•  CD3 Molecule: As a vital component of the T cell receptor (TCR) complex, CD3 is responsible for transducing antigen recognition signals into the cell interior. The engagement and ligation of CD3 can directly induce T cell activation.
• CDH17 Molecule: CDH17 (cadherin-17) is a cell-cell adherent junctional molecule that is strictly restricted to normal intestinal and colonic epithelium but is highly overexpressed in colorectal, gastric, and pancreatic adenocarcinomas, as well as colorectal liver metastases. It drives cancer progression—specifically cell adhesion, proliferation, migration, and invasion—by activating the Wnt/β-catenin, Ras/Raf/MEK/ERK, and α2β1 integrin pathways (via its RGD motif), making its knockdown a promising therapeutic strategy to inhibit tumor malignancy.
• CDH3 Molecule: CDH3 (P-cadherin) is a classical calcium-dependent homophilic cell adhesion molecule essential for tissue architecture, epithelial integrity, and cell fate determination. CDH3 physiologically anchors to the cytoskeleton via β-catenin and p120-catenin to maintain adhesion, but when overexpressed in cancers (e.g., breast, lung), it competes with E-cadherin for these catenins, disrupting junctions and enhancing migration and invasion.
• T-Cell Engagement (TCE): CD3-redirecting bispecific antibodies bind tumor-associated antigens (TAAs) on cancer cells and CD3 on recruited T cells to elicit cancer cell cytotoxicity independent of T cell receptor specificity. Cancer cell cytotoxicity occurs through perforin and granzyme B release, resulting in apoptosis through caspase pathway induction.
• CD3/CDH17/CDH3 Antibody: The bispecific antibody binds to CDH17 on cancer cells and CD3 on recruited T cells, inducing cancer cell cytotoxicity independent of T cell receptor specificity.

CD3EDG

• The chimeric human CD3EDG was expressed, while mouse Cd3edg were knocked out in B-hCD3EDG/hCDH17/hCDH3 mice.

CDH17

• The extracellular region (except signal peptide) of mouse Cdh17 was replaced by human CDH17 extracellular region sequences in B-hCD3EDG/hCDH17/hCDH3 mice.

CDH3

• The exons 4-13 of mouse Cdh3 gene that encode the extracellular domain were replaced by human CDH3 exons 4-13 in B-hCD3EDG/hCDH17/hCDH3 mice.

Strain specific analysis of CDH17 mRNA expression in wild-type C57BL/6JNifdc mice and B-hCD3EDG/hCDH3/hCDH17 mice by RT-PCR. Small intestine and colon RNA were isolated from C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCDH3/hCDH17 (H/H), and then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human CDH17 primers.

Strain specific analysis of CDH3 mRNA expression in wild-type C57BL/6JNifdc mice and B-hCD3EDG/hCDH3/hCDH17 mice by RT-PCR. Skin RNA were isolated from C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCDH3/hCDH17 (H/H;H/H;H/H), and then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human CDH3 primers.

Species-specific analysis of CDH17 and CDH3 gene expression in wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/hCDH17/hCDH3 mice by RT-qPCR. Small intestine, colon, and skin RNA was isolated from wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/hCDH17/hCDH3 mice (female, 7-week-old, n=4). cDNA was synthesized by reverse transcription, followed by RT-qPCR with CDH17 and CDH3 primers.

Strain-specific CD3E expression analysis in homozygous B-hCD3EDG/hCDH17/hCDH3 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCDH17/hCDH3 mice (HO) and analyzed by flow cytometry with a species-specific anti-mouse CD3E antibody (Biolegend, 100312) and an anti-human CD3E antibody (Biolegend, 317344).

Strain-specific CD3E expression analysis in homozygous B-hCD3EDG/hCDH17/hCDH3 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCDH17/hCDH3 mice (HO) and analyzed by flow cytometry with a species-specific anti-mouse CD3E antibody (Biolegend, 100312) and an anti-human CD3E antibody (Biolegend, 317344).

Western blot analysis of CDH17 protein expression in homozygous B-hCD3EDG/hCDH17/hCDH3 mice. Tissue lysates from the small intestine, colon, and skin were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCDH17/hCDH3 mice (H/H) and analyzed by western blot with an anti-human CDH17 antibody (Abcam, ab109190) and an anti-mouse CDH17 antibody (R&D Systems, MAB45191-100). 40 μg of total protein was loaded for each sample. Note: The anti-human CDH17 antibody exhibits weak binding to mouse CDH17.

Western blot analysis of CDH3 protein expression in homozygous B-hCD3EDG/hCDH17/hCDH3 mice. Tissue lysates from the small intestine, colon, and skin were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCDH17/hCDH3 mice (H/H) and analyzed by western blot with an anti-human CDH3 antibody (R&D Systems, MAB861-SP) and an anti-mouse CDH3 antibody (R&D Systems, AF761-SP). 40 μg of total protein was loaded for each sample.