Protein expression analysis

Strain specific PD-1, PD-L1 and CTLA4 expression analysis in homozygous B-hPD-1/hPD-L1/hCTLA4 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-1/hPD-L1/hCTLA4 (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-PD-1, anti-PD-L1 and anti-CTLA4 antibody. Mouse PD-1, PD-L1 and CTLA4 were detectable in WT mice. Human PD-1, PD-L1 and CTLA4 were exclusively detectable in homozygous B-hPD-1/hPD-L1/hCTLA4 but not WT mice.

Analysis of spleen leukocytes cell subpopulations in B-hPD-1/hPD-L1/hCTLA4 mice

Analysis of blood, spleen and lymph node leukocytes cell subpopulations in B-hPD-1/hPD-L1/hCTLA4 mice

Analysis of blood, spleen and lymph node leukocytes cell subpopulations by FACS. Blood, spleen and lymph node leukocytes cell were isolated from female mice in the panel(n=3, 6 week-old). Flow cytometry analysis was performed to assess leukocyte subpopulations. Percent of T, B, NK, Granulocytes, Monocyte, DC and macrophage cells in homozygous B-hPD-1/hPD-L1/hCTLA4 mice were similar to those in the C57BL/6 mice at rest, demonstrating that the humanized mouse does not change the overall development, differentiation or distribution of these cell types in blood, spleen and lymph node.

Analysis of blood, spleen, lymph node T cell subpopulations in B-hPD-1/hPD-L1/hCTLA4 mice

Analysis of blood, spleen and lymph node T cell subpopulations in B-hPD-1/hPD-L1/hCTLA4 mice

Analysis of blood, spleen and lymph node T cell subpopulations by FACS. Blood, spleen and lymph node leukocytes cell were isolated from female mice in the panel(n=3, 6 week-old). Flow cytometry analysis was performed to assess leukocyte subpopulations. Percent of CD4+T, CD8+T and Tre cells in homozygous B-hPD-1/hPD-L1/hCTLA4 mice were similar to those in the C57BL/6 mice at rest, demonstrating that the humanized mouse does not change the overall development, differentiation or distribution of these cell types in blood, spleen and lymph node.

Blood routine test of B-hPD-1/hPD-L1/hCTLA4 mice

Blood chemistry of B-hPD-1/hPD-L1/hCTLA4 mice

Blood chemistry tests of B-hPD-1/hPD-L1/hCTLA4 mice. Serum from C57BL/6 and B-hPD-1/hPD-L1/hCTLA4 mice (n=5, 6 week-old, female) were collected and analyzed for levels of ALT, AST and other indicators in the panel. There was no differences on either measurement between C57BL/6 and humanized mouse, indicating that humanized mouse does not change ALT and AST levels or health of liver. Values are expressed as mean ± SEM.

In vivo efficacy of pembrolizumab, atezolizumab and ipilimumab 

Antitumor activity of pembrolizumab, atezolizumab and ipilimumab in B-hPD-1/hPD-L1/hCTLA4 mice. (A) Anti-human PD-1, PD-L1 and CTLA4 antibody inhibited MC38 tumor growth in B-hPD-1/hPD-L1/hCTLA4 mice. Murine colon cancer hPD-L1 MC38 cells were subcutaneously implanted into homozygous B-hPD-1/hPD-L1/hCTLA4 mice (female, 9-10 week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with pembrolizumab, atezolizumab and ipilimumab with doses and schedules indicated in panel A. (B) Body weight changes during treatment. As shown in panel A, anti-human PD-1, PD-L1 and CTLA4 antibody inhibited MC38 tumor growth in B-hPD-1/hPD-L1/hCTLA4 mice. Values are expressed as mean ± SEM. (All antibodies were made in house)

Combination therapy of pembrolizumab, atezolizumab and ipilimumab 

Antitumor activity of pembrolizumab, atezolizumab and ipilimumab in B-hPD-1/hPD-L1/hCTLA4 mice. 

(A) Anti-human PD-1/PD-L1 antibody combined with anti-human CTLA4 antibody inhibited MC38 tumor growth in B-hPD-1/hPD-L1/hCTLA4 mice. Murine colon cancer hPD-L1 MC38 cells were subcutaneously implanted into homozygous B-hPD-1/hPD-L1/hCTLA4 mice (female, 9-10 week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with pembrolizumab, atezolizumab and ipilimumab with doses and schedules indicated in panel A. (B) Body weight changes during treatment. As shown in panel A, combination of pembrolizumab/ipilimumab or atezolizumab/ipilimumab shows more inhibitory effects than individual groups, demonstrating that the B-hPD-1/hPD-L1/hCTLA4 mice provide a powerful preclinical model for in vivo evaluating combination therapy efficacy of hPD-1/hPD-L1 antibodies and hCTLA4 antibodies. Values are expressed as mean ± SEM (All antibodies were made in house).