mRNA and protein expression analysis
Strain specific analysis of 4-BB and 4-1BBL gene expression in B-h4-1BB(H/H)/h4-1BBL(H/+) mice by RT-PCR and FACS. (A) Mouse 4-1BBL mRNA was detectable in splenocytes of wild-type (+/+) mice and B-h4-1BB(H/H)/h4-1BBL(H/+) mice. Human 4-1BBL mRNA was only detectable in B-h4-1BB(H/H)/h4-1BBL(H/+) mice but not in wild-type C57BL/6 mice. (B) Mouse 4-1BB was detectable in splenocytes of wild-type (+/+) mice. Human 4-1BB was detectable in B-h4-1BB(H/H)/h4-1BBL(H/+) mice but not in wild-type wild-type (+/+) mice.
Hepatotoxicity evaluation-urelumab(in house)
Day 21, collect serum and detect ALT and AST. The results as followed:
- a) In B-h4-1BB mice, compared with the control group, ALT was significantly increased by 20mg/kg Urelumab treatment, but there was no significant change in AST.
- b) In B-h4-1BB/h4-1BBL mice, compared with the control group, AST was significantly increased by 20mg/kg Urelumab treatment.
- c) Both in B-h4-1BB mice and B-h4-1BB/h4-1BBL mice, there was no significant change in ALT and AST in 1mg/kg dose group.
- d) 20mg/kg Urelumab(in house) have no significant effect in ALT between the two mice. But, AST was significantly increased in B-h4-1BB/h4-1BBL mice.
- In B-h4-1BB Mice (G1-G3), no obvious abnormal changes were observed in the liver when the urelumab dose was 1mg/kg (G2). At a dose of 20 mg/kg (G3) changes were observed in 5/5 of the animals, manifested as perivascular cell infiltration or chronic inflammation in the liver, with mild lesions.
- In B-h4-1BB /4-1BBL mice (G4-G6), 1 mg/kg group(G5) 3/5 of mice show pathological changes(slight 2/5, mild 1/5), but in 20 mg/kg dose (G6), all of the experimental animals could see moderate changes in liver(5/5) . Overall, the degree and incidence of liver lesions in the 20 mg/kg group (G6) were significantly higher than that in the 1 mg/kg group (G5).
- The above results suggested that urelumab at 20 mg/kg was more likely to perivascular cell infiltration or chronic inflammation in the liver than that at 1 mg/kg. B-h4-1BB/h4-1BBL mice were more sensitive to urelumab toxicity effects than B-h4-1BB mice.
- B-h4-1BB/h4-1BBL mice was a good preclinical toxicity evaluation model.