Gene targeting strategy for B-hPD-1/hSIRPA/hCD47 mice. The exon 2 of mouse Pd-1 gene that encode the IgV 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.

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.

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.