Protein expression analysis

Strain specific CD3E expression analysis in homozygous B-hCD3E mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hCD3E mice, and analyzed by flow cytometry with species-specific anti-CD3E antibody. Mouse CD3E was exclusively detectable in WT mice. Human CD3E were exclusively detectable in homozygous B-hCD3E but not WT mice.

Phenotypic analysis 

Thymus and spleen were isolated and weighed from C57BL/6 and B-hCD3E mice (n=6). There was no significant difference in spleen weight between C57BL/6 and B-hCD3E mice. However, the thymus weight in B-hCD3E mice is slightly lower than that of WT C57BL/6 mice.

Thymocytes were isolated from 4-week-old C57BL/6 and B-hCD3E mice (n=4). A. Representative FACS plots. Single live CD45+ cells were gated for CD3 T cell population and used for further analysis as shown here. B. Results of FACS analysis. The Percentage of CD4, CD8 T cells in B-hCD3E mice was similar to that of C57BL/6 mice, indicating that T cell subpopulation in thymus of B-hCD3E mice was not significantly affected by Cd3e gene humanization.

Analysis of spleen lymphocyte subpopulation in B-hCD3E mice

Analysis of lymphocyte subpopulations by FACS.

The lymphocyte were isolated from C57BL/6 and B-hCD3E mice (n=4). Single live cells were gated for CD45 population and used for further analysis as indicated here. Percent of T cell , B cell and Treg cell in homozygous B-hCD3E mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD3E in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in spleen.

Analysis of B and T cell subpopulation by FACS – Spleen, PBMC, Lymph node

Lymphocytes were isolated from spleen, peripheral blood and lymph node of C57BL/6 and B-hCD3E mice (n=4). Flow cytometry analysis was performed to assess Lymphocytes subpopulations. Single live cells were gated for CD45 population and used for further analysis as shown here. The Percentage of T, B, cells in B-hCD3E mice was similar to that of C57BL/6 mice.

Analysis of T cell activation stimulated with anti-CD3 antibody in vitro

T cells (2.5×10⁶) were isolated from splenocytes of C57BL/6 and B-hCD3E mice (n=4), and were incubated in the presence of anti-CD3E antibody (2ug/ml) and anti-mCD28 antibody (5ug/ml) for 48h. T cell proliferation was tested by flow cytometry. T cell activation in B-hCD3E 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 (2.5×10⁶) were isolated from the splenocytes of C57BL/6 and B-hCD3E mice (n=4), and incubated in the presence of anti-CD3E antibody (2ug/ml) and anti-mCD28 (5ug/ml) for 24h, 48h and 72h. T cell proliferation was measured by flow cytometry. T cell activation in B-hCD3E mice was significantly up-regulated by anti-hCD3 antibody, similar to the activation level shown in the anti-mCD3 antibody-treated C57BL/6 mice, indicating that Cd3e humanization in B-hCD3E mice does not affect T cell activation in spleen.

Analysis of T cell activation stimulated with anti-CD3 antibody in vitro

T cells (2.5×10⁶) were isolated from the splenocytes of C57BL/6 and B-hCD3E mice (n=4), and 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 production were tested using ELISA method. Concentration of IFN-γ and IL-2 in B-hCD3E mice was similar to that of C57BL/6 mice, indicating that Cd3e humanization in B-hCD3E mice does not affect T cell activation in spleen.

Analysis of T cell activation stimulated with anti-CD3 antibody ex vivo

C57BL/6 and B-hCD3E mice were injected intraperitoneally with anti-CD3E antibody (10ug/mouse). After 24h, T cells were isolated from splenocytes of C57BL/6 and B-hCD3E mice (n=4). T cell proliferation was measured by flow cytometry. T cell activation in B-hCD3E mice was significantly up-regulated by anti-hCD3E antibody, similar to the activation level in the anti-mCD3E antibody-treated C57BL/6 mice, demonstrating that introduction of hCD3E in place of its mouse counterpart does not affect T cell activation in spleen.

