Analysis of thymus leukocyte subpopulations by FACS

Thymocytes were isolated from female C57BL/6 and B-hCD3EDG mice (n=3, 7 week-old). Flow cytometry analysis of the thymocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for CD3 T cell population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8, CD4, and Treg cells in homozygous B-hCD3EDG mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD3EDG in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in thymus. Values are expressed as mean ± SEM.

Analysis of spleen leukocytes cell subpopulations in homozygous B-hCD3EDG mice

Analysis of T cell activation stimulated with anti-CD3E antibody in vitro

T cells (2×10⁵) were isolated from splenocytes of C57BL/6 and B-hCD3EDG mice (n=3,16 week-old), and were incubated in the presence of anti-CD3E antibody (2ug/ml) and anti-mCD28 antibody (5ug/ml) for 24h. T cell proliferation was tested by flow cytometry. T cell activation in B-hCD3EDG mice was significantly up-regulated by anti-hCD3E antibody, similar to the activation level shown in C57BL/6 mice treated with anti-mCD3E antibody.

Analysis of T cell activation stimulated with anti-CD3E antibody in vitro

T cells (2×10⁵) were isolated from splenocytes of C57BL/6 and B-hCD3EDG mice (n=3,16 week-old), and were incubated in the presence of anti-CD3E antibody (2ug/ml) and anti-mCD28 antibody (5ug/ml) for 72h. T cell proliferation was tested by flow cytometry. T cell activation in B-hCD3EDG mice was significantly up-regulated by anti-hCD3E antibody, similar to the activation level shown in C57BL/6 mice treated with anti-mCD3E antibody.

T cells (2×10⁵) were isolated from the splenocytes of C57BL/6 and B-hCD3EDG mice (n=3, 16 week-old), and incubated in the presence of anti-CD3E antibody (2ug/ml) and anti-mCD28 antibody (5ug/ml) for 24h, 48h and 72h. T cell proliferations were measured by flow cytometry. As a result, the T cell activation in B-hCD3EDG mice was specifically up-regulated by anti-hCD3E antibody, similar to the level of activation in the anti-mCD3E antibody-treated C57BL/6 mice. 

Analysis of T cell activation stimulated with anti-CD3E antibody in vitro

T cells (2×10⁵) were isolated from the splenocytes of C57BL/6 and B-hCD3EDG mice (n=3, 16 week-old), incubated in the presence of anti-CD3E antibody (2ug/ml) and anti-mCD28 antibody (5ug/ml) for 24h, 48h and 72h. IFN-γ and IL-2 productions were then tested using ELISA method. As a result, there is no significant difference regarding the production of IFN-γ and IL-2 between B-hCD3EDG mice and C57BL/6 mice. ND: not detectable. 

Cytotoxicity evaluation of CD3/BCMA bispecific antibody in vitro

B-hCD3EDG mice (n=5, 6 week-old) were immunized three times with OVA, 2 weeks apart. Blood samples were collected a week after immunization. (A) Quantification of serum subtypes of mice before immunization. (B) Serum titer test of mice after the second and third immunizations. The levels of OVA-specific antibodies titers of B-hCD3EDG mice before immunization were similar to those in C57BL/6 mice, and the specific antibody titers in the serum of each mouse were significantly increased after the third immunizations, demonstrating that introduction of hCD3EDG instead of its mouse counterpart did not affect the humoral immune response of mice. Values are expressed as mean ± SEM.

Antitumor activity of anti-hCD3ε antibody and anti-mPD-1 antibody in B-hCD3EDG mice. Murine colon cancer MC38 cells were subcutaneously implanted into B-hCD3EDG mice (female, 8 week-old, n=5). Mice were grouped when the tumor size was approximately 100±50mm³ , at which time they were treated with different antibodies with doses and schedules indicated in panel. (A) Tumor volume changes during treatment. (B) Body weight changes during treatment. As shown in panel A, anti-mPD-1 antibody significantly inhibited tumor growth in B-hCD3EDG mice, indicating their T cells function normally. However, in B-hCD3EDG mice, tumor growth was faster after anti-hCD3ε antibody treatment, which may be caused by activation induced cell death (AICD). As a result, the B-hCD3EDG mouse model is a powerful tool for in vivo CD3 antibody pharmacological efficacy studies. Values are expressed as mean ± SEM.