Strain specific TNFA expression analysis in wild type (WT) mice and B-hTNFA/hTNFR2/hTNFR1 mice by ELISA. Serum were collected from WT mice (+/+) and homozygous B-hTNFA/hTNFR2/hTNFR1 mice (H/H;H/H;H/H) stimulated with LPS in vivo, and analyzed by ELISA with species-specific TNFA ELISA kit. Mouse TNFA was detectable in WT mice. Human TNFA was exclusively detectable in homozygous B-hTNFA/hTNFR2/hTNFR1 mice (H/H;H/H;H/H) but not WT mice (+/+) . Values are expressed as mean ± SEM. ND: not detectable.

Strain specific TNFR2 expression analysis in homozygous B-hTNFA/hTNFR2/hTNFR1 mice by flow cytometry. Splenocytes were collected from wild type (WT) mice (+/+) and homozygous B-hTNFA/hTNFR2/hTNFR1 mice (H/H;H/H;H/H) stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-TNFR2 antibody. Mouse TNFR2 was detectable in WT mice (+/+). Human TNFR2 was exclusively detectable in homozygous B-hTNFA/hTNFR2/hTNFR1 mice (H/H;H/H;H/H) but not in WT mice (+/+).

Frequency of leukocyte subpopulations in spleen

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6N mice and homozygous B-hTNFA/hTNFR2/hTNFR1 mice (female, 7-week-old, n=3). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells and Tregs in B-hTNFA/hTNFR2/hTNFR1 mice were similar to those in C57BL/6N mice, demonstrating that humanization of TNFA, TNFR2 and TNFR1 do not change the frequency or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in blood

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood cells were isolated from wild-type C57BL/6N mice and homozygous B-hTNFA/hTNFR2/hTNFR1 mice (female, 7-week-old, n=3). A. Flow cytometry analysis of the blood cells was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells and Tregs in B-hTNFA/hTNFR2/hTNFR1 mice were similar to those in C57BL/6N mice, demonstrating that humanization of TNFA, TNFR2 and TNFR1 do not change the frequency or distribution of these cell types in blood. Values are expressed as mean ± SEM.

Hematology analysis

Biochemistry analysis

CIA Mouse Model Introduction

Experimental Animals：B-hTNFA/hTNFR2/hTNFR1 mice, 10 weeks old, 6 male and 10 female; 
                                         C57BL/6 mice, 10 weeks old.
Modeling reagent：CII emulsion.
Modeling method：Sensitization, 0 days; Challenge, 21 days.

Establishment of CIA Mouse Model

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 anti-human TNFA antibody in CIA model

In vivo efficacy of anti-human TNFA antibody in CIA model