Targeting strategy

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

Analysis of peritoneal leukocytes of B-hSIRPA/hCD47 mice by flow cytometry. Leukocytes were isolated from female B-hSIRPA/hCD47 mice (n=2, 8-week-old). Flow cytometry analysis of the peritoneal leukocyte 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 leukocytes of B-hSIRPA/hCD47 mice as evidenced by anti-human SIRPα antibody binding vs isotype control. 

Blood routine test in 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.

Comparison of hCD47 expression level on red cells between B-hSIRPA/hCD47 mice and human blood

Analysis of hCD47 expression level on red cells in B-hSIRPA/hCD47 mice and human blood by flow cytometry. Red cells were collected from blood of B-hSIRPA/hCD47 mice and human (n=4). The results showed that there was no significant difference in the expression level of human CD47 between B-hSIRPA/hCD47 mice and human red blood cells.

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.

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.