Gene targeting strategy for B-hCTLA4 mice.
The exon 2 of mouse Ctla4 gene that encode the extracellular domain was replaced by human CTLA4 exon 2 in B-hCTLA4 mice.

Strain specific analysis of CTLA4 gene expression in WT and hCTLA4 mice by RT-PCR. Mouse Ctla4 mRNA was detectable only in splenocytes of wild-type (+/+) mice. Human CTLA4 mRNA was detectable only in H/H, but not in +/+ mice.

Strain specific CTLA4 expression analysis in homozygous B-hCTLA4 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hCTLA4 (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-CTLA4 antibody. Mouse CTLA4 was detectable in WT mice. Human CTLA4 was exclusively detectable in homozygous B-hCTLA4 but not WT mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (female, n=5, 9-week-old) and homozygous B-hCTLA4 mice (female, n=5, 9-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hCTLA4 mice were similar to those in C57BL/6 mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood cells were isolated from wild-type C57BL/6 mice (female, n=5, 9-week-old) and homozygous B-hCTLA4 mice (female, n=5, 9-week-old). A. Flow cytometry analysis of the blood cells was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hCTLA4 mice were similar to those in C57BL/6 mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in lymph nodes by flow cytometry. Lymph nodes cells were isolated from wild-type C57BL/6 mice (female, n=5, 9-week-old) and homozygous B-hCTLA4 mice (female, n=5, 9-week-old). A. Flow cytometry analysis of the lymph nodes cells was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells and Tregs in B-hCTLA4 mice were similar to those in C57BL/6 mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

Complete blood count (CBC). Blood from female C57BL/6 and B-hCTLA4 mice (n=3, 6-week-old) was collected and analyzed for CBC. There was no differences among any measurement between C57BL/6 and B-hCTLA4 mice, indicating that introduction of hCTLA4 in place of its mouse counterpart does not change blood cell composition and morphology. Values are expressed as mean ± SEM.

Blood chemistry tests of B-hCTLA4 mice. Serum from the C57BL/6 and B-hCTLA4 mice (n=3, 6-week-old) was collected and analyzed for levels of ALT and AST. There was no differences on either measurement between C57BL/6 and B-hCTLA4 mice, indicating that introduction of hCTLA4 in place of its mouse counterpart does not change ALT and AST levels or health of liver. Values are expressed as mean ± SEM.

Antitumor activity of anti-human CTLA4 antibody in B-hCTLA4 mice. (A) Anti-human CTLA4 antibody inhibited MC38 tumor growth in B-hCTLA4 mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hCTLA4 mice (female, 6-7 week-old, n=5). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with ipilimumab with doses and schedules indicated in panel. (B) Body weight changes during treatment. As shown in panel A, ipilimumab (in house) was efficacious in controlling tumor growth in B-hCTLA4 mice, demonstrating that the B-hCTLA4 mice provide a powerful preclinical model for in vivo evaluation of anti-human CTLA4 antibody. Values are expressed as mean ± SEM.

Antitumor activity of anti-human CTLA4 antibody (in-house) in B-hCTLA4 mice. (A) Anti-human CTLA4 antibody inhibited MC38 tumor growth in B-hCTLA4 mice. Murine colon cancer MC38 cells (5×10⁵) were subcutaneously implanted into homozygous B-hCTLA4 mice (female, 6-9-week-old, n=8). Mice were grouped when tumor volume reached approximately 100-150 mm³, at which time they were intraperitoneally injected with anti-human CTLA4 antibody indicated in panel. (B) Body weight changes during treatment. As shown in panel A, anti-human CTLA4 antibody was efficacious in controlling tumor growth in B-hCTLA4 mice with a dose dependent, demonstrating that the B-hCTLA4 mice provide a powerful preclinical model for in vivo evaluation of anti-human CTLA4 antibodies. Values are expressed as mean ± SEM.

The overage of this tumor model is 40%.