• Background: CD3 consists of four protein chains (CD3E, CD3D, CD3G and CD3Z), which are important biological markers on the T cell membrane. CD3 can form a TCR/CD3 complex with the T cell receptor, participating in the regulation of T cell antigen recognition, signal transduction and T cell development. CD19 is a biomarker for normal and neoplastic B cells, as well as follicular dendritic cells. 4-1BB (TNFRSF9/CD137) is a key member of the immune co-stimulatory pathway. It is primarily induced and expressed on activated T cells, and its activation potently enhances the anti-tumor immune functions of CD8+ T cells and NK cells. CD19 is critically involved in establishing intrinsic B cell signaling thresholds through modulating both B cell receptor-dependent and independent signaling.
• Targeting strategy: B-hCD3EDG/h4-1BB/hCD19 mice were obtained by mating B-hCD3EDG/h4-1BB mice(112646) and B-hCD3EDG/hCD19 mice(112902). In B-hCD3EDG/h4-1BB/hCD19 mice, chimeric human CD3EDG was expressed, while mouse Cd3edg were knocked out. The exons 2-7 of mouse Tnfrsf9 gene that encode the extracellular domain were replaced by human TNFRSF9 exons 2-7. The chimeric CD19 coding sequence containing the extracellular region of human CD19 gene is inserted into mouse Cd19 gene.
• Protein expression: Human CD3E and CD19 were exclusively detectable respectively in T cells and B cells from spleen and blood of homozygous B-hCD3EDG/h4-1BB/hCD19 mice, but not in wild-type (WT) mice. Human 4-1BB was exclusively detectable in T cells of homozygous B-hCD3EDG/h4-1BB/hCD19 mice, but not in wild-type C57BL/6 mice. Mouse 4-1BB was not detectable in homozygous B-hCD3EDG/h4-1BB/hCD19 mice.
• Application: This product is used for the pharmacological and safety evaluation of antibodies in oncology.
• Note: These is a risk in B cells depletion in vivo. Please see the data from page 11 to 23.

CD3E expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hCD3EDG/h4-1BB/hCD19 mice by flow cytometry. Spleen and blood cells were collected from wild-type C57BL/6 mice (+/+) (Female, 10-week-old, n=2) and homozygous B-hCD3EDG/h4-1BB/hCD19 mice (H/H) (Female, 10-week-old, n=2). Protein expression was analyzed with anti-human CD3E antibody (BD Horizon™, 562426) and anti-mouse CD3E antibody (Biolegend, 100312) by flow cytometry. Human CD3E was exclusively detectable in T cells of homozygous B-hCD3EDG/h4-1BB/hCD19 mice, but not in wild-type C57BL/6 mice.

CD19 expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hCD3EDG/h4-1BB/hCD19 mice by flow cytometry. Spleen and blood cells were collected from wild-type C57BL/6 mice (+/+) (Female, 10-week-old, n=2) and homozygous B-hCD3EDG/h4-1BB/hCD19 mice (H/H) (Female, 10-week-old, n=2). Protein expression was analyzed with anti-human CD19 antibody (Biolegend, 302234) and anti-mouse CD19 antibody (Biolegend, 115507) by flow cytometry. Human CD19 was exclusively detectable in B cells of homozygous B-hCD3EDG/h4-1BB/hCD19 mice, but not in wild-type C57BL/6 mice.

4-1BB expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hCD3EDG/h4-1BB/hCD19 mice by flow cytometry. Spleen cells were collected from wild-type C57BL/6 mice (+/+) (Female, 10-week-old, n=2) and homozygous B-hCD3EDG/h4-1BB/hCD19 mice (H/H) (Female, 10-week-old, n=2), stimulated with anti-m/hCD3ε in vivo (7.5 μg/mice for 24 hours, i.p.; BioXcell, BE0001-1/BE0001-2). Protein expression was analyzed with anti-human 4-1BB antibody (Biolegend, 309804) and anti-mouse 4-1BB antibody (Biolegend, 106110) by flow cytometry. Human 4-1BB was exclusively detectable in T cells of homozygous B-hCD3EDG/h4-1BB/hCD19 mice, but not in wild-type C57BL/6 mice. Mouse 4-1BB was not detectable in homozygous B-hCD3EDG/h4-1BB/hCD19 mice.

4-1BB expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hCD3EDG/h4-1BB/hCD19 mice by flow cytometry. Spleen cells were collected from wild-type C57BL/6 mice (+/+) (Female, 10-week-old, n=2) and homozygous B-hCD3EDG/h4-1BB/hCD19 mice (H/H) (Female, 10-week-old, n=2), stimulated with anti-m/hCD3ε in vivo (7.5 μg/mice for 24 hours, i.p.; BioXcell, BE0001-1/BE0001-2). Protein expression was analyzed with anti-human 4-1BB antibody (Biolegend, 309804) and anti-mouse 4-1BB antibody (Biolegend, 106110) by flow cytometry. Human 4-1BB was exclusively detectable in CD4+ T cells of homozygous B-hCD3EDG/h4-1BB/hCD19 mice, but not in wild-type C57BL/6 mice. Mouse 4-1BB was not detectable in homozygous B-hCD3EDG/h4-1BB/hCD19 mice.

4-1BB expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hCD3EDG/h4-1BB/hCD19 mice by flow cytometry. Spleen cells were collected from wild-type C57BL/6 mice (+/+) (Female, 10-week-old, n=2) and homozygous B-hCD3EDG/h4-1BB/hCD19 mice (H/H) (Female, 10-week-old, n=2), stimulated with anti-m/hCD3ε in vivo (7.5 μg/mice for 24 hours, i.p.; BioXcell, BE0001-1/BE0001-2). Protein expression was analyzed with anti-human 4-1BB antibody (Biolegend, 309804) and anti-mouse 4-1BB antibody (Biolegend, 106110) by flow cytometry. Human 4-1BB was exclusively detectable in CD8+ T cells of homozygous B-hCD3EDG/h4-1BB/hCD19 mice, but not in wild-type C57BL/6 mice. Mouse 4-1BB was not detectable in homozygous B-hCD3EDG/h4-1BB/hCD19 mice.

4-1BB expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hCD3EDG/h4-1BB/hCD19 mice by flow cytometry. Spleen cells were collected from wild-type C57BL/6 mice (+/+) (Female, 10-week-old, n=2) and homozygous B-hCD3EDG/h4-1BB/hCD19 mice (H/H) (Female, 10-week-old, n=2), stimulated with anti-m/hCD3ε in vivo (7.5 μg/mice for 24 hours, i.p.; BioXcell, BE0001-1/BE0001-2). Protein expression was analyzed with anti-human 4-1BB antibody (Biolegend, 309804) and anti-mouse 4-1BB antibody (Biolegend, 106110) by flow cytometry. Human 4-1BB was exclusively detectable in Tregs of homozygous B-hCD3EDG/h4-1BB/hCD19 mice, but not in wild-type C57BL/6 mice. Mouse 4-1BB was not detectable in homozygous B-hCD3EDG/h4-1BB/hCD19 mice.