• CD3 consists of four protein chains (CD3E, CD3D, CD3G and CD3Z), which are important biological markers on the T cell membrane. CD3 can form a TCR/CD3 complex with the T cell receptor, participating in the regulation of T cell antigen recognition, signal transduction and T cell development. EPCAM (Epithelial Cell Adhesion Molecule) is a gene encoding a transmembrane glycoprotein with a molecular weight of approximately 40 kDa. It primarily participates in cell adhesion, signal transduction (e.g., PI3K/AKT and Notch signaling pathways), and DNA damage repair (via homologous recombination). Under normal physiological conditions, EPCAM is predominantly expressed on the surface cells of epithelial tissues; however, its expression becomes aberrant (either overexpressed or lost) in various epithelium-derived tumors (e.g., colorectal cancer and breast cancer), correlating with tumor invasion, metastasis, and drug resistance. EPCAM has emerged as a promising therapeutic target. Current research and development efforts focus on targeted monoclonal antibodies, antibody-drug conjugates (ADCs), and combinatorial immunotherapy.
• B-hCD3EDG/hEPCAM mice were obtained by mating B-hCD3EDG mice (110039) and B-hEPCAM mice (110826). In B-hCD3EDG/hEPCAM mice, chimeric human CD3EDG were expressed, while mouse Cd3edg were knocked out. The exons 3-8 of mouse Epcam genes that encode the extracellular protein were replaced by human EPCAM exons 2-7 in B-hCD3EDG/hEPCAM mice.
• Human CD3E was exclusively detectable in homozygous B-hCD3EDG/hEPCAM mice, but not in wild-type C57BL/6 mice. Human EPCAM mRNA was exclusively detectable in small intestine of homozygous B-hCD3EDG/hEPCAM mice. Human EPCAM protein was detectable in kidney, skin, small intestine, and large intestine, but not in heart, liver, and spleen. Humanization of CD3EDG and EPCAM does not change the overall frequency or distribution of immune cell types in spleen, blood, and lymph nodes.
• This product is used for the pharmacological and safety evaluation of targets related TCE drugs.

Strain specific analysis of EPCAM mRNA expression in wild-type C57BL/6 mice and B-hCD3EDG/hEPCAM mice by RT-PCR. Small intestine RNA were isolated from wild-type C57BL/6 mice (female, 6-week-old, n=1) and homozygous B-hCD3EDG/hEPCAM mice (female, 6-week-old, n=1), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human EPCAM primers. Mouse Epcam mRNA was only detectable in wild-type mice (+/+). Human EPCAM mRNA was exclusively detectable in homozygous B-hCD3EDG/hEPCAM mice (H/H) but not in wild-type mice (+/+).

CD3E expression analysis in wild-type C57BL/6 mice and homozygous B-hCD3EDG/hEPCAM mice by flow cytometry. Spleen and blood T cells were collected from wild-type C57BL/6 mice (female, 6-week-old, n=1) and homozygous B-hCD3EDG/hEPCAM mice (female, 6-week-old, n=1). Protein expression was analyzed with anti-human CD3E antibody (BD Horizon™, 562426) and anti-mouse CD3E antibody (Biolegend, 100312) by flow cytometry. Human CD3E was exclusively detectable in homozygous B-hCD3EDG/hEPCAM mice, but not in wild-type C57BL/6 mice.

Immunohistochemical (IHC) analysis of EPCAM expression in B-hCD3EDG/hEPCAM mice. The kidney, heart, lung, liver, skin, small intestine, large intestine and spleen were collected from wild-type C57BL/6 mice and B-hCD3EDG/hEPCAM mice (female, 6-week-old), analyzed by IHC with anti-EPCAM antibody (anti-mEPCAM: abcam, ab213501; anti-hEPCAM: abcam, ab223582). Mouse EPCAM was detectable in C57BL/6 mice. Human EPCAM was detectable in B-hCD3EDG/hEPCAM mice. The arrow   indicates tissue cells with positive EPCAM staining (brown). “+” indicates that the tissue is positive, and “-” indicates that the tissue is negative. The antibody ab223582 exhibited strong non-specific binding in the small intestine and liver, with no obvious membrane staining signals observed in the bronchial epithelial cells of the lung.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (female, n=3, 6-week-old) and homozygous B-hCD3EDG/hEPCAM mice (female, n=3, 8-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hCD3EDG/hEPCAM mice were similar to those in C57BL/6 mice, demonstrating that humanization does not change the frequency or distribution of these cell types in spleen. The frequency of leukocyte subpopulations in blood and lymph node of B-hCD3EDG/hEPCAM mice were also comparable to wild-type C57BL/6 mice (Data not shown).

Antitumor Activity of Anti-Human CD3/EPCAM Bispecific Antibodies (Solitomab, Commercialized) in B-hCD3EDG/hEPCAM Mice.​​ B-hEPCAM MC38 mouse colon carcinoma cells were subcutaneously implanted into homozygous B-hCD3EDG/hEPCAM mice (male, 9-week-old, n=6). Mice were grouped once tumor volume reached approximately 100 mm³, at which time they were intravenously injected with anti-human CD3/EPCAM bispecific antibodies (indicated in the panel). (A) Anti-human CD3/EPCAM bispecific antibodies inhibited B-hEPCAM MC38 tumor growth in B-hCD3EDG/hEPCAM mice. (B) Body weight changes during treatment. Values are expressed as mean ± SEM.