• CD134, also known as OX40 and TNFRSF4, is a 50 kD type I transmembrane protein, which is a member of the TNF receptor family.
• OX40 is expressed on activated T lymphocytes including Th1, Th2, Th17, and Treg cells and the interaction of OX40 with OX40L results in B cell proliferation and antibody secretion, regulation of primary T cell expansion, and T cell survival.
• CD252 is a 35 kD member of the TNF superfamily and is also known as OX40 ligand, OX40L, and CD134L. The OX40L is expressed on activated B cells and antigen presenting cells.
• 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.
• Application: This product is used for pharmacodynamics and safety evaluation of  drugs.

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.

Strain specific analysis of OX40 mRNA expression in wild-type C57BL/6JNifdc mice and homozygous B-hOX40/hOX40L mice by RT-PCR. Spleen RNA was isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hOX40/hOX40L mice (H/H;H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human OX40 primers. Mouse OX40 mRNA was only detectable in wild-type mice Human OX40 mRNA was exclusively detectable in homozygous B-hOX40/hOX40L mice  but not in wild-type mice.

Strain specific analysis of OX40L gene expression in wild-type C57BL/6JNifdc mice and homozygous B-hOX40/hOX40L mice by RT-PCR. Bone marrow cells were collected from wild-type C57BL/6JNifdc mice(+/+) and homozygous B-hOX40/hOX40L (H/H;H/H) mice. DCs were induced from bone marrow cells. Mouse Ox40l mRNA was detectable in DC cell of wild-type mice. Human OX40L mRNA was detectable only in B-hOX40/hOX40L but not in wild-type mice.

Strain specific OX40 expression analysis in homozygous B-hOX40/hOX40L mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc(+/+) and homozygous B-hOX40/hOX40L mice (H/H;H/H) stimulated with anti-CD3ε in vivo, and protein expression was analyzed with anti-mouse OX40 antibody (Biolegend, 119414)  and anti-human OX40 antibody (Biolegend, 350004) by flow cytometry. Mouse OX40 was only detectable in T cells of wild-type C57BL/6JNifdc mice. Human OX40 was only detectable in T cells of homozygous B-hOX40/hOX40L mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific OX40L expression analysis in homozygous B-hOX40/hOX40L mice by flow cytometry. Bone marrow cells were collected from WT and homozygous B-hOX40/hOX40L (H/H;H/H) mice. DCs were induced from bone marrow cells and stimulated with LPS. Then DCs were analyzed by flow cytometry with anti-OX40L antibodies. Mouse OX40L was detectable in WT mice. Human OX40L was exclusively detectable in homozygous B-hOX40/hOX40L (H/H;H/H) but  not  WT 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 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 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 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 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 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.

Strain specific cytokines expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hOX40/0X40L mice by ELISA. Serum was collected from wild-type C57BL/6JNifdc mice (+/+) (female, n=3, 7-week-old) and homozygous B-hOX40/0X40L mice (H/H;H/H)  (female, n=3, 7-week-old) stimulated with anti-mouse CD3ε antibody (37.5 μg/mL, 200 μL/mouse, i.p.) for 2 hrs in vivo. Expression level of mouse IL4, IL13, IL2, IL22, IL17A, and IFN-γ were analyzed by ELISA. After mCD3ε stimulation, a significant increase of mouse IL4, IL13, IL2, IL22, IL17A, and IFN-γ were detected in C57BL/6JNifdc mice (n=3) and homozygous B-hOX40/0X40L mice  (n=3). Under the stimulation of mCD3ε, the downstream cytokines of the humanized mice can fully respond, which proves that the signaling pathway of humanized mice is complete. Values are expressed as mean ± SEM.

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 (provided by a client) was administered by intraperitoneal injection twice a week on days -1 to 23. Serum was collected at the endpoint on day 26. AD: atopic dermatitis; OXA: oxazolone.

Note: This experiment is a collaboration with the client.

Efficacy of anti-human OX40L antibody in B-hOX40/hOX40L mice. Mice in each group were intraperitoneally injected with anti-hOX40L antibody (provided by a client, n=5). (A) Body weight changes during the treatment. (B) Statistical analysis of ear thickness in each group. (C&D) Total IgE levels in serum. The results showed that compared to the untreated group (G2), the group of mice treated with anti-OX40L antibody 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 anti-OX40L antibody 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.

Note: This experiment is a collaboration with the client.

Efficacy of anti-human OX40L antibody in B-hOX40/hOX40L mice. Ear tissues were collected at the study endpoint and analyzed with H&E. The results showed that compared to the untreated group (G2), the group of mice treated with anti-OX40L antiboday (provided by a client). showed a significant reduction in epidermal thickness and pathological score of ear skin. 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.

Note: This experiment is a collaboration with the client.

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. Bronchoalveolar 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. 

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.

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. 

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.

Antitumor activity of anti-human TA1 antibody in B-hOX40/hOX40L mice. Anti-human TA1 antibody inhibited B16-F10 tumor growth in homozygous B-hOX40/hOX40L mice. B16-F10 cells were subcutaneously implanted into both sides of homozygous B-hOX40/hOX40L mice (female, 6-8 weeks-old, n=7). Mice were grouped when tumor volume reached approximately 50-150 mm3, at which time they were treated with Anti-human TA1 antibody (TA1 provided by client) via intrathecal injection (i.t.) indicated in panel. The tumor volumes on the administration side (A) and the non-administration side (B)  were statistically analyzed respectively. (C) Body weight changes during treatment. As shown in panel A and B, anti-human TA1 antibody was efficacious in controlling tumor growth in B-hOX40/hOX40L mice and also triggered the bystander effect, demonstrating that the B-hOX40/hOX40L mice provide a powerful preclinical model for in vivo evaluation of anti-human TA1 antibodies. Values are expressed as mean ± SEM.

Note: This experiment is a collaboration with the client.