Biological Features and Therapeutic Potential of CD3 and CEACAM5

•  Gene Information: CD3 is a multi-subunit T-cell receptor complex essential for antigen-induced T-cell signaling. CEACAM5, also known as CEA, is a GPI-anchored cell-surface glycoprotein and a well-established tumor-associated antigen. 
• Protein Expression: CD3 is expressed on T cells and is essential for T-cell receptor signaling. CEACAM5 is normally expressed in gastrointestinal epithelium and is frequently overexpressed in colorectal, gastric, pancreatic, and other epithelial cancers. 
• Signaling Pathway: CD3 engagement activates the TCR signaling cascade through Lck, ZAP-70, LAT, and downstream pathways, leading to T-cell activation and cytotoxicity. CEACAM5 provides a tumor-cell surface target that enables CD3-mediated T-cell redirection by T-cell engagers. 
• Therapeutic Inhibition: CEACAM5×CD3 bispecific antibodies recruit CD3⁺ T cells to CEACAM5-positive tumor cells and induce targeted tumor-cell killing. The dual-humanized model supports in vivo evaluation of T-cell engagers, including efficacy, pharmacodynamics, and safety.

CD3EDG 

• The chimeric human CD3EDG was expressed, while mouse Cd3edg were knocked out in B-hCD3EDG/hCEACAM5 mice. 

CEACAM5

•  The full coding sequences of human CEACAM5 gene, including the promoter, 5’UTR and 3’UTR are inserted into mouse Hipp11 (H11) locus in B-hCD3EDG/hCEACAM5 mice.

mRNA CD3D and CD3G were detectable in B-hCD3EDG/hCEACAM5 mice by RT-PCR and sequencing. Spleen tissues were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCEACAM5 mice (H/H). Primers were designed to detect the mouse and human CD3D and CD3G. Sequencing of the PCR products confirmed that the amplified sequences were consistent with database reference sequences.

Mouse and human CD3E expression analysis in splenocytes. Splenocytes were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/hCEACAM5 mice (Female, 5-week-old, n=1). CD3E expression on T cells was analyzed by flow cytometry using species-specific anti-CD3E antibodies (anti-human CD3E antibody, BD Horizon, 562426; anti-mouse CD3E antibody, Biolegend, 100312 ).

Human CEACAM5 expression analysis in colon. Colon was collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCEACAM5 mice (H/H, H/H) and analyzed by flow cytometry with species-specific anti-human CD66a/c/e(CEACAM5) Antibody (Biolegend, 342308).

Western blot analysis of CEACAM5 protein expression in homozygous B-hCD3EDG/hCEACAM5 mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCEACAM5 mice (H/H, H/H), and then analyzed by western blot with human-specific anti-CEACAM5 antibody (Invitrogen, PA5-88533). 40 μg total proteins were loaded for western blotting analysis.

Immunohistochemical analysis of organs in B-hCD3EDG/hCEACAM5 mice. Major tissues were collected from wild-type mice and homozygous B-hCD3EDG/hCEACAM5 mice and analyzed by IHC with anti-human CEACAM5 antibody (Abcam, ab133633). The arrow indicates tissue cells with positive CEACAM5 staining (brown). "+" and "-" indicate positive and negative tissues, respectively.