mRNA expression analysis
Strain specific analysis of 4-1BB (TNFRSF9) and HER2 (ERBB2) gene expression in wild-type mice and B-h4-1BB/hHER2 mice by RT-PCR. Mouse Tnfrsf9 mRNA was detectable only in spleen of wild-type mice (+/+). Human TNFRSF9 mRNA was detectable only in spleen of homozygous B-h4-1BB/hHER2 mice (H/H;H/H), but not in wild-type mice. Mouse Erbb2 mRNA was detectable only in liver of wild-type mice (+/+). Human ERBB2 mRNA was detectable only in liver of homozygous B-h4-1BB/hHER2 mice (H/H;H/H), but not in wild-type mice.
Protein expression analysis
Strain specific 4-1BB expression analysis in homozygous B-h4-1BB/hHER2 mice by flow cytometry.Splenocytes were collected from wild type (WT) mice (+/+) and homozygous B-h4-1BB/hHER2 mice (H/H;H/H) stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-4-1BB antibody. Mouse 4-1BB was detectable in WT mice (+/+). Human 4-1BB was exclusively detectable in homozygous B-h4-1BB/hHER2 mice (H/H;H/H) but not in WT mice (+/+).
IHC analysis of HER2 expression
Immunohistochemical (IHC) analysis of HER2 expression in homozygous B-h4-1BB/hHER2 mice.The liver and mammary gland were collected from WT mice and homozygous B-h4-1BB/hHER2 mice and analyzed by IHC with anti-HER2 antibody. HER2 was detectable in WT mice and homozygous B-h4-1BB/hHER2 mice due to the cross-reactivity of the antibody. The arrow indicates tissue cells with positive HER2 staining (brown).
In vivo efficacy of anti-human HER2 ADC Trastuzumab analog-MMAE (in-house)
Antitumor activity of anti-human HER2 ADC (Trastuzumab analog-MMAE, in-house) in B-h4-1BB/hHER2 mice. (A) Anti-human HER2 ADC inhibited B-hHER2 MC38 plus tumor growth in B-h4-1BB/hHER2 mice. Murine colon cancer B-hHER2 MC38 plus cells were subcutaneously implanted into homozygous B-h4-1BB/hHER2 mice (female, 8-week-old, n=6). Mice were grouped when tumor volume reached approximately 100-150 mm3, at which time they were intravenously injected with anti-human HER2 ADC Trastuzumab analog-MMAE (in-house) indicated in panel. (B) Body weight changes during treatment. As shown in panel A, 10mg/kg anti-human HER2 ADC was efficacious in controlling tumor growth in B-h4-1BB/hHER2 mice, demonstrating that B-hHER2 MC38 plus provide a powerful preclinical model for in vivo evalsuation of anti-human HER2 ADC. Values are expressed as mean ± SEM.
* When publishing results obtained using this animal model, please acknowledge the source as follows: The animal model [B-h4-1BB/hHER2 mice] (Cat# 111869) was purchased from Biocytogen.
