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B-hPD-1/hSIRPA/hCD47 mice
Strain Name C57BL/6-Pdcd1tm1(PDCD1)Sirpatm1(SIRPA)Cd47tm1(CD47)/Bcgen Common Name  B-hPD-1/hSIRPA/hCD47 mice
Background C57BL/6 Catalog number  130562
Related Genes 
PDCD1 (Programmed death-1, as known as PD-1) 
CD47
(CD47 molecule) 
SIRPA
(Signal regulatory protein alpha)
Gene 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


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Gene targeting strategy for B-hPD-1/hSIRPA/hCD47 mice. 

The exon 2 of mouse Pd-1 gene that encode the extracellular domain was replaced by human PD-1 exon 2 in B-hPD-L/hSIRPA/hCD47 mice. 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 in the B-hSIRPA/hCD47 mice. This triple knock-in mouse model was developed by mating the B-hPD-1 mice, B-hSIRPA mice and B-hCD47 mice together.


Protein expression analysis


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Strain specific PD-1, CD47 and SIRPα expression analysis in homozygous B-hPD-1/hSIRPA/hCD47 mice by flow cytometry. 

Splenocytes from both wild type (+/+) C57BL/6  and  homozygous B-hPD-1/hSIRPA/hCD47 (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry. Mouse PD-1+ and CD47+ T cells were only detectable in the WT C57BL/6 mice, human PD-1+ and CD47+ T cells were only detectable in the homozygous B-hPD-1/SIRPA/CD47mice. Mouse SIRPα was detectable in WT mice. This anti-mouse SIRPα antibody also cross reacts with hSIRPα. Human PD-1, CD47 and SIRPα were exclusively detectable in homozygous B-hPD-1/hSIRPa/hCD47 mice but not in WT mice.


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


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Antitumor activity of anti-human PD-1 antibody pembrolizumab combined with anti-human CD47 antibody in B-hPD-1/hSIRPA/hCD47 mice. 

(A) Pembrolizumab combined with hCD47 antibody inhibited MC38-hCD47 tumor growth in B-hPD-1/hSIRPA/hCD47 mice. Murine colon cancer MC38-hCD47 cells (5×105) were subcutaneously implanted into homozygous B-hPD-1/hSIRPA/hCD47 mice (female, 5-7 week-old, n=6). Mice were grouped when tumor volume reached approximately 150 mm3, at which time they were treated with pembrolizumab and hCD47 antibody with doses and schedules indicated in panel. (B) Body weight changes during treatment. As shown in panel A, combination of pembrolizumab and CD47  antibody shows more inhibitory effects than individual groups, demonstrating that the B-hPD-1/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.