Gene targeting strategy for B-hCD3E/h4-1BB mice.
The exons 2-6 of mouse Cd3e gene that encode the extracellular domain were replaced by human CD3E exons 2-7 in B-hCD3E/h4-1BB mice. The exons 2-7 of mouse Tnfrsf9 gene that encode the extracellular domain were replaced by human TNFRSF9 exons 3-8 in B-hCD3E/h4-1BB mice.

Strain specific CD3E expression analysis in homozygous B-hCD3E/h4-1BB mice by flow cytometry.

Splenocytes were collected from wild type (WT) mice (+/+) and homozygous B-hCD3E/h4-1BB mice (H/H;H/H), and analyzed by flow cytometry with species-specific anti-CD3E antibody. Mouse CD3E was detectable in WT mice (+/+). Human CD3E was exclusively detectable in homozygous B-hCD3E/h4-1BB mice (H/H;H/H) but not in WT mice (+/+) .

Strain specific 4-1BB expression analysis in homozygous B-hCD3E/h4-1BB mice by flow cytometry.

Splenocytes were collected from wild type (WT) mice (+/+) and homozygous B-hCD3E/h4-1BB mice (H/H;H/H) stimulated with anti-CD3ε antibody in vivo, and analyzed by flow cytometry with species-specific anti-CD3E antibody. Mouse 4-1BB was detectable in WT mice (+/+). Human 4-1BB was exclusively detectable in homozygous B-hCD3E/h4-1BB mice (H/H;H/H) but not in WT mice (+/+) .

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6JNifdc mice and homozygous B-hCD3E/h4-1BB mice (female, n=3, 8-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hCD3E/h4-1BB mice were similar to those in C57BL/6JNifdc. The frequency of leukocyte subpopulations in blood and lymph nodes of B-hCD3E/h4-1BB mice were also comparable to wild-type C57BL/6JNifdc (Data not shown).

Antitumor activity of Ab1 in B-hCD3E/h4-1BB mice. B-hCDCP1 MC38 tumor cells (1×10⁶) were subcutaneously implanted into female homozygous B-hCD3E/h4-1BB mice (6-week-old, n=5). Mice were grouped when tumor volume reached approximately 80 mm³ and treated with antibody Ab1 provided by the client. (A) Tumor volume changes during treatment. (B) Body weight changes during treatment. Antibody Ab1 effectively inhibited tumor growth in B-hCD3E/h4-1BB mice, demonstrating that this model is a powerful tool for the in vivo efficacy evaluation of antibodies. Values are expressed as mean ± SEM.