B-hCD3E mice

Basic Information

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.

Details

Phenotype

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 stimulated with anti-CD3ε in vivo, 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 

Weight of thymuses and spleens

(A) Thymus and spleens were weighed after isolated from C57BL/6 and B-hCD3E mice (n=6). (B) The result shows that there is no significant difference regarding the spleen weight between B-hCD3E mice and C57BL/6 mice. However, the thymus weight in B-hCD3E is slightly lower than that in the wild type C57BL/6 mice.

Analysis of thymus lymphocyte subpopulation in B-hCD3E mice

Analysis of  lymphocyte subpopulations by FACS.

lymphocyte were isolated from 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 indicated here. B. Results of FACS analysis. Percent of CD4, CD8 T 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 T cell development, differentiation or distribution in thymus.

Analysis of spleen lymphocyte subpopulation in B-hCD3E mice

Analysis of  lymphocyte subpopulations by FACS.

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 spleen, peripheral blood and lymph node leukocyte subpopulations by FACS

 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. 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 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. A. Representative FACS plots. B. Results of FACS analysis. 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.

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-CD3 antibody (C57BL/6, anti-mCD3; B-hCD3E, anti-hCD3) and anti-mCD28 for 24h, 48h and 72h. IFN-γ and IL-2 productions were then tested using ELISA method. Concentration of IFN-γ and IL-2 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 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.

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 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.

Application

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 the humanized mouse model, mPD-1 antibody significantly inhibited tumor growth, indicating normal T cell function. More aggressive tumor growth after anti-hCD3 antibody treatment was observed. This may result from 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

The ratio of B cells and T cells in the blood of mice was detected by flow cytometry. Lymphocytes were isolated from peripheral blood at the 48 hours after the 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 hCD3 Ab treatment in the humanized mice.

Percentage of CD4+ and CD8+ cells after hCD3 antibody treatment

(A) Compared with hlgG Ab, CD4+ % decreased after hCD3 Ab treatment in Blood. (B) Compared with hlgG Ab, CD8+ % decreased after hCD3 Ab treatment in Blood.

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 the 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 cells 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 proportion of T cells was significantly decreased due to the activation induced cell death (AICD) effect caused by CD3E antibody.

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 cells 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 proportion of T cells and B cells were no significant change.

Blinatumomab efficacy evaluation

Murine colon cancer MC38-hCD19 cells were subcutaneously implanted into B-hCD3E mice. Mice were divided into control and treatment group dose(n=6) when the tumor size was approximately 150±50 mm3. High doses of hCD3E antibodies (Blinatumomab) significantly inhibited tumor growth, confirming that the B-hCD3E mouse model is a powerful tool for in vivo anti-hCD3E based Bi-specific antibody efficacy evaluation. (A) Tumor average volume ± SEM, (B) Mice average weight ±SEM.

CD3E BsAb  Efficacy Evaluation

Collaboration Data

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

Murine colon cancer  MC38-hEpCAM cells were subcutaneously implanted into B-hCD3E mice. Mice were grouped  when the average  tumor size reached approximately 100±20 mm³.  The result shows  a moderate antitumor activity by the anti-human CD3/hEpCAM bispecific antibody compared with the vehicle control group. The data also indicated that anti-murine CD4, a CD4-depleting antibody, potentiated the antitumor activity of the bispecific anti-human CD3/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

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.