C57BL/6 and B-hCD3E mice were injected intraperitoneally with anti-CD3E antibody (10ug/mouse). After 48h, T cells were isolated from splenocytes of C57BL/6 and B-hCD3E mice (n=4). T cell proliferation was measured by flow cytometry. T cell activation in B-hCD3E mice was significantly down-regulated, similar to the activation level in the anti-mCD3 antibody-treated C57BL/6 mice, demonstrating that introduction of hCD3E in place of its mouse counterpart does not affect T cell activation in spleen.

B-hCD3E mouse spleen cells were mixed with MC38-hPD-L1 and various concentrations of CD3-PD-L1 bispecific antibodies were added. The killing activity was detected after 48 hours. When effector cells : target  cells (E:T) =10:1，the EC50 of CD3-PD-L1 bispecific antibodies activity was 452.4 ng/mL; When E:T=20:1，the EC50 of CD3-PD-L1 bispecific antibodies activity was 144.2 ng/mL

Blood routine test in B-hCD3E mice

CD3 Abs efficacy evaluation 

Murine colon cancer MC38 cells were subcutaneously implanted into C57BL/6 (A) and B-hCD3E (B) mice. Mice were grouped when the tumor size was approximately 150±50mm³ (n=5).In B-hCD3E mice, mPD-1 antibody significantly inhibited tumor growth, indicating their T cells function normally. However, in B-hCD3E mice, tumor growth was faster after anti-hCD3E antibody treatment, which may be caused by activation induced cell death (AICD). As a result, the B-hCD3E mouse model is a powerful tool for in vivo CD3 antibody pharmacological efficacy studies.

the proportion of T cells was significantly decreased due to the activation induced cell death (AICD) effect caused by CD3E antibody treatment. However, the proportion of T cells has no significant change in the anti-mPD-1 antibody group. (A) Compared with hlgG Ab , there is no significant difference in the percentage of CD19+ cells in total CD45+ cells after hCD3 Ab or mCD3 Ab treatment. (B) Compared with hlgG Ab, the percentage of TCR-β positive cells was significantly decreased after treatment with hCD3 Ab in the humanized mice.

(A) Compared with hlgG Ab, the percentage of CD4+ T cells in the blood decreased significantly after hCD3E Ab treatment. (B) Compared with hlgG Ab, the percentage of CD8+ T cells in the blood was significantly reduced after hCD3E Ab treatment. 

Murine colon cancer MC38 cells were subcutaneously implanted into B-hCD3E mice. Mice were divided into control and treatment groups(n=5) when tumon size was approximately 150±50 mm³. High doses of hCD3E antibodies resulted in faster tumor growth due to activation induced cell death (AICD), confirming that the B-hCD3E mouse model is a powerful tool for in vivo anti-hCD3 antibody pharmacological efficacy study. (A) Tumor average volume ±SEM, (B) Mice average weight±SEM.

Antitumor activity of Antibody X in B-hCD3E mice. (A) High-dose Antibody X inhibited MC38 tumor growth in B-hCD3E mice(n=5). Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hCD3E mice. Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with Antibody X with doses and schedules indicated in panel ; (B) Body weight changes during treatment. As shown in panel A, high-dose Antibody X were efficacious in controlling tumor growth in B-hCD3E mice, demonstrating that the B-hCD3E mice provide a powerful preclinical model for in vivo evaluation of Antibody X . Values are expressed as mean ±SEM

The ratio of B and T cells in the blood of mice was detected by flow cytometry. Lymphocytes were isolated from peripheral blood at the experimental end point. In the treatment group, the ratio of B and T cells did not change significantly. 

Antitumor activity of CD3E BsAb in B-hCD3E mice. (A) CD3E BsAb inhibited tumor growth in B-hCD3E mice. (B) Body weight changes during treatment. As shown in panel A, CD3E BsAb with different doses were efficacious in controlling tumor growth in B-hCD3E mice, demonstrating that the B-hCD3E mice provide a powerful preclinical model for in vivo evaluation of CD3E BsAb. Values are expressed as mean ± SEM.

MC38-hEpCAM cells were implanted subcutaneously into B-hCD3E mice. The mice were divided into control and treatment groups when the tumor size was about 100±20 mm³. The results show that the anti-hCD3E/hEpCAM bispecific antibody has a moderate degree of antitumor activity compared to the control group. The data also show that anti-mouse CD4 (a CD4-depleting antibody) enhances the antitumor activity of the bispecific anti-hCD3E/hEpCAM antibody.