B-hPD-1/hPD-L1/hCTLA4 mice

Basic Information

Strain Name
C57BL/6-Pdcd1tm1(PDCD1) Cd274tm1(CD274)Ctla4tm1(CTLA4)/Bcgen
Stock Number
130571
Common Name
B-hPD-1/hPD-L1/hCTLA4 mice
Source/Investigator
Bcgen (Beijing Biocytogen Co., Ltd)
Related Genes
PD-1 (Programmed death-1) ; CD274 (CD274 antigen) CTLA4 (Cytotoxic T-lymphocyte-associated protein 4,CD152)
Species
C57BL/6
Appearance
Black
Genotypes
Homozygous

Description

PD-1 (Programmed death-1) is mainly expressed on the surface of T cells and primary B cells. The two PD-1 ligands, PD-L1 and PD-L2, are widely expressed on antigen-presenting cells (APCs). PD-L1 expression is favorable for tumorigenesis and growth, for induction of anti-tumor T cell apoptosis, and for escaping responses by the immune system. Inhibition of PD-1 binding to its ligand can result in tumor cells that are exposed to the killing version of the immune system, and thus is a target for cancer treatments. PD-L1 (Programmed cell death ligand-1), also known as B7-H1 and CD274, is mainly expressed in antigen-presenting cells (APCs) and activated T cells, and is one of the two ligands of PD-1. The interaction between PD1 and PD-L1 plays an important role in the negative regulation of the immune response. PD-L1 is highly expressed in a variety of solid tumors. PD-1 and PD-L1 interactions can reduce T cell activation and promote tumor immune escape. The PD-1/PD-L1 signaling pathway can be blocked and antitumor immune response can be restored by using by anti-PD-1 or anti-PD-L1 antibodies to block the binding of PD1 to PD-L1. CTLA4 (Cytotoxic T-lymphocyte-associated protein 4), also known as CD152, competitively binds to B7-1 (CD80) and B7-2 (CD86) on Antigen-Presenting Cells (APCs) to block the T cell activating signal by B7 and CD28 (on T cells) interaction. The inhibition of CTLA4 by its inhibitory antibodies enhances T cell activity. The CTLA4 antibody is the first FDA-approved antibody to treat advanced melanoma.

Targeting strategy

Gene targeting strategy for B-hPD-1/hPD-L1/hCTLA4 mice. The exon 2 of mouse Pd-1 gene that encodes the extracellular domain was replaced by human PD-1 exon 2 in B-hPD-1/hPD-L1/hCTLA4 mice. The exon 3 of mouse Pd-l1 gene that encodes the extracellular domain was replaced by human PD-L1 exon 3 in B-hPD-1/hPD-L1/hCTLA4 mice. The exon 2 of mouse Ctla4 gene that encodes the extracellular domain was replaced by human CTLA4 exon 2 in B-hPD-1/hPD-L1/hCTLA4 mice.

Details

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, 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.

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 blood leukocytes cell subpopulations in B-hPD-1/hPD-L1/hCTLA4 mice

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

Analysis of lymph node 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, 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 results

Complete blood count (CBC). Blood from C57BL/6 and B-hPD-1/hPD-L1/hCTLA4 mice (n=5, 6 week-old, female) were collected and analyzed for CBC. Any measurement of B-hPD-1/hPD-L1/hCTLA4 mice in the panel were similar to C57BL/6, and there was no differences between male and female mice, indicating that humanized mouse does not change blood cell composition and morphology. Values are expressed as mean ± SEM.

Blood chemistry results

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)

 

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