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Strain Name: BALB/cJGpt-Tg(hCD3E BAC)102/Gpt

Strain Type: Transgenic

Strain ID: T001550

Background: BALB/cJGpt



1. Study on the development and activation of T cells

2. T cell bispecific antibody screening



CD3E encoded the CD3-epsilon polypeptide, which together with CD3-gamma, -delta and -zeta, and the T-cell receptor alpha/beta and gamma/delta heterodimers, forms the T-cell receptor-CD3 complex. This complex plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. The CD3 complex as an external signal transducer, is one of the target molecules to modulate T cell functions. Defects in this gene cause immunodeficiency. Part of the TCR-CD3 complex is present on the surface of T lymphocytes, receives antigen-presenting cell signals, activates intracellular downstream signaling pathways, and plays an important role in adaptive immune responses.

CD3-target-based bispecific antibodies (CD3-TCB) shine in the treatment of lymphoma.CD3-TCB specifically recognizes the target molecule CD3 on T cells, while at the same time being able to recognize tumor-specific targets, thereby stimulating a targeted immune response of T cells and bringing about specific killing.

The hCD3E mouse model is a transgenic line obtained by injecting modified human BAC (Bacterial Artificial Chromosome) containing the human CD3E gene and its entire regulatory sequence into fertilized eggs of BALB/c mice. hCD3E is co-expressed with mCD3e on the surface of T cells. This strain is used to screen specific antibodies against human CD3E, and facilitates development of bispecific antibody drugs.

Data Support

1. The hCD3E expression and immune system analysis in the BALB/c-hCD3E mice


Fig.2 Detection of hCD3E expression in BALB/c-hCD3E.

hCD3E was expressed on the surface of T cells, and 96% CD3+ T cells are hCD3E and mCD3E co-expressing cells in BALB/c-hCD3E mice.

2. T/B/NK cell ratio assay in BALB/c-hCD3E mice



Fig.3 Detection of T/B/NK cell ratio in BALB/c-hCD3E mice.

No significant differences of T/B/NK cell ratio were observed between BALB/c-hCD3E and wild type mice.

3. Cytokine release assay in BALB/c-hCD3E mice




Fig.4 Detection of cytokine release assay in BALB/c-hCD3E mice.

BALB/c-hCD3E mice respond to anti-hCD3E antibody and anti-mCD3E antibody, and BALB/c widy-type mice just respond to the stimulation of anti-mCD3E antibody. IL2, IFN-γ and TNF level was elevated strikingly after the stimulation with anti-hCD3 or anti-mCD3 in BALB/c-hCD3E mice, and cytokine levels were elevated similarly with BALB/c wide type mice after the stimulation with anti-mCD3.

4. Anti-tumor efficacy test in BALB/c-hCD3E mice



Fig.5 In vivo efficacy test based on BALB/c-hCD3E mice (Data collected cooperating with CrownBio). (A)Tumor size of A20 in hCD3E mice (Mean volume ± SEM), (B) B cell depletion by CD3/CD20 bi-specific antibody in blood.

A20-CD20 cells in logarithmic growth phase were inoculated into BALB/c-hCD3E mice (6-8 weeks old) by tail vein injection. When the average volume of tumors reached to 100 mm3, all mice were randomly allocated to three different study groups(n=8), mice were treated with PBS(black), 1mg/kg Bispecific mCD3xmCD20(red), 1mg/kg Bispecific hCD3xmCD20(green) every 3 days for a total of 4 times. The results showed that Bispecific hCD3xmCD20(TGI=42%) and Bispecific mCD3xmCD20(TGI=50%) inhibited tumor growth when used alone. The B cells in the blood of the mice were destroyed.

The immune system analysis in the BALB/c-hCD3E/mCd3e KO mice

1. T/B/NK cell ratio assay in BALB/c-hCD3E/mCd3e KO mice


Fig.6 Detection of T/B/NK cell ratio in BALB/c-hCD3E/mCd3e mice.

Compare to wildtype and BALB/c-hCD3E mice, BALB/c-hCD3E/mCd3e-KO mice displayed a marked reduction in the number of splenic T cells and also reduced percentages and numbers of CD4+ T and CD8+ T cells.

2. Anti-tumor efficacy test

In vivo Efficacy Study of anti-mCTLA4 in BALB/c-hCD3E and BALB/c-hCD3E/mCd3e KO Mouse Model Bearing Subcutaneous Mouse CT26 Tumor


Fig.7 In vivo efficacy test in BALB/c-hCD3E and BALB/c-hCD3E/mCd3e KO mice.

BALB/c-hCD3E and BALB/c-hCD3E/mCd3e KO mice were inoculated subcutaneously with Murine colon carcinoma CT26 cells. When tumors reached an average volume of 100 mm3, mice were treated with vehicle or anti-mCTLA4 every two day for 4 times and followed by every three day for 3 times. The results showed that anti-mCTLA4 antibodies strongly inhibited the growth of subcutaneously inoculated CT26 tumor cells in BALB/c-hCD3E(TGI=61.68) but not BALB/c-hCD3E/mCd3e-KO mice.

The results demonstrate that mCD3ε is indispensable for T-cell development and function in BALB/c-hCD3E transgenic mice and these mice are a novel and valuable model to assessing the therapeutic efficacy of TCBs.


Fig.8 In vivo efficacy test based on BALB/c-hCD3E.

CD20 humanized mouse B lymphoma cells A20-HCD20 cells were cultured to logarithmic growth phase and inoculated subcutaneously into 6-8 weeks old BALB/c-hCD3E mice. Mice were divided into Vehicle groups (n=8),  Anti-hCD3/hCD20 (0.02mpk、0.1 mpk、0.5 mpk、2 mpk and 5 mpk)groups.When the tumor size was about 105 mm3, mice were treated with the appropriate medication, and administrated twice a week for a total of 6 times. Data are presented in Mean ± SEM format. Resolution: Anti-hCD3/hCD20 groups with different doages(0.02mpk、0.1 mpk、0.5 mpk、2 mpk and 5 mpk)have different tumor inhibitory effect

Indication: BALB/c-hCD3E mice are powerful tools to evaluate the efficacy of Bispecific antibodies in vivo.

Humanized tumor cell lines available at GemPharmatech 



1. Yamazaki, Tetsuo, et al. "CAST, a novel CD3ε-binding protein transducing activation signal for interleukin-2 production in T cells." Journal of Biological Chemistry 274.26 (1999): 18173-18180.

2. Kuhn, Chantal, et al. "Human CD3 transgenic mice: preclinical testing of antibodies promoting immune tolerance." Science Translational Medicine 3.68 (2011): 68ra10-68ra10.

3. Soudais, Claire, et al. "Independent mutations of the human CD3–ε gene resulting in a T cell receptor/CD3 complex immunodeficiency." Nature genetics 3.1 (1993): 77.

4. de Saint Basile, Geneviève, et al. "Severe combined immunodeficiency caused by deficiency in either the δ or the ε subunit of CD3." The Journal of clinical investigation 114.10 (2004): 1512-1517.

5. Dreier, Torsten, et al. "T cell costimulus-independent and very efficacious inhibition of tumor growth in mice bearing subcutaneous or leukemic human B cell lymphoma xenografts by a CD19-/CD3-bispecific single-chain antibody construct." The Journal of Immunology 170.8 (2003): 4397-4402.


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