SIRPA: A key immune checkpoints in macrophages phagocytosis and its therapeutic intervention

• Gene Information: Signal-regulatory protein alpha(SIRPA) is a protein-coding gene located on chromosome 20p13. It encodes a transmembrane receptor-type glycoprotein belonging to the immunoglobulin superfamily (IgSF). Cluster of differentiation 47 (CD47) is a protein-coding gene located on human chromosome 3q13.12. It encodes a heavily glycosylated, five-pass transmembrane protein that is a prominent member of the immunoglobulin superfamily (IgSF).
• Protein Expression: SIRPα is predominantly expressed on the surface of myeloid cells, stem cells and neurons. CD47 is also widely expressed in multiple tissue cells.
• Signaling Pathway: SIRPα binds to its ligand CD47 through the variable IgV-like domains. CD47+ cells activate SIRPα on macrophage surface to prevent its phagocytosis.
• Therapeutic Inhibition: Blocking the SIRPα-CD47 interaction via monoclonal antibodies or fusion proteins abrogates this inhibitory signal. This intervention successfully restores macrophage-mediated tumor phagocytosis, facilitates antigen cross-presentation, and improves anti-tumor clinical outcomes.

SIRPA

• The exon 2 of mouse Sirpa gene that encodes the IgV domain was replaced by exon 2 of human SIRPA gene.
• The endogenous mouse promoter, 5′ UTR, and 3′ UTR regions are retained, allowing chimeric SIRPA expression to be driven by the native mouse Sirpa promoter, while endogenous mouse Sirpa transcription and translation are abolished.

CD47

• The exon 2 of mouse Cd47 gene that encodes the IgV domain was replaced by exon 2 of human CD47 gene.
• The endogenous mouse promoter, 5′ UTR, and 3′ UTR regions are retained, allowing chimeric CD47 expression to be driven by the native mouse Cd47 promoter, while endogenous mouse Cd47 transcription and translation are abolished.

• Mouse SIRPA was detected in wild-type C57BL/6 mice. This anti-mouse SIRPα antibody also reacts crossly with human SIRPα.
• Human SIRPA was detected in homozygous B-hSIRPA/hCD47 mice, but not in wild-type C57BL/6 mice.

Mouse and human SIRPα expression analysis in Splenocytes. Splenocytes were collected from wild-type C57BL/6 mice and homozygous B-hSIRPA/hCD47 mice stimulated with or without anti-mouse CD3ε in vivo, and analyzed by flow cytometry with anti-mouse SIRPα antibody (Biolegend, 144011) and anti-human SIRPα antibody (Biolegend, 323810).

• Human SIRPα was detected on the peritoneal lymphocytes of B-hSIRPA/hCD47 mice, as evidenced by anti-human SIRPα antibody binding vs. isotype control.

Human SIRPα expression analysis on peritoneal lymphocytes. Peritoneal lymphocytes were isolated from female B-hSIRPA/hCD47 mice (n=2, 8-week-old). Flow cytometry analysis of the peritoneal lymphocytes was performed to assess the expression of human SIRPα. Single live cells were gated for the CD45 population and used for further analysis as indicated here.

• Mouse CD47 was exclusively detected in wild-type C57BL/6 mice.
• Human CD47 was detected in homozygous B-hSIRPA/hCD47 mice, but not in wild-type C57BL/6 mice.

Mouse and human CD47 expression analysis in Splenocytes. Splenocytes were collected from wild-type C57BL/6 mice and homozygous B-hSIRPA/hCD47 mice stimulated with or without anti-mouse CD3ε in vivo, and analyzed by flow cytometry with anti-mouse CD47 antibody (Biolegend, 127514) and anti-human antibody (Biolegend, 323108).

• Frequencies of B cell, T cell, NK cell, CD4+ T cell, CD8+ T cell, granulocyte, DC, macrophage and monocyte in homozygous B-hSIRPA/hCD47 mice were comparable to those in C57BL/6 mice, demonstrating that introduction of human SIRPA and CD47 in place of their mouse counterparts does not change the overall development, differentiation or distribution of these T cell subtypes in spleen or blood.

Analysis of leukocyte subpopulations by flow cytometry in Spleen. Splenocytes were collected from wild-type C57BL/6 mice and homozygous B-hSIRPA/hCD47 mice (female, 6-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated.

• Frequencies of CD4+ T, CD8+ T, and Tregs in homozygous B-hSIRPA/hCD47 mice were comparable to those in C57BL/6 mice, demonstrating that introduction of human SIRPA and CD47 in place of their mouse counterparts does not change the overall development, differentiation or distribution of these T cell subtypes in spleen or blood.

Analysis of T-cell subpopulations by flow cytometry in Spleen and blood. Splenocytes and blood were collected from wild-type C57BL/6 mice and homozygous B-hSIRPA/hCD47 mice (female, 6-week-old, n = 3). Single live cells were gated on the CD3⁺ T-cell population and analyzed by flow cytometry as indicated.

• No significant differences were observed compared with wild-type mice.

Complete blood count (CBC) of B-hSIRPA/hCD47 mice. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Blood biochemical parameters of B-hSIRPA/hCD47 mice are shown. Values are expressed as mean ± SD.

• No abnormalities were observed.

Organs of female B-hSIRPA/hCD47 mice(8-week-old, n = 10).

• No abnormalities were observed.

Organs of Male B-hSIRPA/hCD47 mice(8-week-old, n = 10).

• No obvious abnormalities were observed in any organs examined (heart, liver, spleen, lung, kidney, brain, stomach, small intestine, large intestine, testis, uterus, ovary).

Histopathological analysis of organs in B-hSIRPA/hCD47 mice. Major organs from B-hSIRPA/hCD47 mice were collected at 8 weeks of age and analyzed by H&E staining (male, n = 10; female, n = 10).

Establishment of a B-hCD47 MC38 model and in vivo efficacy study of anti-human CD47 antibodies. B-hCD47 MC38 melanoma cells were implanted subcutaneously into homozygous B-hSIRPA/hCD47mice (female, 6-8 weeks old, n=5). When the average tumor volume reached approximately 150 mm³, mice were randomized and subsequently administered the anti-human CD47 antibody via intraperitoneal injection.

Efficacy of CD47 Antibodies in B-hSIRPA/hCD47 mice. (A) Tumor growth curves. (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.

Establishment of a B-hCD47 MC38 model and in vivo efficacy study of anti-human SIRPα antibodies. B-hCD47 MC38 melanoma cells were implanted subcutaneously into homozygous B-hSIRPA/hCD47 mice (female, 6-8 weeks old, n=5). When the average tumor volume reached approximately 150 mm³, mice were randomized and subsequently administered the anti-human CD47 antibody via intraperitoneal injection.

Efficacy of SIRPα Specific Antibodies in B-hSIRPA/hCD47 mice. (A) Tumor growth curves. (B) Body weight changes during treatment. As shown in panel A, three human SIRPα 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.