Targeting strategy
Gene targeting strategy for B-hTREM2 mice. The exons 1~5 and 3’UTR of mouse Trem2 gene that encode the full-length protein were replaced by human TREM2 exons 1~5 and 3’UTR in B-hTREM2 mice.
mRNA expression analysis
Strain specific analysis of TREM2 gene expression in wild-type mice and B-hTREM2 mice by RT-PCR. Mouse Trem2 mRNA was detectable in wild-type mice (+/+). Human TREM2 mRNA was detectable only in homozygous B-hTREM2 mice(H/H) but not in wild-type mice.
Protein expression analysis
Strain specific TREM2 expression analysis in homozygous B-hTREM2 mice by flow cytometry.Microglia were collected from wild-type mice (+/+) and homozygous B-hTREM2 mice(H/H), and analyzed by flow cytometry with anti-TREM2 antibody. TREM2 was detectable in microglia of wild-type mice and homozygous B-hTREM2 mice, as the antibody is crossly reactive with TREM2 in human and mice.
Strain specific TREM2 expression analysis in homozygous B-hTREM2 mice by flow cytometry.MC38 cells were inoculated into wild-type C57BL/6(+/+) and homozygous B-hTREM2 mice(H/H). Tumor tissues were harvested when tumor volume reached approximately 1000 mm3, and analyzed by flow cytometry with anti-TREM2 antibody. TREM2 was detectable in tumor macrophage of wild-type mice and homozygous B-hTREM2 mice, as the antibody is crossly reactive with TREM2 in human and mice.
Strain specific TREM2 expression analysis in heterozygous B-hTREM2 mice by western blot.Brain were collected from wild-type mice (+/+) and heterozygous B-hTREM2 mice (H/+), and analyzed by western blot with species-specific anti-hTREM2 antibody. Mouse TREM2 was not detectable in wild-type mice. Human TREM2 was exclusively detectable in heterozygous B-hTREM2 mice.
In vivo efficacy of anti-human TREM2 antibody
Antitumor activity of anti-human TREM2 antibody in B-hTREM2 mice. (A) Anti-human TREM2 antibody inhibited MC38 tumor growth in B-hTREM2 mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hTREM2 mice (male, 6-7 week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm3, and treated with anti-hTREM2 antibody at doses and schedules in panel A. (B) Body weight changes during treatment. As shown in panel A, anti-human TREM2 antibody (in house) were efficacious in controlling tumor growth in B-hTREM2 mice, demonstrating they provide a powerful preclinical model for in vivo evaluation of anti-human TREM2 antibody. Values are expressed as mean ± SEM.
