General Information

Development

Injecting a linearized DNA fragment composed of the human CD3E gene CDS sequence and the mouse CD3E gene promoter and enhancer sequences into the pronucleus of C57BL/6NCrl mice, results in transgenic positive mice. Through phenotypic analysis, hCD3E transgenic mice with appropriate expression levels are selected as the founder mice, which are then bred with wild-type C57BL/6NCrl to establish a commercialized hCD3E NPG mouse strain. The CD3E gene encodes a protein that is the CD3-epsilon polypeptide, which, along with CD3-gamma, CD3-delta, and CD3-zeta, forms the T cell receptor-cd3 complex with the T cell receptor alpha/beta or gamma/delta heterodimer, and is the signal transducer for T cells upon antigen stimulation. This hCD3E mouse strain retains the expression of the mouse-derived CD3 complex subunits (CD3εδ) and expresses physiological levels of hCD3E on the normally developing T cells in mice. hCD3E can randomly incorporate into the mouse T cell CD3 complex molecules, perform normal signal transduction functions, and can be bound by hCD3E-specific antibodies and activate the CD3 molecule-mediated T cell activation signals. The CD3E humanized mouse can be used to verify the antitumor effects of bispecific antibody targeting hCD3E and tumor antigens.

Phenotype

1. Peripheral blood hCD3E assay

Fig 1. hCD3E expression detected in peripheral blood (C57BL/6N mice on the left, hCD3E mice on the right)

Expression of hCD3E in peripheral blood was detected by flow cytometry. hCD3E was not expressed by wild-type C57BL/6N mice, and hCD3E was detected in 90% of mCD3⁺ positive T cells expressing hCD3E in hCD3E mice.

2. The levels of B, CD4⁺ T, CD8+ T and NK cells in peripheral blood of hCD3E mice

Fig2. B, CD4+ T, CD8+ T and NK cells in the peripheral blood of hCD3E mice

Results: hCD3E mice undergo normal B, T and NK cell differentiation

3. hCD3E mice (wild-type C57BL/6 as control) immune cell levels

Fig3. hCD3E mice (wild-type C57BL/6 as control) immune cell levels

hCD3E mice in which T cells can be effectively activated, and wild-type C57BL/6 mice in which only anti-mouse CD3E antibody can effectively activate T cells.

Application Cases

1. Construct colon cancer model and evaluate therapeutic efficacy

Fig4. Transplantation of CT26-TAA and Keytruda efficacy in hCD3E mice

The hCD3E mice were subcutaneously inoculated with BALB/c mouse-derived colon cancer cell line CT26-TAA and grouped (n=7) when the tumor volume was close to 100±50 mm³, and injected with five different types of CD3E bispecific antibody drugs, CS4, Y6, Y7, M-1, and M-K, twice a week for 3 weeks. The results showed that 1~2 mg/kg hCD3e bispecific antibodies CS4, Y6 and Y7 could significantly inhibit tumor growth. It was demonstrated that hCD3E mice could effectively evaluate the in vivo efficacy of the bispecific antibodies.

hCD3E Mice Applications

1. Validation of the anti-tumor effects of bispecific antibodies against hCD3E and tumor antigens

2. Research on T-cell development and activation

3. Research on the pathogenesis and treatment of type I diabetes mellitus.

Reference

1. Clevers HC, Dunlap S, Wileman TE, et al. Human CD3-epsilon gene contains three miniexons and is transcribed from a non-TATA promoter. Proceedings of the National Academy of Sciences of the United States of America. 1988, 85(21): 8156–8160.

2. J Autoimmun. Mucosal administration of CD3-specific monoclonal antibody inhibits diabetes in NOD mice and in a preclinical mouse model transgenic for the CD3 epsilon chain. 2017, 115-122.

3. Ueda, Otoya, et al. "Entire CD3ε, δ, and γ humanized mouse to evaluate human CD3–mediated therapeutics." Scientific Reports7 (2017).