Basic Information
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Details
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mRNA expression analysis
Strain specific analysis of IL2RA gene expression in WT and hIL2RA mice by RT-PCR. Mouse Il2ra mRNA was detectable only in splenocytes of wild-type (+/+) mice. Human IL2RA mRNA was detectable only in H/H, but not in +/+ mice.
Protein expression analysis
Strain specific IL2RA expression analysis in homozygous B-hIL2RA mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hIL2RA (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-IL2RA antibody. Mouse IL2RA was detectable in WT mice. Human IL2RA was exclusively detectable in homozygous B-hIL2RA but not WT mice.
Phenotypic analysis
Analysis of spleen leukocytes cell subpopulations in B-hIL2RA mice
Analysis of spleen leukocyte subpopulations by FACS
Splenocytes were isolated from female C57BL/6 and B-hIL2RA 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 CD45 population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T, B, NK, Monocyte, DC and macrophage cells in homozygous B-hIL2RA mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL2RA in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in spleen.
Analysis of spleen T cell subpopulations in B-hIL2RA mice
Analysis of spleen T cell subpopulations by FACS
Splenocytes were isolated from female C57BL/6 and B-hIL2RA 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 CD3 T cell population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8, CD4, and Treg cells in homozygous B-hIL2RA mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL2RA in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in spleen. Values are expressed as mean ± SEM.
Analysis of lymph node leukocytes cell subpopulations in B-hIL2RA mice
Analysis of lymph node leukocyte subpopulations by FACS
Leukocytes were isolated from female C57BL/6 and B-hIL2RA 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 CD45 population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T, B and NK cells in homozygous B-hIL2RA mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL2RA in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in lymph node.
Analysis of lymph node T cell subpopulations in B-hIL2RA mice
Analysis of lymph node T cell subpopulations by FACS
Leukocytes were isolated from female C57BL/6 and B-hIL2RA 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 CD3 T cell population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8, CD4, and Treg cells in homozygous B-hIL2RA mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL2RA in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in lymph node. Values are expressed as mean ± SEM.
Data provided by client
STAT-5 Phosphorylation is blocked by hIL2RA Ab1, non-blocked by hIL2RA Ab2
STAT-5 Phosphorylation is blocked by hIL2RA Ab1, non-blocked by hIL2RA Ab2
Splenocytes were isolated from C57BL/6 and B-hIL2RA mice (n=3), treated with 100 U/mL mouse IL2 and then analyzed by flow cytometry. STAT-5 phosphorylation is blocked by hIL2RA Ab1, but not hIL2RA Ab2.
In vivo efficacy of anti human IL2RA antibodies
Antitumor activity of anti-human IL2RA antibodies in B-hIL2RA mice. (A) Anti-human IL2RA antibodies inhibited MC38 tumor growth in B-hIL2RA mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hIL2RA mice (female, 6-7 week-old, n=5). Mice were grouped according to body weight differences, at which time they were treated with anti-hIL2RA Ab4 and Ab5 with doses and schedules indicated with red arrows in panel. (B) Body weight changes during treatment. As shown in panel A, anti-human IL2RA antibodies were efficacious in controlling tumor growth in B-hIL2RA, demonstrating that the B-hIL2RA mice provide a powerful preclinical model for in vivo evaluation of anti-human IL2RA antibodies. Values are expressed as mean ± SEM.
Lymphocytes analysis in blood
Blood were harvested at the endpoint of experiment, and the lymphocytes were analyzed by flow cytometry.
Ratio of blood lymphocytes
Flow cytometry analysis of blood lymphocytes. Blood were harvested at the endpoint of experiment (n=5). Flow cytometry analysis of the lymphocytes were performed to assess cell number and proportion changes compared to the group with no anti-hIL2RA antibody treated. CD4+ T cells were significantly reduced when treated with anti-hIL2RA antibodies, while the ratio of CD8+ T cells/Treg cells were significantly increased in the group treated with anti-hIL2RA Ab4 compared to the hIgG1. Values are expressed as mean ± SEM.
Tumor infiltrating lymphocytes (TILs）analysis
Tumor cells were harvested at the endpoint of experiment, and the lymphocytes were analyzed by flow cytometry.
Ratio of tumor infiltrating lymphocytes (TILs）
Flow cytometry analysis of tumor infiltrating lymphocytes (TILs). Tumor cells were harvested at the endpoint of experiment (n=5). Flow cytometry analysis of the lymphocytes were performed to assess cell number and proportion changes compared to the group with no anti-hIL2RA antibody treated. CD4+ T cells, Treg cells and hCD25+mFoxP3+ T cells were significantly reduced when treated with anti-hIL2RA antibodies, while the ratio of CD8+ T cells/Treg cells were significantly increased in the all groups treated with anti-hIL2RA antibodies compared to the hIgG1. Values are expressed as mean ± SEM.
In vivo efficacy of anti-human IL2RA antibodies
Antitumor activity of anti-human IL2RA antibodies in B-hIL2RA mice. (A) Anti-human IL2RA antibodies inhibited MC38 tumor growth in B-hIL2RA mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hIL2RA mice (female, 6-7 week-old, n=8). Mice were grouped according to body weight differences, at which time they were treated with four anti-human IL2RA antibodies with doses. (B) Body weight changes during treatment. As shown in panel A, anti-human IL2RA antibodies (211At-7G7/B6 and M-A251) were efficacious in controlling tumor growth in B-hIL2RA, demonstrating that the B-hIL2RA mice provide a powerful preclinical model for in vivo evaluation of anti-human IL2RA antibodies. Values are expressed as mean ± SEM. (All antibodies were made in house)
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Publications
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Song, D., Liu, X., Dong, C. et al. Two novel human anti-CD25 antibodies with antitumor activity inversely related to their affinity and in vitro activity. Sci Rep 11, 22966 (2021). https://doi.org/10.1038/s41598-021-02449-y
