Basic Information
TNFRSF4(Tumor necrosis factor receptor superfamily, member 4, also known as OX40);
TNFSF4 ( tumor necrosis factor(TNF) superfamily member 4, also known as OX40L)
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Gene Targeting Strategy
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Gene targeting strategy for B-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-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-hOX40/hOX40L mice .
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mRNA Expression Analysis
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Strain specific analysis of OX40L gene expression in WT and B-hOX40/hOX40L mice by RT-PCR. (B)Mouse Ox40l mRNA was detectable in DC cell of wild-type (+/+) . Human OX40L mRNA was detectable only in B-hOX40/hOX40L (H/H) but not in +/+ mice.
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Protein Expression Analysis
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Species–specific OX40 expression analysis in double humanized B-hOX40/hOX40L mice. Following anti-CD3ε stimulation in vivo, splenocytes were collected from wild-type C57BL/6 (+/+) and homozygous B-hOX40/hOX40L (H/H) mice and analyzed by flow cytometry using species-specific anti-OX40 antibodies. Human OX40 was exclusively detected in B-hOX40/hOX40L mice compared to wild-type mice.
Species–specific OX40L expression analysis in double humanized B-hOX40/hOX40L mice. Bone marrow cells or bone marrow-derived DCs stimulated with LPS were collected from wild-type C57BL/6 (+/+) and homozygous B-hOX40/hOX40L (H/H) mice and analyzed by flow cytometry using species-specific anti-OX40L antibodies. Human OX40L was exclusively detected in B-hOX40/hOX40L mice compared to wild-type mice.
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Analysis of Immune Cells
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Analysis of spleen leukocytes cell subpopulations in B-hOX40/hOX40L mice
Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hOX40/hOX40L mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was 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 cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hOX40/hOX40L mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hOX40/hOX40L in place of its mouse counterpart 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 in B-hOX40/hOX40L mice
Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hOX40/hOX40L mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. (B) Results of FACS analysis. The percent of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hOX40/hOX40L mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hOX40/hOX40L in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. Values are expressed as mean ± SEM.
Analysis of blood leukocytes cell subpopulations in B-hOX40/hOX40L mice
Analysis of blood leukocyte subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hOX40/hOX40L mice (n=5, 6-week-old). Flow cytometry analysis of the blood cell was 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 cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hOX40/hOX40L mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hOX40/hOX40L in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in blood. Values are expressed as mean ± SEM.
Analysis of blood leukocytes cell subpopulations in B-hOX40/hOX40L mice
Analysis of blood T cell subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hOX40/hOX40L mice (n=3, 6-week-old). Flow cytometry analysis of the blood was performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. (B) Results of FACS analysis. The percent of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hOX40/hOX40L mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hOX40/hOX40L in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in blood. Values are expressed as mean ± SEM.
Analysis of lymph node leukocytes cell subpopulations in B-hOX40/hOX40L mice
Analysis of lymph node leukocyte subpopulations by FACS. Lymph nodes were isolated from female C57BL/6 and B-hOX40/hOX40L mice (n=3, 6-week-old). Flow cytometry analysis of the leukocytes was 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 cells, B cells, NK cells in homozygous B-hOX40/hOX40L mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hOX40/hOX40L in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.
Analysis of lymph node T cell subpopulations in B-hOX40/hOX40L mice
Analysis of lymph node T cell subpopulations by FACS. Lymph nodes were isolated from female C57BL/6 and B-hOX40/hOX40L mice (n=3, 6-week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. (B) Results of FACS analysis. The percent of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hOX40/hOX40L mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hOX40/hOX40L in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in lymph node. Values are expressed as mean ± SEM.
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Experimental design for induction of AD-like skin lesions and in vivo efficacy of anti-human OX40L antibody
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Experimental schedule for induction of AD-like skin lesions and in vivo efficacy of anti-human OX40L antibody. OXA was applied to dorsal and ear skin of mice on day 0, and then challenge to the same site of skin nine times from days 7 to 25. Anti-human OX40L antibody Amlitelimab (in-house) was administered by intraperitoneal injection twice a week on days 6 to 23. Serum was collected at the endpoint on day 27. AD: atopic dermatitis; OXA: oxazolone.
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In vivo efficacy of anti-human OX40L antibody in OXA induced AD-like mouse model
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Efficacy of anti-human OX40L antibody in B-hOX40/hOX40L mice. Mice in each group were intraperitoneally injected with anti-hOX40L antibody Amlitelimab analog (in-house, n=6). (A&B) Body weight changes during the treatment. (C) Statistical analysis of ear thickness in each group. Epidermis of ear began to desquamate from day 18. So the ear thicknesses were decreased from day 18 as shown in figure. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001.
Efficacy of anti-human OX40L antibody in B-hOX40/hOX40L mice. Mice in each group were intraperitoneally injected with anti-hOX40L antibody Amlitelimab analog (in-house, n=6). (A) Total IgE level in serum. Serum was collected on day 27 and total IgE level was measured by ELISA. The results showed that the level of total IgE in mice treated with Amlitelimab (in-house) was lower than that in untreated mice. (B) Total score of ear epidermis. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001. AD: Atopic dermatitis.
