B-hPD-1/hPD-L1/h4-1BB mice

Basic Information

Strain name
C57BL/6-Pdcd1tm1(PDCD1) Cd274tm1(CD274) Tnfrsf9tm1(TNFRSF9)/Bcgen
Common name
B-hPD-1/hPD-L1/h4-1BB mice
Background
C57BL/6
Catalog number
130569
Related genes
  PD-1(Programmed death-1) ; TNFRSF9(TNF receptor superfamily member 9, also known as 4-1BB)

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-1 interacts with its ligands and plays an important role in the negative regulation of the immune response. PD-L1 protein expression is detected in many human tumor tissues. PD-L1 expression in tumor cells could be induced by the microenvironment of tumor cells. 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.

TNFRSF9 (Tumor necrosis factor receptor super family, member 9), also called CD137 and 4-1BB, is a co-stimulatory molecule and is mainly expressed on the surface of T, NK and mononuclear cells. CD137 is activated by its ligand CD137L or activating anti-CD137 antibodies, resulting in T Cell activation, proliferation and cytokine production. CD137L is expressed on antigen presenting cells such as dendritic cells and macrophages and activated B cells. In vivo studies show that CD137 activation increases anti-tumor immune responses, providing a new target for immunotherapy.

Targeting strategy

Gene targeting strategy for B-hPD-1/hPD-L1/h4-1BB mice. The exon 2 of mouse PD-1 gene that encode the IgV domain were replaced by human PD-1 exon 2 in B-hPD-1/hPD-L1/h4-1BB mice. The exon 3 of mouse Pd-l1 gene that encode the IgV domain were replaced by human PD-L1 exon 3 in B-hPD-L1/hPD-L1/h4-1BB mice. The exons 2-7 of mouse 4-1bb gene that encode the extracellular domain were replaced by human 4-1BB exons 2-7 in B-hPD-1/hPD-L1/h4-1BBmice.

Details

Protein expression analysis

Strain specific PD-1 expression analysis in homozygous B-hPD-1/hPD-L1/h4-1BB mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-1/hPD-L1/h4-1BB (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-hPD-1 antibody. Mouse PD-1 was detectable in WT mice but not homozygous B-hPD-1/hPD-L1/h4-1BB mice. Human PD-1 was exclusively detectable in homozygous B-hPD-1/hPD-L1/h4-1BB mice but not WT mice.

Strain specific PD-L1 expression analysis in homozygous B-hPD-1/hPD-L1/h4-1BB mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-1/hPD-L1/h4-1BB (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-hPD-L1 antibody. Mouse PD-L1 was detectable in WT mice but not homozygous B-hPD-1/hPD-L1/h4-1BB mice. Human PD-L1 was exclusively detectable in homozygous B-hPD-1/hPD-L1/h4-1BB mice but not WT mice.

Strain specific 4-1BB expression analysis in homozygous B-hPD-1/hPD-L1/h4-1BB mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-1/hPD-L1/h4-1BB (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-h4-1BB antibody. Mouse 4-1BB was detectable in WT mice but not homozygous B-hPD-1/hPD-L1/h4-1BB mice. Human 4-1BB was exclusively detectable in homozygous B-hPD-1/hPD-L1/h4-1BB mice but not WT mice.

Analysis of blood leukocytes cell subpopulations –no anti-mCD3

Analysis of blood leukocytes cell subpopulations –anti-mCD3

Analysis of spleen leukocytes cell subpopulations –no anti-mCD3

Analysis of spleen leukocytes cell subpopulations –anti-mCD3

Analysis of lymph node leukocytes cell subpopulations –no anti-mCD3

Analysis of lymph node leukocytes cell subpopulations –anti-mCD3

Analysis of blood,spleen and lymph node leukocytes cell subpopulations

Analysis of blood,spleen and lymph node leukocytes cell subpopulations

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, B-hPD-1/hPD-L1/h4-1BB and B-hPD-L1/h4-1BB mice were similar to those in the C57BL/6 mice both at rest and in stimulated with anti-CD3ε in vivo, 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 T cell subpopulations

Analysis of spleen T cell subpopulations

Analysis of lymph node T cell subpopulations

Analysis of blood,spleen and lymph node T cell subpopulations

Analysis of blood,spleen and lymph node T cell subpopulations

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, B-hPD-1/hPD-L1/h4-1BB and B-hPD-L1/h4-1BB mice were similar to those in the C57BL/6 mice both at rest and in stimulated with anti-CD3ε in vivo, 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, B-hPD-L1/h4-1BB and B-hPD-1/hPD-L1/h4-1BB mice (n=5, 6 week-old, female and male) ) were collected and analyzed for CBC. Any measurement of B-hPD-L1/h4-1BB and B-hPD-1/hPD-L1/h4-1BB 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-L1/h4-1BB and B-hPD-1/hPD-L1/h4-1BB mice. Serum from C57BL/6, B-hPD-L1/h4-1BB and B-hPD-1/hPD-L1/h4-1BB mice (n=5, 6 week-old, female and male) 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.

Combination therapy of anti-human PD-L1 antibody and anti-human 4-1BB antibody

Antitumor activity of anti-human PD-L1 antibody combined with anti-human 4-1BB antibody in B-hPD-1/hPD-L1/h4-1BB mice. (A) Anti-human PD-L1 antibody (in house) combined with anti-human 4-1BB antibody (in house) inhibited MC38 tumor growth in B-hPD-1/hPD-L1/h4-1BB mice. Murine colon cancer MC38-hPD-L1 cells were subcutaneously implanted into homozygous B-hPD-1/hPD-L1/h4-1BB 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 human PD-L1 and human 4-1BB antibodies in panel A. (B) Body weight changes during treatment. Values are expressed as mean ± SEM.

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