B-hPD-1/hPD-L1/hSIRPA/hCD47 mice

Basic Information

Strain Name
C57BL/6-Pdcd1tm1(PDCD1)Cd274tm1(CD274)Sirpatm1(SIRPA)Cd47tm1(CD47)/Bcgen
Common Name
B-hPD-1/PD-L1/hSIRPA/hCD47 mice
Background
C57BL/6
Catalog number
140577
Related Genes
PDCD1 (Programmed death-1, as known as PD-1) CD274 (CD274 molecule,Also known as PD-L1); CD47 (CD47 molecule) ; SIRPA (Signal regulatory protein alpha)

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. SIRPA (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.SIRPA (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.

Targeting strategy

Gene targeting strategy for B-hPD-1/PD-L1/hSIRPA/hCD47 mice. The exon 2 of mouse Pd-1 gene that encode the extracellular domain was replaced by human PD-1 exon 2. The exon 3 of mouse Pdl1 gene that encode the extracellular domain was replaced by human PD-L1 exon 3. The exon 2 of mouse Sirpα gene that encode the extracellular domain was replaced by human SIRPα exon 2. The exon 2 of mouse Cd47 gene that encode the extracellular domain was replaced by human CD47 exon 2. This triple knock-in mouse model was developed by mating the B-hPD-1 mice, B-hPD-L1 mice, B-hSIRPA mice and B-hCD47 mice together.

Details

Protein expression analysis

Strain specific PD-1, PD-L1, CD47 and SIRPα expression analysis in homozygous B-hPD-1/PD-L1/hSIRPA/hCD47 mice by flow cytometry. Splenocytes from both wild type (+/+) C57BL/6  and  homozygous B-hPD-1/PD-L1/hSIRPA/hCD47 (H/H) mice were analyzed by flow cytometry. Mouse PD-1+, PD-L1 and CD47+ T cells were only detectable in the WT C57BL/6 mice.Human PD-1+, PD-L1+ and CD47+ T cells were  only detectable in the homozygous B-hPD-1/PD-L1/hSIRPA/hCD47 mice. Mouse SIRPα was detectable in WT mice. This anti-mouse SIRPα antibody also cross reacts with hSIRPα. Human SIRPα was exclusively detectable in homozygous B-hPD-1/PD-L1/hSIRPA/hCD47 mice but not in WT mice.

 

Strain specific PD-1, PD-L1, CD47 and SIRPα expression analysis in homozygous B-hPD-1/PD-L1/hSIRPA/hCD47 mice by flow cytometry. Splenocytes from both wild type (+/+) C57BL/6  and  homozygous B-hPD-1/PD-L1/hSIRPA/hCD47 (H/H) mice were stimulated with anti-CD3ε in vivo and analyzed by flow cytometry. Mouse PD-1+, PD-L1 and CD47+ T cells were only detectable in the WT C57BL/6 mice.Human PD-1+, PD-L1+ and CD47+ T cells were  only detectable in the homozygous B-hPD-1/PD-L1/hSIRPA/hCD47 mice. Mouse SIRPα was detectable in WT mice. This anti-mouse SIRPα antibody also cross reacts with hSIRPα. Human SIRPα was exclusively detectable in homozygous B-hPD-1/PD-L1/hSIRPA/hCD47 mice but not in WT mice.

 

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hPD-1/hPD-L1/hSIRPA/hCD47 (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-hPD-1/hPD-L1/hSIRPA/hCD47 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-hPD-1/PD-L1/hSIRPA/hCD47

Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hPD-1/PD-L1/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 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-hPD-1/PD-L1/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.

 

Analysis of lymph node T cell subpopulations in B-hPD-1/PD-L1/hSIRPA/hCD47

Analysis of lymph node T cell and NK cell subpopulations by FACS. Lymphocyte were isolated from female C57BL/6 and B-hPD-1/PD-L1/hSIRPA/hCD47 mice (n=3, 6-week-old).  Flow cytometry analysis of the lymphocyte was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for TCRβ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8, CD4, NK and Treg cells in homozygous B-hPD-1/PD-L1/hSIRPA/hCD47 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of  hPD-1, hPD-L1, 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-hPD-1/PD-L1/hSIRPA/hCD47 mice

Complete blood count (CBC) Blood from female C57BL/6 and B-hPD-1/PD-L1/hSIRPA/hCD47 mice (n=5, 6 weeks-old) was collected and analyzed by CBC. Except PLT, RBC, Hb and HCT, C57BL/6 and B-hPD-1/PD-L1/hSIRPA/hCD47 mice showed no difference in other test results. Values are expressed as mean ± SEM.

Blood chemistry of B-hPD-1/PD-L1/hSIRPA/hCD47 mice

Blood chemistry tests of B-hPD-1/PD-L1/hSIRPA/hCD47 mice Serum from the C57BL/6 and B-hPD-1/PD-L1/hSIRPA/hCD47 (n=3, 6 week-old) was collected and analyzed for levels of biochemistry. There was no differences on AST, ALB, CHOL, CREA measurement between C57BL/6 and B-hPD-1/hPD-L1 mice. Values are expressed as mean ± SEM.

Combination therapy of anti-human PD-1 Ab and CD47 Ab

Antitumor activity of anti-human PD-1 antibody pembrolizumab (in house) combined with anti-human CD47 antibody Hu5F9 (in house) in B-hPD-1/hPD-L1/hSIRPA/hCD47 mice. (A) Pembrolizumab combined with Hu5F9 inhibited MC38-hPD-L1/hCD47 tumor growth in B-hPD-1/hPD-L1/hSIRPA/hCD47 mice. Murine colon cells (5E5) were subcutaneously implanted into homozygous B-hPD-1/hPD-L1/hSIRPA/hCD47 mice  (female, 8week-old, n=5). Mice were grouped when tumor volume reached approximately 150 mm3, at which time they were treated with antibodies with doses and schedules indicated in panel. (B) Body weight changes during treatment. As shown in panel A, combination of pembrolizumab and Hu5F9 shows more inhibitory effects than individual groups, demonstrating that the B-hPD-1/hPD-L1/hSIRPA/hCD47 mice provide a powerful preclinical model for in vivo evaluation of combination therapy of anti-human PD-1 and anti-human CD47 antibodies. Values are expressed as mean ± SEM.

Combination therapy of anti-human PD-L1 Ab and CD47 Ab

Antitumor activity of anti-human PD-L1 antibody Atezolizumab (in house) combined with anti-human CD47 antibody Hu5F9 (in house) in B-hPD-1/hPD-L1/hSIRPA/hCD47 mice. (A) Atezolizumab combined with Hu5F9 inhibited MC38-hPD-L1/hCD47 tumor growth in B-hPD-1/hPD-L1/hSIRPA/hCD47 mice. Murine colon cells (5E5) were subcutaneously implanted into homozygous B-hPD-1/hPD-L1/hSIRPA/hCD47 mice  (female, 8week-old, n=5). Mice were grouped when tumor volume reached approximately 150 mm3, at which time they were treated with antibodies with doses and schedules indicated in panel. (B) Body weight changes during treatment. As shown in panel A, combination of Atezolizumab and Hu5F9 shows more inhibitory effects than individual groups, demonstrating that the B-hPD-1/hPD-L1/hSIRPA/hCD47 mice provide a powerful preclinical model for in vivo evaluation of combination therapy of anti-human PD-L1 and anti-human CD47 antibodies. Values are expressed as mean ± SEM.

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