Strain Name: BALB/cJGpt-Tg(hCD3E BAC)102/Gpt
Strain Type: Transgenic
Strain ID: T001550
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.
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.
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.
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
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.
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.
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