B-hSIRPα/hCD47 mice

Basic Information

Strain Name
C57BL/6-SIRPα tm1(SIRPα) Cd47 tm1(CD47) /Bcgen
Stock Number
120525
Common Name
B-hSIRPα/hCD47 mice
Source/Investigator
Bcgen (Beijing Biocytogen Co., Ltd)
Related Genes
CD47 (CD47 molecule); SIRPα(Signal regulatory protein alpha)
Species
C57BL/6
Appearance
Black
Genotypes
Homozygous

Description

SIRPα (Signal-regulatory protein alpha) is a transmembrane protein widely expressed in myeloid cells , stem cells and neurons. Its extracellular part include 3 Immunoglobulinlike domains. SIRPα binds to its ligand CD47 through the variable IgV-like domains. CD47 is also widely expressed in multiple tissue cells. CD47+ cells activate SIRPα on macrophage surface to prevent its phagocytosis. Previous studies reveal that the diversity of SIRPα is the key to human hematopoietic stem cell suppression, especially tumor suppression. The interruption of SIRPα-CD47 interaction substantially inhibits a variety of tumors. SIRPα/CD47 antibodies are considered as the next star target for tumor immunosuppression following PD1/PD-L1 antibodies.

CD47 is an integrin associated protein (IAP) and is an immunoglobulin superfamily member. CD47 is widely expressed on cell surface and interacts with the signal regulatory protein α (SIRPα), thrombospondin-1 (TSP1) and integrins to mediate a series of responses including apoptosis, proliferation and immunity. CD47 is an important “self” mark on the cell surface and inhibits macrophagocytosis by interaction with SIRPα on macrophage surface. Animal studies have shown that a CD47 antibody is an effective treatment for multiple types of tumors. CD47 is another target for tumor immunity following PD-1/PD-L1.

Targeting Strategy

Gene targeting strategy for B-hSIRPA/hCD47 mice. The exon 2 of mouse CD47 gene that  encode the extracellular domain was replaced by exon2 of human CD47 gene. The exon 2 of mouse  Sirpα  gene that  encode the extracellular domain was replaced by exon2 of human SIRPα in the B-hSIRPA/hCD47 mice. This double knock-in model was developed by mating the B-hSIRPA mice and the B-hCD47 mice together.

Details

Phenotype

Protein Expression Analysis

Strain specific CD47 and SIRPα expression analysis in homozygous B-hSIRPA/hCD47 mice by flow cytometry. Splenocytes were collected from WT (+/+) and homozygous B-hSIRPA/hCD47 (H/H) mice and analyzed by flow cytometry with species-specific anti-CD47 and anti-SIRPα antibodies. Mouse CD47 was exclusively detectable in WT mice. Mouse SIRPα was detectable in WT mice. This anti-mouse SIRPα antibody also reacts crossly with human SIRPα. Human CD47 and SIRPα were exclusively detectable in homozygous B-hSIRPA/hCD47 mice but not in WT mice.

Strain specific CD47 and SIRPα expression analysis in homozygous B-hSIRPA/hCD47 mice by flow cytometry. Splenocytes were collected from WT (+/+) and homozygous B-hSIRPA/hCD47 (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-CD47 and anti-SIRPα antibodies. Mouse CD47 was exclusively detectable in WT mice. Mouse SIRPα was detectable in WT mice. This anti-mouse SIRPα antibody also reacts crossly with human SIRPα. Human CD47 and SIRPα were exclusively detectable in homozygous B-hSIRPA/hCD47 mice but not in WT mice.

Peritoneal lymphocyte in B-hSIRPA/hCD47 mice bind to anti-human SIRPα antibody

Analysis of peritoneal lymphocyte of B-hSIRPA/hCD47 mice by flow cytometry. T cells were isolated from female B-hSIRPA/hCD47 mice (n=2, 8-week-old). Flow cytometry analysis of the peritoneal lymphocyte was performed to assess expression of human SIRPα. Single live cells were gated for CD45 population and used for further analysis as indicated here. Human SIRPα was detectable on peritoneal lymphocyte of B-hSIRPA/hCD47 mice as evidenced by anti-human SIRPα antibody binding vs isotype control.

T cells in B-hSIRPA/hCD47 mice bind to anti-human CD47 antibody

Analysis of Splenocytes of B-hSIRPA/hCD47 mice by flow cytometry. Splenocytes were isolated from female B-hSIRPA/hCD47 mice (n=2, 8-week-old).  Flow cytometry analysis of the splenocytes was performed to assess human CD47 expression on T cells. Single live cells were gated for CD45 population and used for further analysis as indicated here. Human CD47 expression was detectable on T cells in B-hSIRPA/hCD47 mice as evidenced by anti-human CD47 antibody Hu5F9 (in house) binding vs isotype control.

