Basic Information
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Gene targeting strategy
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Gene targeting strategy for B-hTNFA/hTNFR2/hTNFR1 mice. The targeted mouse Tnf whole genomic sequence including 5’UTR, 3’UTR and coding region was replaced by human TNF whole genomic sequence in B-hTNFA/hTNFR2/hTNFR1 mice. The exons 2~6 of mouse Tnfr2 gene that encode the extracellular domain was replaced by human TNFR2 exons 2~6 in B-hTNFA/hTNFR2/hTNFR1 mice. The exons 2~6 of mouse Tnfr1 gene that encode the extracellular domain was replaced by human TNFR1 exons 2~6 in B-hTNFA/hTNFR2/hTNFR1 mice.
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Protein expression analysis
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Species-specific TNFA expression analysis in wild-type and humanized B-hTNFA/hTNFR2/hTNFR1 mice. Serum was collected from wild-type C57BL/6 (+/+) and homozygous B-hTNFA/hTNFR2/hTNFR1 (H/H;H/H;H/H) mice stimulated with LPS in vivo, and analyzed by ELISA using species-specific TNFA ELISA kits. Mouse TNFA was detected in wild-type mice, while human TNFA was detected in B-hTNFA/hTNFR2/hTNFR1 mice. Values are expressed as mean ± SEM. ND: not detectable.
Species-specific TNFR1 expression analysis in wild-type and humanized B-hTNFA/hTNFR2/hTNFR1 mice. Splenocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hTNFA/hTNFR2/hTNFR1 (H/H;H/H;H/H) mice, and analyzed by flow cytometry using species-specific anti-TNFR1 antibodies. Mouse TNFR1 was detected in wild-type mice, while human TNFR1 was detected in B-hTNFA/hTNFR2/hTNFR1 mice.
Species-specific TNFR2 expression analysis in wild-type and humanized B-hTNFA/hTNFR2/hTNFR1 mice. Splenocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hTNFA/hTNFR2/hTNFR1 (H/H;H/H;H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry using species-specific anti-TNFR2 antibodies. Mouse TNFR2 was detected in wild-type mice, while human TNFR2 was detected in B-hTNFA/hTNFR2/hTNFR1 mice.
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In vivo efficacy of anti-human TNFR2 antibodies
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Antitumor activity of anti-human TNFR2 antibodies in B-hTNFA/hTNFR2/hTNFR1 mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hTNFA/hTNFR2/hTNFR1 mice (female, 8 week-old, n=6). Mice were grouped according to body weight differences, at which time they were treated with anti-TNFR2 Ab1 provided by the client with doses and schedules indicated in panel A. (A) Anti-human TNFR2 antibodies inhibited MC38 tumor growth in B-hTNFA/hTNFR2/hTNFR1 mice. (B) Body weight changes during treatment. As shown in panel A, anti-human TNFR2 antibodies were efficacious in controlling tumor growth in B-hTNFA/hTNFR2/hTNFR1 mice in a dose-dependent manner, demonstrating that the B-hTNFA/hTNFR2/hTNFR1 mice provide a powerful preclinical model for in vivo evaluation of anti-human TNFR2 antibody. Values are expressed as mean ± SEM.
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Establishment and treatment of collagen induced arthritis in B-hTNFA/hTNFR2/hTNFR1 mice
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Establishment of a CIA mouse model
Experimental animals: B-hTNFA/hTNFR2/hTNFR1 mice, 10-weeks-old, 6 male and 10 female; C57BL/6, 10-weeks-old
Modeling reagent: CII emulsion
Modeling method: Sensitization, day 0; challenge, day 21
The arthritis model was induced in B-hTNFA/hTNFR2/hTNFR1 mice and C57BL/6 mice using collagen (CII). (A) mouse body weight change; (B) clinical score. The results showed that the clinical score of B-hTNFA/hTNFR2/hTNFR1 mice was significantly increased, suggesting that the arthritis model was successfully established.
Pathological analysis after the establishment of arthritis in B-hTNFA/hTNFR2/hTNFR1 mice and C57BL/6 mice. (A) Pathological score; (B) H&E staining of pathological sections. In the model group, subcutaneous mixed inflammatory cell infiltration, periarticular stenosis, articular cartilage and bone tissue destruction and other arthritic lesions were observed in all or part of the limb joints, suggesting that the arthritis model was successfully established.
In vivo efficacy of an anti-human TNFA antibody in mice with CIA
Efficacy of anti-human TNFA antibody in B-hTNFA/hTNFR2/hTNFR1 mice with collagen induced arthritis (CIA) model. Arthritis was induced by the subcutaneous injection of CII emulsion into B-hTNFA/hTNFR2/hTNFR1 mice on Day 0 and Day 21 (female, n=9-10 in each group). The development of arthritis was monitored and the arthritis score was evaluated every day. The mice were divided into groups at the moment of inflammation onset (defined as day 0, Clinical score ＞1 or the continuous score =1). The treatment group was intraperitoneally injected with different doses of anti-human TNFA antibody adalimumab (in house). Body weight change(A) and clinical score (B) were evaluated daily during treatment. Mice were euthanized at 2 days after the last treatment, and the paws were removed. Joint pathology was evaluated on decalcified H&E-stained sections. There was no significant change in body weight, while total clinical score increased in the groups except control during treatment. It indicated that the arthritis model was successfully established. Dose-dependent reduction in clinical score in the adalimumab (in house) treatment groups. The results indicated the B-hTNFA/hTNFR2/hTNFR1 mice provide a powerful preclinical CIA mouse model for in vivo evaluation of anti-human TNFA antibody.
