Background:

• CD3 is a key component of the T cell receptor (TCR) complex, composed of γ, δ, ε, and ζ chains that form dimers (e.g., CD3γε, ζζ). Its intracellular domains contain ITAMs essential for signal transduction. When the TCR engages an MHC–peptide complex, CD3 transmits the signal into the cell, initiating cascades that drive T cell activation, proliferation, and effector functions.
• GUCY2C is a receptor expressed on intestinal epithelial cells, activated by ligands including the hormone guanylin, bacterial enterotoxin ST, and drugs like linaclotide. Ligand binding increases intracellular cGMP, activating downstream effectors to regulate fluid secretion, barrier function, and metabolism. Therapeutic antibodies targeting GUCY2C can activate this pathway to induce apoptosis and inhibit tumor growth and metastasis.

Targeting strategy:

• The targeting strategy of humanized CD3EDG are undisclosed. The exons 1-11 of mouse Gucy2c gene that encode signal peptide and extracellular domain and part of transmembrane domain are replaced by human counterparts in B-hCD3EDG/hGUCY2C mice. The genomic region of mouse Gucy2c gene that encodes cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also replaced by human counterparts. The chimeric GUCY2C expression is driven by endogenous human Gucy2c promoter, while mouse Gucy2c gene transcription and translation will be disrupted.

Validation:

• Mouse Cd3d and Cd3g mRNA was detectable only in wild-type mice. Human CD3D and CD3G mRNA was detectable only homozygous B-hCD3EDG/hGUCY2C mice but not in wild-type mice. Mouse Gucy2c mRNA was detectable only in wild-type mice. Human GUCY2C mRNA was detectable only homozygous B-hCD3EDG/hGUCY2C mice but not in wild-type mice.
• Human CD3E was exclusively detectable in homozygous B-hCD3EDG/hGUCY2C mice, but not in wild-type C57BL/6JNifdc mice. Human GUCY2C was detectable in ileum and jejunum of B-hCD3EDG/hGUCY2C mice.

Application: This product is used for the pharmacological and safety evaluation of CD3EDG/hGUCY2C bispecific antibodies.

Gene targeting strategy for B-hCD3EDG/hGUCY2C mice. The targeting strategy of humanized CD3EDG were undisclosed. The exons 1-11 of mouse Gucy2c gene that encode signal peptide and extracellular domain and part of transmembrane domain are replaced by human counterparts in B-hCD3EDG/hGUCY2C mice. The genomic region of mouse Gucy2c gene that encodes cytoplasmic portion is retained. The promoter and 5’UTR region of the mouse gene are replaced by human counterparts. The chimeric GUCY2C expression is driven by endogenous human Gucy2c promoter, while mouse Gucy2c gene transcription and translation will be disrupted.

Strain specific analysis of CD3D and CD3G mRNA expression in wild-type C57BL/6JNifdc mice and B-hCD3EDG/hGUCY2C mice by RT-PCR. Spleen RNA were isolated from wildtype C57BL/6 mice (+/+) and homozygous B-hCD3EDG/hGUCY2C mice (H/H; H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human CD3D and CD3G primers. Mouse Cd3d and Cd3g mRNA was detectable only in wild-type mice. Human CD3D and CD3G mRNA was detectable only homozygous B-hCD3EDG/hGUCY2C mice but not in wild-type mice.

Strain specific analysis of GUCY2C mRNA expression in wild-type C57BL/6JNifdc mice and B-hCD3EDG/hGUCY2C mice by RT-PCR. Ileum RNA were isolated from wildtype C57BL/6 mice (+/+) and homozygous B-hCD3EDG/hGUCY2C mice (H/H; H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human GUCY2C primers. Mouse Gucy2c mRNA was detectable only in wild-type. Human GUCY2C mRNA was detectable only homozygous B-hCD3EDG/hGUCY2C mice but not in wild-type mice.

CD3E expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hGUCY2C mice by flow cytometry. Spleen T cells were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hGUCY2C mice (H/H; H/H). 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/hGUCY2C mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific GUCY2C expression analysis in homozygous B-hCD3EDG/hGUCY2C mice by flow cytometry. Ileum and Jejunum were collected from homozygous B-hCD3EDG/hGUCY2C mice (H/H; H/H) and analyzed by flow cytometry with anti-human GUCY2C Antibody (R&D, FAB2157R-100UG). Human GUCY2C was detectable in ileum and jejunum of B-hCD3EDG/hGUCY2C mice.