Analysis of spleen leukocytes cell subpopulations in B-hSIRPA/hCD47 mice

Analysis of spleen leukocyte subpopulations by FACS

Splenocytes were isolated from female C57BL/6 and B-hSIRPA/hCD47 mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for CD45 population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells, monocytes, DCs,  granulocytes and macrophages in homozygous B-hSIRPA/hCD47 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hSIRPα and hCD47 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 spleen T cell subpopulations in B-hSIRPA/hCD47 mice

Analysis of spleen T cell subpopulations by FACS

Splenocytes were isolated from female C57BL/6 and B-hSIRPA/hCD47 mice mice (n=3, 6-week-old).  Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for CD3 T cell population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8, CD4, and Treg cells in homozygous B-hSIRPA/hCD47 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of  hSIRPα and hCD47 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.

Blood routine test in B-hSIRPA/hCD47 mice

Complete blood count (CBC). Blood from female C57BL/6 and B-hSIRPA/hCD47 mice (n=3, 6-week-old) was collected and analyzed by CBC. There was no differences among any measurement between C57BL/6 and B-hSIRPA/hCD47 mice, indicating that introduction of hSIRPα and hCD47 in place of its mouse counterpart does not change blood cell composition and morphology. Values are expressed as mean ± SEM.

Blood chemistry of B-hSIRPA/hCD47 mice

Blood chemistry tests of B-hSIRPA/hCD47 mice. Plasma from the C57BL/6 and B-hSIRPA/hCD47 mice (n=3, 6 week-old) was collected and analyzed for levels of ALT and AST. There was no differences on either measurement between C57BL/6 and B-hSIRPA/hCD47 mice, indicating that introduction of hSIRPα and hCD47 in place of its mouse counterpart does not change ALT and AST levels or health of liver. Values are expressed as mean ± SEM.

In vivo efficacy of anti-human CD47 antibody

Antitumor activity of anti-human CD47 antibody Hu5F9 (in house) in B-hSIRPA/hCD47 mice. (A) anti-human CD47 antibody inhibited MC38-hCD47 tumor growth in B-hSIRPA/hCD47 mice. Murine colon cancer MC38-hCD47 cells were subcutaneously implanted into homozygous B-hSIRPA/hCD47 mice (female, 6-8 weeks old, n=5). Mice were grouped when tumor volume reached approximately 150 mm3, at which time they were treated with anti-human CD47 antibody with doses and schedules indicated in panel. (B) Body weight changes during treatment. As shown in panel A, anti-human CD47 antibody was efficacious in controlling tumor growth in B-hSIRPA/hCD47 mice, demonstrating that the B-hSIRPA/hCD47 mice provide a powerful preclinical model for in vivo evaluation of anti-human CD47 antibodies. Values are expressed as mean ± SEM.

In vivo efficacy and toxicity evaluation of anti-human CD47 antibody Hu5F9 (in house)

Different doses of anti-human CD47 antibody caused anemia in B-hSIRPA/hCD47 mice. Homozygous B-hSIRPA/hCD47 mice were treated with PBS or Hu5F9 (in house) as indicated on the graph (female, 6-8 week-old, n=5). Blood were collected at different time points and analyzed by blood routine test. The group of Hu5F9 3mg/kg showed a significant decrease in hemoglobin and RBC levels in the short term. Values are expressed as mean ± SEM.

In vivo efficacy of anti-human CD47 antibodies

Antitumor activity of anti-human CD47 antibody in B-hSIRPA/hCD47 mice. (A) anti-human CD47 antibody inhibited MC38-hCD47 tumor growth in B-hSIRPA/hCD47 mice. Murine colon cancer MC38-hCD47 cells were subcutaneously implanted into homozygous B-hSIRPA/hCD47 mice (female, 6-8 week-old, n=5). Mice were grouped when tumor volume reached approximately 150 mm3, at which time they were treated with anti-human CD47 antibody with doses and schedules indicated in panel. (B) Body weight changes during treatment. As shown in panel A, four anti-human CD47 antibodies differently inhibited tumor growth in B-hSIRPA/hCD47 mice, demonstrating that the B-hSIRPA/hCD47 mice provide a powerful preclinical model for in vivo evaluation of anti-human CD47 antibodies. Values are expressed as mean ± SEM.

In vivo efficacy of anti-human SIRPα antibodies

Antitumor activity of anti-human SIRPα antibody in B-hSIRPA/hCD47 mice. (A) anti-human SIRPα antibody inhibited MC38-hCD47 tumor growth in B-hSIRPA/hCD47 mice. Murine colon cancer MC38-hCD47 cells were subcutaneously implanted into homozygous B-hSIRPA/hCD47 mice (female, 6-8 week-old, n=5). Mice were grouped when tumor volume reached approximately 150 mm3, at which time they were treated with anti-human SIRPα antibody with doses and schedules indicated in panel. (B) Body weight changes during treatment. As shown in panel A, three human SIRPA antibodies differently inhibited tumor growth in B-hSIRPA/hCD47 mice, demonstrating that the B-hSIRPA/hCD47 mice provide a powerful preclinical model for in vivo evaluation of anti-human SIRPα antibodies. Values are expressed as mean ± SEM.

References

  1. PNAS July 2, 2013 110 (27) 11103-11108; doi: 10.1073/pnas.1305569110
  2. Curr Opin Immunol. 2012 April ; 24(2): 225–232. doi:10.1016/j.coi.2012.01.010
  3.  Genes to Cells (2015) 20, 451–463 doi:10.1111/gtc.12238
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