PD-1

•A chimeric CDS including human PD-1 gene encoding the signal peptide and extracellular region, mouse PD-1 gene encoding the transmembrane and cytoplasmic region, a stop sequence WPRE were inserted after the initiation codon ATG of mouse PD-1 gene in B-hPD-1 mice plus.

•The endogenous mouse promoter and 5′ UTR are retained. The chimeric PD-1 expression is driven by the endogenous mouse PD-1 promoter, while endogenous mouse PD-1 transcription and translation are disrupted.

•Mouse PD-1 was detected in wild-type C57BL/6 mice.

•Human PD-1 was detected in B-hPD-1 mice plus but not in wild-type mice.

Strain-specific analysis of PD-1 protein expression in wild-type C57BL/6 mice and homozygous B-hPD-1 mice plus by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hPD-1 mice plus (H/H) and stimulated with or without anti-mouse CD3ε antibody. PD-1 expression on splenocytes was analyzed by flow cytometry using anti-mouse PD-1 antibody (Biolegend, 109104) and anti-human PD-1 antibody (Biolegend, 329904).

•Major splenic leukocyte subpopulations were comparable between B-hPD-1 mice plus and C57BL/6 mice.

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were collected 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. The percentages of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes, and macrophages in homozygous B-hPD-1 mice plus were comparable to those in C57BL/6 controls, indicating that PD-1 humanization does not alter the overall development, differentiation, or distribution of these leukocyte populations in spleen. Values are expressed as mean ± SEM.

•CD4+ T cells, CD8+ T cells and Tregs in spleen were comparable between B-hPD-1 mice plus and C57BL/6 mice.

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). Single live CD45+ cells were gated for the TCRβ+ T-cell population and used for further analysis. The percentages of CD4+ T cells, CD8+ T cells, and Tregs in homozygous B-hPD-1 mice plus were comparable to those in C57BL/6 controls, indicating that replacement of mouse PD-1 with human PD-1 does not alter the overall development, differentiation, or distribution of these T-cell subtypes in spleen. Values are expressed as mean ± SEM.

•Major lymph node leukocyte subpopulations were comparable between B-hPD-1 mice plus and C57BL/6 mice.

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). Single live cells were gated for the CD45+ population and used for further analysis. The percentages of T cells, B cells, CD4+ T cells, CD8+ T cells, Tregs, and NK cells in homozygous B-hPD-1 mice plus were comparable to those in C57BL/6 controls, indicating that PD-1 humanization does not alter the overall development, differentiation, or distribution of these immune-cell populations in lymph nodes. Values are expressed as mean ± SEM.

•Human PD-1 on T cells from B-hPD-1 mice plus is specifically recognized by anti-human PD-1 antibody analogs.

Analysis of splenocytes from B-hPD-1 mice plus by flow cytometry. Splenocytes were isolated from female B-hPD-1 mice plus and analyzed for human PD-1 expression on T cells. Single live cells were gated for the CD45+ population and used for further analysis. Human PD-1 expression was detectable on T cells from B-hPD-1 mice plus, as demonstrated by nivolumab analog (in-house) and pembrolizumab analog (in-house) binding compared with isotype control.

•Pembrolizumab analog inhibited B-hPD-L1 MC38 plus tumor growth without obvious body-weight loss.

Antitumor activity of anti-human PD-1 antibody pembrolizumab analog (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.

•Nivolumab analog inhibited B-hPD-L1 MC38 plus tumor growth without obvious body-weight loss.

Antitumor activity of anti-human PD-1 antibody nivolumab analog (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.