Strain specific 4-1BB protein expression analysis in BALB/c mice and homozygous B-h4-1BB mice(C) by flow cytometry. Splenocytes were collected from wild type BALB/c mice (+/+) and homozygous B-h4-1BB mice(C) (H/H) that stimulated with or without anti-CD3ε treatment in vivo, and analyzed by flow cytometry with species-specific anti-4-1BB antibodies. Mouse 4-1BB was detectable in different T cell subtypes of BALB/c mice, while human 4-1BB was exclusively detectable in T cell subtypes of B-h4-1BB mice(C).

Strain specific 4-1BB protein expression analysis in BALB/c mice and homozygous B-h4-1BB mice(C) by flow cytometry. Splenocytes were collected from wild type BALB/c mice (+/+) and homozygous B-h4-1BB mice(C) (H/H), and analyzed by flow cytometry with species-specific anti-4-1BB antibodies. Mouse 4-1BB was detectable in B cells, NK cells, dendritic cells and monocytes of BALB/c mice but not in macrophages and neutrophils. While human 4-1BB was exclusively detectable in B cells, NK cells, dendritic cells and monocytes of B-h4-1BB mice(C) but not in macrophages and neutrophils. 

Strain specific 4-1BB protein expression analysis in BALB/c mice and homozygous B-h4-1BB mice(C) by flow cytometry. Splenocytes were collected from wild type BALB/c mice (+/+) and homozygous B-h4-1BB mice(C) (H/H) that stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-4-1BB antibodies. Mouse 4-1BB was detectable in B cells, NK cells, dendritic cells and monocytes of BALB/c mice but not in macrophages and neutrophils. While human 4-1BB was exclusively detectable in B cells, NK cells, dendritic cells and monocytes of B-h4-1BB mice(C) but not in macrophages and neutrophils. 

Analysis of spleen leukocyte subpopulations by flow cytometry. Splenocytes were isolated from female BALB/c and B-h4-1BB mice(C) (n=3, 8-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, neutrophils, monocytes and macrophages in homozygous B-h4-1BB mice(C) were similar to those in the BALB/c mice, demonstrating that 4-1BB humanized 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 flow cytometry. Splenocytes were isolated from female BALB/c and B-h4-1BB mice(C) (n=3, 8-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-h4-1BB mice(C) were similar to those in the BALB/c mice, demonstrating that introduction of h4-1BB 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 flow cytometry. Blood cells were isolated from female BALB/c and B-h4-1BB mice(C) (n=3, 8-week-old). Flow cytometry analysis of the blood 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, neutrophils, monocytes and macrophages in homozygous B-h4-1BB mice(C) were similar to those in the BALB/c mice, demonstrating that 4-1BB humanized 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 flow cytometry. Blood were isolated from female BALB/c and B-h4-1BB mice(C)(n=3, 8-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-h4-1BB mice(C) were similar to those in the BALB/c mice, demonstrating that introduction of h4-1BB 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 flow cytometry. Lymph node cells were isolated from female BALB/c and B-h4-1BB mice(C) (n=3, 8-week-old). Flow cytometry analysis of the lymph node 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, and NK cells in homozygous B-h4-1BB mice(C) were similar to those in the BALB/c mice, demonstrating that 4-1BB humanized 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 flow cytometry. Lymph node were isolated from female BALB/c and B-h4-1BB mice(C) (n=3, 8-week-old). Flow cytometry analysis of the lymph node 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-h4-1BB mice(C) were similar to those in the BALB/c mice, demonstrating that introduction of h4-1BB 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.

Antitumor activity of anti-human 4-1BB antibody (Urelumab analog, in-house) in B-h4-1BB mice(C). Murine colon cancer CT26 cells were subcutaneously implanted into homozygous B-h4-1BB mice(C) (male, 9 week-old, n=6). Mice were grouped when tumor volume reached approximately 50 mm³, at which time they were treated with antibodies with doses and schedules indicated in panel. (A) Tumor growth curve. (B) Body weight changes during treatment. As shown, anti-human 4-1BB antibodies were efficacious in controlling tumor growth in B-h4-1BB mice(C). B-h4-1BB mice(C) provide a powerful preclinical model for in vivo evaluation of anti-human 4-1BB antibodies. Values are expressed as mean ± SEM.