Gene targeting strategy for B-hCD94/hNKG2A mice.
The exons 3~6 of mouse Cd94 gene that encode the extracellular domain were replaced by human CD94 exons 3~6 in B-hCD94/hNKG2A mice. The exons 2~6 of mouse Nkg2a gene that encode the extracellular domain were replaced by human NKG2A exons 2~6 in B-hCD94/hNKG2A mice.

Strain specific analysis of CD94 and NKG2A gene expression in WT and homozygous B-hCD94/hNKG2A mice by RT-PCR. Mouse Cd94 and Nkg2a mRNA were detectable in splenocytes of wild-type (+/+) mice. Human CD94 and NKG2A mRNA were detectable only in the homozygous B-hCD94/hNKG2A, but not in +/+ mice.

Strain specific CD94 and NKG2A expression analysis in homozygous B-hCD94/hNKG2A mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hCD94/hNKG2A (H/H) mice, and analyzed by flow cytometry with species-specific CD94 or NKG2A antibody. Mouse CD94 and NKG2A were detectable in WT mice. Human CD94 and NKG2A were exclusively detectable in homozygous B-hCD94/hNKG2A but not WT mice. (Monalizumab was used to detect the human NKG2A protein in WT and homozygous B-hCD94/hNKG2A)

Strain specific NKG2A expression analysis in homozygous B-hCD94/hNKG2A mice by flow cytometry. Splenocytes were collected from homozygous B-hCD94/hNKG2A (H/H) mice, and analyzed by flow cytometry with species-specific NKG2A antibody. Human NKG2A were exclusively detectable in activated CD8+ T cells of homozygous B-hCD94/hNKG2A mice after treated with mCD3e 24h.

Strain specific CD94 expression analysis in homozygous B-hCD94/hNKG2A mice by flow cytometry. Splenocytes were collected from homozygous B-hCD94/hNKG2A (H/H) mice, and analyzed by flow cytometry with species-specific CD94 antibody. Human CD94 were exclusively detectable in activated CD8+ T cells of homozygous B-hCD94/hNKG2A mice after treated with mCD3e 24h.

Analysis of splenic leukocyte subpopulations by FACS
Splenocytes were isolated from female C57BL/6 and B-hCD94/hNKG2A mice (n=3, 6 weeks-old) and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative FACS plots gated on single live CD45+ cells for further analysis. (B) Results of FACS analysis. Percentages of T, B, NK cells, monocytes/macrophages, and DC were similar in homozygous B-hCD94/hNKG2A mice and C57BL/6 mice, demonstrating that introduction of hCD94/hNKG2A in place of its mouse counterpart does not change the overall development, differentiation, or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Analysis of splenic T cell subpopulations by FACS
Splenocytes were isolated from female C57BL/6 and B-hCD94/hNKG2A mice (n=3, 6 weeks-old) and analyzed by flow cytometry for T cell subsets. (A) Representative FACS plots gated on TCRβ+ T cells and further analyzed. (B) Results of FACS analysis. Percentages of CD8+, CD4+, and Treg cells were similar in homozygous B-hCD94/hNKG2A and C57BL/6 mice, demonstrating that introduction of hCD94/hNKG2A 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 lymph node leukocyte subpopulations by FACS
Lymph node were isolated from female C57BL/6 and B-hCD94/hNKG2A mice (n=3, 6 weeks-old) and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative FACS plots gated on single live CD45+ cells for further analysis. (B) Results of FACS analysis. Percentages of T, B, NK cells, monocytes/macrophages, and DC were similar in homozygous B-hCD94/hNKG2A mice and C57BL/6 mice, demonstrating that introduction of hCD94/hNKG2A in place of its mouse counterpart does not change the overall development, differentiation, or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.

Analysis of lymph node T cell subpopulations by FACS
Lymph node were isolated from female C57BL/6 and B-hCD94/hNKG2A mice (n=3, 6 weeks-old) and analyzed by flow cytometry for T cell subsets. (A) Representative FACS plots gated on TCRβ+ T cells and further analyzed. (B) Results of FACS analysis. Percentages of CD8+, CD4+, and Treg cells were similar in homozygous B-hCD94/hNKG2A and C57BL/6 mice, demonstrating that introduction of hCD94/hNKG2A in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in lymph node. Values are expressed as mean ± SEM.

Analysis of blood leukocyte subpopulations by FACS
Blood were isolated from female C57BL/6 and B-hCD94/hNKG2A mice (n=3, 6 weeks-old) and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative FACS plots gated on single live CD45+ cells for further analysis. (B) Results of FACS analysis. Percentages of T, B, NK cells, monocytes/macrophages, and DC were similar in homozygous B-hCD94/hNKG2A mice and C57BL/6 mice, demonstrating that introduction of hCD94/hNKG2A in place of its mouse counterpart does not change the overall development, differentiation, or distribution of these cell types in blood. Values are expressed as mean ± SEM.

Analysis of blood T cell subpopulations by FACS. Blood were isolated from female C57BL/6 and B-hCD94/hNKG2A mice (n=3, 6 weeks-old) and analyzed by flow cytometry for T cell subsets. (A) Representative FACS plots gated on TCRβ+ T cells and further analyzed. (B) Results of FACS analysis. Percentages of CD8+, CD4+, and Treg cells were similar in homozygous B-hCD94/hNKG2A and C57BL/6 mice, demonstrating that introduction of hCD94/hNKG2A in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in blood. Values are expressed as mean ± SEM.

Antitumor activity of anti-human NKG2A antibody in B-hCD94/hNKG2A mice. (A) Anti-human NKG2A antibody inhibited hHLA-E MC38 tumor growth in B-hCD94/hNKG2A mice. Murine colon cancer hHLA-E MC38 cells were subcutaneously implanted into homozygous B-hCD94/hNKG2A mice (female, 6-7 week-old, n=5). Mice were grouped when tumor volume reached approximately 100 mm³, and treated with anti-hNKG2A antibody at doses and schedules in panel A. (B) Body weight changes during treatment. As shown in panel A, anti-human NKG2A antibody (monalizumab, in house) were efficacious in controlling tumor growth in B-hCD94/hNKG2A mice, demonstrating they provide a powerful preclinical model for in vivo evaluation of anti-human NKG2A antibody. Values are expressed as mean ± SEM.

Anti-tumor activity of tecentriq and monalizumab in B-hCD94/hNKG2A mice. (A) NKG2A antibody monalizumab(from partner) combined with PD-L1 antibody tecentriq(from partner) inhibited B-hHLA-E MC38 tumor growth in B-hCD94/hNKG2A mice. Murine colon cancer B-hHLA-E MC38 cells were subcutaneously implanted into homozygous B-hCD94/hNKG2A mice (female, 6-7 week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with antibodies at schedules in panel A. (B) Body weight changes during treatment. As shown in panel A, the combination of monalizumab and tecentriq showed more inhibitory effects than individual groups, demonstrating they provide a powerful preclinical model for in vivo evaluating combination therapy for PD-L1 and NKG2A. Values are expressed as mean ± SEM.