Basic Information

Strain Name
C57BL/6-CD3etm2(CD3e)Bcgen/Bcgen
Stock Number
110008
Common Name
B-hCD3E mice
Source/Investigator
Bcgen (Beijing Biocytogen Co., Ltd)
Related Genes
CD3e (CD3e molecule)
Species
C57BL/6
Appearance
Black
Genotypes
Homozygous
NCBI Gene ID

Targeting Strategy

Gene targeting strategy for B-hCD3E 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 mice.

mRNA expression analysis

Strain specific analysis of CD3E gene expression in WT and hCD3E mice by RT-PCR. Mouse Cd3e mRNA was detectable only in splenocytes of wild-type. Human CD3E mRNA was detectable only in homozygous B-hCD3E mice.

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.

Weight of thymus and spleens and the total cell number of thymocytes and splenocytes in B-hCD3E mice

(A,B) 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. (C) The number of splenocytes in C57BL/6 and B-hCD3EDG mice was similar. (C) The number of thymocytes in B-hCD3E mice was significantly lower than that in C57BL/6.

Analysis of spleen leukocytes subpopulation in B-hCD3E mice

Analysis of leukocytes subpopulation in B-hCD3E mice -spleen. Splenocytes were isolated from female C57BL/6 and B-hCD3E mice (n=3, 6 week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for CD45 population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T, B, NK, Monocyte, DC and macrophage cells 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 spleen T cell subpopulations in B-hCD3E mice

Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hCD3E mice (n=3, 6 week-old). Flow cytometry analysis of the splenocytes 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-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 T cell sub types in spleen. Values are expressed as mean ± SEM.

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 thymus leukocytes subpopulation in B-hCD3E mice

Analysis of thymus leukocyte subpopulations by FACS. Thymocytes were isolated from female C57BL/6 and B-hCD3E mice (n=6, 8 week-old). Flow cytometry analysis of the thymocytes was performed to assess leukocyte subpopulations. Representative FACS plots. Single live cells were gated for CD45 population and used for further analysis as indicated here. Percent of CD4/CD8 cells, B cells 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 thymus. DN: double negative. DP: double positive.

Analysis of thymus T cell subpopulations by FACS. Thymocytes were isolated from female C57BL/6 and B-hCD3E mice (n=6, 6 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-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 T cell sub types in thymus. Values are expressed as mean ± SEM. DN: double negative. DP: double positive.

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 indicated here. The percent of T, B, cells 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, peripheral blood and lymph node.

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-CD3 antibody (C57BL/6, anti-mCD3; B-hCD3E, anti-hCD3) and anti-mCD28 for 24h, 48h and 72h. T cell proliferations were measured by flow cytometry. The T cell activation in B-hCD3E mice was specifically up-regulated by anti-hCD3 antibody, similar to the level of activation 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. A. Representative FACS plots. B. Results of FACS analysis.

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 change the cytokine secretion after T cell activation.

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.

Cytotoxicity evaluation of CD3-PD-L1 bispecific antibody in vitro

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.

Serum titers of OVA-specific antibodies

B-hCD3E 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-hCD3E 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 hCD3E instead of its mouse counterpart did not affect the humoral immune response of mice. Values are expressed as mean ± SEM.

Routine blood test in B-hCD3E mice

Complete blood count (CBC). Blood from female C57BL/6 and B-hCD3E mice (n=8, 7-week-old) was collected and analyzed for CBC. There were no differences among any measurement between C57BL/6 and B-hCD3E mice, indicating that the introduction of hCD3E in place of its mouse counterpart does not change blood cell composition and morphology. Values are expressed as mean ± SEM.

Blood chemistry in B-hCD3E mice

Blood chemistry tests of B-hCD3E mice. Serum from female C57BL/6 and B-hCD3E mice (n=8, 7-week-old) was collected and analyzed for levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase). The results were similar between C57BL/6 and B-hCD3E mice, indicating that the introduction of hCD3E in place of its mouse counterpart does not change ALT and AST levels or the health of the liver. Values are expressed as mean ± SEM.

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.

T cell activation in CD3 Abs efficacy evaluation

Lymphocytes were isolated from peripheral blood at 48 hours after treatment. In the anti-hCD3 antibody treatment group, 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.

hCD3 Abs efficacy evaluation with two doses

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.

Dose-dependent T cell depletion caused by hCD3 Ab treatment

The ratio of B and T cells in the blood was detected by flow cytometry. Lymphocytes were isolated from peripheral blood at the end of the experiment. In the treatment group, the proportion of T cells was significantly reduced due to the activation induced cell death (AICD) effect caused by CD3E antibody treatment.

Tumor marker Ab (X) efficacy evaluation with two doses

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.

T cell activation in tumor marker Ab (X) efficacy evaluation with two doses

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.

Blinatumomab efficacy evaluation

MC38-hCD19 cells were  implanted subcutaneously into B-hCD3E mice. The mice were divided into control and treatment groups (n=6) when the tumor size was about 150±50 mm3. High-dose hCD3E antibody (Blinatumomab) significantly inhibited tumor growth, confirming that the B-hCD3E mouse model is a powerful tool for evaluating the efficacy of bispecific antibody against hCD3E in vivo.(A) Tumor average volume ± SEM, (B) Mice average weight ±SEM.

CD3E BsAb efficacy evaluation

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.

CD3-hEpCAM BsAb efficacy evaluation

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. 

AICD analysis after antibody treatment in B-hCD3E mice

The ratio of T cells in spleen and blood were analyzed at 24h、72 and 168h by flow cytometry.

Posters

Preclinical CD3-based mouse models for evaluation of bi-specific T-cell engager antibodies

Publications

Zhang R, Zhang J, Zhou X, Zhao A, Yu C. The establishment and application of CD3E humanized mice in immunotherapy. Exp Anim. 2022 May 13. doi: 10.1538/expanim.22-0012. Epub ahead of print. PMID: 35570001.

Reference

1. Sci Transl Med. 2011 Feb 2;3(68):68ra10. doi: 10.1126/scitranslmed.3001830.
2. Sci Rep. 2018 Mar 19;8:46960. doi: 10.1038/srep46960.

Back to top
WordPress Double Opt-in by Forge12