Gene targeting strategy for B-hPD-1/hCTLA4 mice. The exon 2 of mouse Pd-1 gene that encodes the extracellular domain was replaced by human PD-1 exon 2 in B-hPD-1/hCTLA4 mice. The exon 2 of mouse Ctla4 gene that encodes the extracellular domain was replaced by human CTLA4 exon 2 in B-hPD-1/hCTLA4 mice.
mRNA expression analysis
Strain specific analysis of PD-1 and CTLA4 gene expression in WT and hPD-1/hCTLA4 mice by RT-PCR. Mouse Pd-1 and Ctla4 mRNA were detectable only in splenocytes of wild-type (+/+) mice. Human PD-1 and CTLA4 mRNA were detectable only in H/H, but not in +/+ mice.
Protein Expression Analysis
Strain specific PD-1 and CTLA4 expression analysis in homozygous B-hPD-1/hCTLA4 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-1/hCTLA4 (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-PD-1 and anti-CTLA4 antibody. Mouse PD-1 and CTLA4 were detectable in WT mice. Human PD-1 and CTLA4 were exclusively detectable in homozygous B-hPD-1/hCTLA4 but not WT mice.
Combination therapy of anti-human PD-1 antibody and anti-human CTLA4 antibody
Antitumor activity of anti-human PD-1 antibody combined with anti-human CTLA4 antibody in B-hPD-1/hCTLA4 mice. (A) Anti-human PD-1 antibody combined with anti-human CTLA4 antibody inhibited MC38 tumor growth in B-hPD-1/hCTLA4 mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hPD-1/hCTLA4 mice (female, 6-7 week-old, n=5). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with pembrolizumab and ipilimumab with doses and schedules indicated in panel (B) Body weight changes during treatment. As shown in panel A, combination of pembrolizumab and ipilimumab shows more inhibitory effects than individual groups, demonstrating that the B-hPD-1/hCTLA4 mice provide a powerful preclinical model for in vivo evaluating combination therapy efficacy of hPD-1 antibodies and hCTLA4 antibodies. Values are expressed as mean ± SEM.
- 1. Cell Research (2018) 0:1–15; doi: 10.1038/s41422-018-0012-z
- 2. Blood.2005;106:3127-3133
- 3. Scientific Reports volume7, 42913 (2017) doi: 10.1038/srep42913
- Nat Commun. 2017 Feb 6;8:14369. doi: 10.1038/ncomms14369.
- EMBO J. 1992 Nov;11(11):3887-95.
- J Exp Med. 2000 Oct 2;192(7):1027-34.
- Science. 2001 Jan 12;291(5502):319-22.