B-hPD-1 mice Plus

Basic Information

Targeting Strategy

Gene targeting strategy for B-hPD-1 mice plus. The ectodomain of human PD-1 and cytoplasmic domain of mouse PD-1 were inserted after the initiation codon ATG of mouse PD-1 gene in B-hPD-1 mice plus.

Protein Expression Analysis

Strain specific PD-1 expression analysis in homozygous B-hPD-1 mice plus by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-1 mice plus (H/H) stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-PD-1 antibody. Mouse PD-1 was detected in WT mice. Human PD-1 was exclusively detected in homozygous B-hPD-1 mice plus but not WT mice.

Strain specific PD-1 expression analysis in homozygous B-hPD-1 mice plus by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-1 mice plus (H/H) and analyzed by flow cytometry with species-specific anti-PD-1 antibody. Mouse PD-1 was detected in WT mice. Human PD-1 was exclusively detected in homozygous B-hPD-1 mice plus but not WT mice.

Analysis of leukocytes cell subpopulation in spleen

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hPD-1 mice plus (n=3, 7-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-hPD-1 mice plus were similar to those in the C57BL/6 mice, demonstrating that PD-1 humanized does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Analysis of T cell subpopulation in spleen

Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hPD-1 mice plus (n=3, 7-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 CD4+ T cells, CD8+ T cells and Tregs in homozygous B-hPD-1 mice plus were similar to those in the C57BL/6 mice, demonstrating that introduction of hPD-1 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 leukocytes cell subpopulation in lymph node

Analysis of lymph node leukocyte subpopulations by FACS. Lymph nodes were isolated from female C57BL/6 and B-hPD-1 mice plus (n=3, 7-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, CD4+ T cells, CD8+ T cells, Tregs and NK cells in homozygous B-hPD-1 mice plus were similar to those in the C57BL/6 mice, demonstrating that PD-1 humanized does not change the overall development, differentiation or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.

T cells in B-hPD-1 plus mice bind anti-human PD-1 antibody

Analysis of splenocytes of B-hPD-1 mice plus by FACS. Splenocytes were isolated from female B-hPD-1 mice plus. Flow cytometry analysis of the splenocytes was performed to assess human PD-1 expression on T cells. Single live cells were gated for CD45+ population and used for further analysis as indicated here. Human PD-1 expression was detectable on T cells in B-hPD-1 mice plus as evidenced by nivolumab (in house) and pembrolizumab (in house) binding vs isotype control.

In vivo efficacy of anti-human PD-1 antibody

Antitumor activity of anti-human PD-1 antibody pembrolizumab (in house) in B-hPD-1 mice plus. (A) Anti-human PD-1 antibody inhibited MC38 tumor growth in B-hPD-1 mice plus. Murine colon cancer MC38 cells (5×10⁵) were subcutaneously implanted into homozygous B-hPD-1 mice plus (female 7-week-old, n=5). Mice were grouped when tumor volume reached approximately 150 mm³, at which time they were treated with anti-human PD-1 antibody with doses and schedules indicated in panel. (B) Body weight changes during treatment. As shown in panel A, anti-human PD-1 antibody was efficacious in controlling tumor growth in B-hPD-1 mice plus, demonstrating that the B-hPD-1 mice plus provide a powerful preclinical model for in vivo evaluation of anti-human PD-1 antibodies. Values are expressed as mean ± SEM.

Antitumor activity of anti-human PD-1 antibody nivolumab (in house) in B-hPD-1 mice plus. (A) Anti-human PD-1 antibody inhibited MC38 tumor growth in B-hPD-1 mice plus. Murine colon cancer MC38 cells (5×10⁵) were subcutaneously implanted into homozygous B-hPD-1 mice plus (female 7-week-old, n=5). Mice were grouped when tumor volume reached approximately 150 mm³, at which time they were treated with anti-human PD-1 antibody with doses and schedules indicated in panel. (B) Body weight changes during treatment. As shown in panel A, anti-human PD-1 antibody was efficacious in controlling tumor growth in B-hPD-1 mice plus, demonstrating that the B-hPD-1 mice plus provide a powerful preclinical model for in vivo evaluation of anti-human PD-1 antibodies. Values are expressed as mean ± SEM.

References

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publication

Kim HM, Kim KJ, Lee K, Yoon MJ, Choih J, Hong TJ, Cho EJ, Jung HJ, Kim J, Park JS, Na HY, Heo YS, Park CG, Park H, Han S, Bae D. GNUV201, a novel human/mouse cross-reactive and low pH-selective anti-PD-1 monoclonal antibody for cancer immunotherapy. BMC Immunol. 2024 May 11;25(1):29.