B-hPD-L1/hSIRPA/hCD47 mice

Basic Information

Strain Name
C57BL/6-PD-L1 tm1(PD-L1)Sirpa tm1(SIRPA) Cd47 tm1(CD47) /Bcgen
Stock Number
130561
Common Name
B-hPD-L1/hSIRPA/hCD47 mice
Source/Investigator
Bcgen (Beijing Biocytogen Co., Ltd)
Related Genes
CD274(CD274 molecule,Also known as PD-L1); CD47 (CD47 molecule) ; SIRPA (Signal regulatory protein alpha)
Species
C57BL/6
Appearance
Black
Genotypes
Homozygous

Description

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. 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-L1/hSIRPA/hCD47 mice. The exon 3 of mouse Pdl1 gene that encode the extracellular domain was replaced by human PD-L1 exon 3 in B-hPD-L1/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-L1 mice, B-hSIRPA mice and B-hCD47 mice together.

Details

Protein expression analysis

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

Strain specific PD-L1, CD47 and SIRPα expression analysis in homozygous B-hPD-L1/hSIRPA/hCD47 mice by flow cytometry. Splenocytes from both wild type (+/+) C57BL/6 and homozygous B-hPD-L1/hSIRPA/hCD47 (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry. Mouse PD-L1+ and CD47+ T cells were only detectable in the WT C57BL/6 mice. Human PD-L1+ and CD47+ T cells were only detectable in the homozygous B-hPD-L1/SIRPA/CD47mice. Mouse SIRPα was detectable in WT mice. This anti-mouse SIRPα antibody also cross reacts with hSIRPα. Human SIRPα were exclusively detectable in homozygous B-hPD-L1/hSIRPA/hCD47 but not WT mice.

Analysis of spleen leukocytes cell subpopulations in B-hPD-L1/hSIRPA/hCD47 mice

Analysis of spleen leukocyte subpopulations by FACS

Splenocytes were isolated from C57BL/6 and B-hPD-L1/hSIRPA/hCD47 mice (n=3). 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-L1/hSIRPA/hCD47 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hPD-L1, hSIRPα and hCD47 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in spleen.

Analysis of spleen T cell subpopulations in B-hPD-L1/hSIRPA/hCD47 mice

Analysis of spleen T cell subpopulations by FACS

Splenocytes were isolated from female C57BL/6 and B-hPD-L1/hSIRPA/hCD47 mice (n=3). 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+ T cells, CD4+ T cells and Treg cells in homozygous B-hPD-L1/hSIRPA/hCD47 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of 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.

Analysis of blood leukocytes cell subpopulations in B-hPD-L1/hSIRPA/hCD47 mice

Analysis of blood leukocyte subpopulations by FACS

Leukocytes were isolated from C57BL/6 and B-hPD-L1/hSIRPA/hCD47 mice (n=3). 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-L1/hSIRPA/hCD47 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hPD-L1, hSIRPα and hCD47 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in blood.

Analysis of blood T cell subpopulations in B-hPD-L1/hSIRPA/hCD47 mice

Analysis of blood T cell subpopulations by FACS Leukocytes were isolated from female C57BL/6 and B-hPD-L1/hSIRPA/hCD47 mice (n=3). 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+ T cells, CD4+ T cells and Treg cells in homozygous B-hPD-L1/hSIRPA/hCD47 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hPD-L1, hSIRPα and hCD47 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in blood.

In vivo efficacy of anti-human CD47/PD-L1 bispecific antibodies

Antitumor activity of anti-human CD47/PD-L1 bispecific antibodies in B-hPD-L1/hSIRPA/hCD47 mice. (A) Anti-human CD47/PD-L1 bispecific antibody inhibited MC38-hPD-L1-hCD47 tumor growth in B-hPD-L1/hSIRPA/hCD47 mice (female, 6-8 week-old, n=5). Mice were grouped when tumor volume reached approximately 200 mm3, at which time they were treated with different antibodies with doses and schedules indicated in panel. (B) Body weight changes during treatment. As shown in panel A, the bispecific antibody showed better efficacy among these antibodies, demonstrating that the B-hPD-L1/hSIRPA/hCD47 mice provide a powerful preclinical model for in vivo evaluation of anti-human CD47/PD-L1 bispecific antibodies. Values are expressed as mean ± SEM.

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