Basic Information
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Target strategy
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The exon 2 of mouse PD-1 gene that encode the extracellular domain was replaced by human PD-1 exon 2 in B-hPD-1/hPD-L1/hC5AR1 mice. The exon 3 of mouse Pdl1 gene that encode the extracellular domain was replaced by human PD-L1 exon 3 in B-hPD-1/hPD-L1/hC5AR1 mice. The exon 2 of mouse C5ar1 gene that encodes the full-length protein was replaced by human C5AR1 exon 2 in B-hPD-1/hPD-L1/hC5AR1 mice. This treble knock-in model was developed by breeding the B-hPD-1/hPD-L1 mice and the B-hC5AR1 mice together.
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Protein expression analysis
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Species-specific PD-1, PD-L1, C5AR1 protein expression analysis in humanized B-hPD-1/hPD-L1/hC5AR1 mice. Splenocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hPD-1/hPD-L1/hC5AR1 (H/H) mice and analyzed by flow cytometry using species-specific anti-PD-1 and anti-PD-L1 antibodies. Bone marrow was isolated from wild-type C57BL/6 (+/+) and homozygous B-hPD-1/hPD-L1/hC5AR1 (H/H) mice and analyzed by flow cytometry using species-specific anti-C5AR1 antibodies. Murine PD-1, PD-L1, C5AR1 proteins were detected in wild-type mice, while human PD-1, PD-L1, C5AR1 proteins were exclusively detected in B-hPD-1/hPD-L1/hC5AR1 mice.
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Analysis of immune cells
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Analysis of spleen leukocyte subpopulations by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 and humanized B-hPD-1/hPD-L1/hC5AR1 mice (female, n=3, 6-week-old), and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative flow cytometry plots. Single live cells were gated on CD45+ and used for further analysis as indicated. (B) Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in B-hPD-1/hPD-L1/hC5AR1 mice were similar to those in wild-type mice, demonstrating that hPD-1, hPD-L1 and hC5AR1 in place of their murine counterparts does not change the overall development, differentiation or distribution of these spleen cell subtypes. Values are expressed as mean ± SEM.
Analysis of spleen T cell subpopulations by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 and humanized B-hPD-1/hPD-L1/hC5AR1 mice (female, n=3, 6-week-old), and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative flow cytometry plots. Single live CD45+ cells were gated on TCRβ+ T cells and used for further analysis as indicated. (B) Percent of CD4+ T cells, CD8+ T cells and Tregs in B-hPD-1/hPD-L1/hC5AR1 mice were similar to those in wild-type mice, demonstrating that introduction of hPD-1, hPD-L1 and hC5AR1 in place of their murine counterparts does not change the overall development, differentiation or distribution of these spleen T cell subtypes. Values are expressed as mean ± SEM.
Analysis of blood leukocyte subpopulations by flow cytometry. Blood cells were isolated from wild-type C57BL/6 and humanized B-hPD-1/hPD-L1/hC5AR1 mice (female, n=3, 6-week-old), and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative flow cytometry plots. Single live cells were gated on CD45+ and used for further analysis as indicated. (B) Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in B-hPD-1/hPD-L1/hC5AR1 mice were similar to those in wild-type mice, demonstrating that hPD-1, hPD-L1 and hC5AR1 in place of their murine counterparts does not change the overall development, differentiation or distribution of these blood cell subtypes. Values are expressed as mean ± SEM.
Analysis of blood T cell subpopulations by flow cytometry. Blood cells were isolated from wild-type C57BL/6 and humanized B-hPD-1/hPD-L1/hC5AR1 mice (female, n=3, 6-week-old), and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative flow cytometry plots. Single live CD45+ cells were gated on TCRβ+ T cells and used for further analysis as indicated. (B) Percent of CD4+ T cells, CD8+ T cells, and Tregs in B-hPD-1/hPD-L1/hC5AR1 mice were similar to those in wild-type mice, demonstrating that introduction of hPD-1, hPD-L1 and hC5AR1 in place of their murine counterparts does not change the overall development, differentiation or distribution of these blood T cell subtypes. Values are expressed as mean ± SEM.
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In vivo efficacy studies
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Combination therapy using anti-human PD-1 (pembrolizumab, in house) and anti-human C5AR1 antibodies (avdoralimab, in house) in B-hPD-1/hPD-L1/hC5AR1 mice. Humanized murine colon cancer B-hPD-L1 MC38 plus cells were subcutaneously implanted into homozygous B-hPD-1/hPD-L1/hC5AR1 mice (female, 7-9-week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with anti-human PD-1 and anti-human C5AR1 antibodies at doses and schedules indicated. (A) Combination therapy using anti-human PD-1 and anti-human C5AR1 antibodies inhibited B-hPD-L1 MC38 plus tumor growth in B-hPD-1/hPD-L1/hC5AR1 mice, (B) without negatively impacting body weight changes. B-hPD-1/hPD-L1/hC5AR1 mice provide a powerful preclinical model for in vivo evaluation of anti-human PD-1 and anti-human C5AR1 antibodies. Values are expressed as mean ± SEM.
Combination therapy using anti-human PD-1 (pembrolizumab, in house) and anti-human C5AR1 antibodies (avdoralimab, in house) in B-hPD-1/hPD-L1/hC5AR1 mice. Humanized murine colon cancer B-hPD-L1 MC38 plus cells were subcutaneously implanted into homozygous B-hPD-1/hPD-L1/hC5AR1 mice (female, 5-8-week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with anti-human PD-1 and anti-human C5AR1 antibodies at doses and schedules indicated. (A) Combination therapy using anti-human PD-1 and anti-human C5AR1 antibodies inhibited B-hPD-L1 MC38 plus tumor growth in B-hPD-1/hPD-L1/hC5AR1 mice, (B) without negatively impacting body weight changes. B-hPD-1/hPD-L1/hC5AR1 mice provide a powerful preclinical model for in vivo evaluation of anti-human PD-1 and anti-human C5AR1 antibodies. Values are expressed as mean ± SEM.