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In vivo efficacy of anti-human OX40L antibody with asthma model
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Experimental schedule for induction of asthma and in vivo efficacy of anti-human OX40L antibody. OVA + Al(OH)3 was injected intraperitoneally on days 0, 7, and 14; followed by daily nebulization with OVA for the challenge phase from days 21 to 27. . Anti-human OX40L antibody amlitelimab (in-house) was administered by intraperitoneal injection every 3 days on days 0 to 27. Serum was collected at the endpoint on day 28. OVA: ovalbumin.
Analysis of immune cells in BALF by flow cytometry. B-hOX40/hOX40L mice (female, 7-week-old, n=6) were immunized with OVA to induce asthma. Anti-human OX40L antibody (Amlitelimab analog, synthesized in-house) was intraperitoneally injected from day 0 to day 27. Broncheoalveolar fluid (BALF) was collected at the end of the experiment to detect inflammatory cell infiltration in lung tissue. The results showed that the number of CD45+ cells and eosinophils of BALF in the Amlitelimab treated group (G3) decreased significantly compared with the OVA -induced untreated group (G2). Data indicated that anti-human OX40L antibodies could effectively reduce the number and proportion of eosinophils in B-hOX40/hOX40L mice induced with OVA. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Mouse total IgE and OVA-specific IgE in serum were reduced in the mouse asthma model treated with anti-OX40L antibody. Serum was collected at the study endpoint. IgE level was analyzed by ELISA. The results showed that the level of OVA specific IgE in mice treated with Amlitelimab (in-house) was lower than that in untreated mice. Values are expressed as mean ± SEM. Significance was determined by unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
H&E staining of asthma-like model in B-hOX40/hOX40L mice. Lung tissues were collected at the study endpoint and analyzed with H&E staining. The results showed that compared to the untreated group (G2), the group of mice treated with Amlitelimab (in-house) showed a significant reduction in inflammatory infiltration and mucus secretion in lung tissue, indicating that B-hOX40/hOX40L mice provide a powerful preclinical model for in vivo evaluation of anti-human OX40L antibodies. Values are expressed as mean ± SEM. Significance was determined by unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
Analysis of immune cells in BALF and mouse total IgE in serum. B-hOX40/hOX40L mice (male, 11-week-old, n=6) were immunized with mTSLP/OVA to induce asthma. Anti-human OX40L antibody (Amlitelimab analog, synthesized in-house) was intraperitoneally injected from Day -1. (A&B) The number of CD45+ cells and eosinophils of BALF in the Amlitelimab treated group decreased significantly compared with the mTSLP/OVA-induced isotype treated group. (D) 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 Amlitelimab (in-house) 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, ****P < 0.0001.
H&E staining of asthma-like model in B-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 with Amlitelimab (in-house) in inflammatory infiltration and mucus secretion in lung tissue was lower than that in untreated mice, indicating that B-hOX40/hOX40L mice provide a powerful preclinical model for in vivo evaluation of anti-human OX40L antibodies. Values are expressed as mean ± SEM. Significance was determined by unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
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In vivo efficacy of anti-human OX40L antibody with AD and asthma model
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Efficacy of anti-human OX40L antibody in B-hOX40/hOX40L mice. (A&B) Ear thickness and body weight changes during the treatment. (C) Total IgE levels in serum. The results showed that compared to the untreated group (G2), the group of mice treated with Amlitelimab (in-house) showed a significant reduction in ear thickness. 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 Amlitelimab (in-house) was lower than that in 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-hOX40/hOX40L mice. ear tissues were collected at the study endpoint and analyzed with H&E staining. The results showed that compared to the untreated group (G2), the group of mice treated with Amlitelimab (in-house) showed a significant reduction in histopathology score and score of eosinophil infiltration. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. AD: Atopic dermatitis.
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In vivo efficacy of anti-human OX40L antibody with CIA model
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Experimental schedule for induction of CIA and in vivo efficacy of anti-human OX40L antibody.
Experimental schedule for Induction of CIA and in vivo efficacy of anti-human OX40L antibody. 50 μL CⅡ emulsion injection subcutaneously at 2 points-the base of the tail and buttocks on day 0 and day 21 respectively. Animals with disease onset were grouped individually into G1-G3 between day 22 to day 30 while ensuring similar average clinical scores on the day of grouping. Mouse body weight (A) and clinical score (B) post grouping were shown for selected timepoints.
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Summary
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Protein expression analysis:
- Human OX40 was exclusively detectable in T cells of the homozygous B-hOX40/hOX40L mice but not in wild-type mice.
- Human OX40L was exclusively detectable in DCs of the homozygous B-hOX40/hOX40L mice but not in wild-type mice.
Leukocytes subpopulation analysis:
- OX40 and OX40L humanized does not change the overall development, differentiation or distribution of immune cell types in spleen, bone marrow, and blood.
In vivo efficacy:
- Anti-human OX40L antibody was efficacious in controlling atopic dermatitis like symptoms in B-hOX40/hOX40L mice, the concentrations of total serum IgE was decreased after treat with Anti-human OX40L antibody.
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Poster
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AACR 2023: Humanized OX40/OX40L Mice as a Tool for Evaluating Novel Therapeutics