissue were lower than that in untreated mice, indicating that B-hTSLP/hTSLPR plus/hOX40/hOX40L mice provide a powerful preclinical model for in vivo evaluation of anti-human TSLP antibodies and anti-human OX40L antibodies. Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, ***P < 0.001, ns，No significance.