• The CD3 complex is an important marker on the surface of T cells. CD3 has four peptide chains, namely, the γ chain, the δ chain, the ε chain, and the ζ chain. The transmembrane region of CD3 molecules is connected to the transmembrane region of TCRα and β chains through salt Bridges to form the TCR-CD3 complex, which jointly participates in the recognition of antigens by T cells. In this process, TCR recognizes and binds to antigenic peptides presented by MHC molecules, resulting in the phosphorylation of the immune receptor tyrosine activation motifs ITAM in the CD3 cytoplasmic region, which can then recruit other tyrosine protein kinases (such as ZAP-70), thereby causing activation of downstream pathways such as MAPK, NF-κB and other signaling pathways. Under the action of multiple transcription factors, it can cause T cell proliferation, migration, cytokine production and effector function.
• EGFR is expressed in various tissues. Upon binding of ligands like EGF to the EGFR receptor, EGFR dimers are formed. The activation of EGFR dimers stimulates intracellular tyrosine kinases, resulting in phosphorylation that triggers downstream signaling cascades, leading to cancer cell proliferation and the proliferation of new blood vessels within cancer cells. The mechanism of action of EGFR-targeting drugs primarily involves blocking or inhibiting the activity of EGFR, thereby preventing the growth and spread of cancer cells.

Gene targeting strategy for B-hCD3EDG/hEGFR mice.Chimeric human CD3EDG were expressed, while mouse Cd3edg were knocked out in B-hCD3EDG/hEGFR mice.The exons 2-17 of mouse Egfr gene that encode extracellular domain were replaced by human counterparts in B-hCD3EDG/hEGFR mice. The genomic region of mouse Egfr gene that encodes transmembrane domain and cytoplasmic portion was retained. The promoter, 5’UTR, signal peptide and 3’UTR region of the mouse gene were also retained. The chimeric EGFR expression was driven by endogenous mouse Egfr promoter, while mouse Egfr gene transcription and translation will be disrupted.

CD3E expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hCD3EDG/hEGFR mice by flow cytometry. Spleen T cells were collected from wild-type C57BL/6 mice (+/+) (Female, 6-week-old, n=1) and homozygous B-hCD3EDG/hEGFR mice (H/H; H/H; H/H; H/H) (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/hEGFR mice, but not in wild-type C57BL/6 mice.

CD3E expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hCD3EDG/hEGFR mice by flow cytometry. Blood T cells were collected from wild-type C57BL/6 mice (+/+) (Female, 6-week-old, n=1) and homozygous B-hCD3EDG/hEGFR mice (H/H; H/H; H/H; H/H) (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/hEGFR mice, but not in wild-type C57BL/6 mice.

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

Immunohistochemical (IHC) analysis of EGFR expression in homozygous B-hCD3EDG/hEGFR mice. The ovary, testis, heart, lung, liver, spleen, skin, thyroid, esophagus, kidney and uterus were collected from C57BL/6 mice (+/+) (G1: female, 5-week-old, n=2; G3: male, 5-week-old , n=2) and homozygous B-hCD3EDG/hEGFR mice (H/H; H/H; H/H; H/H) (G2: female, 5-week-old, n=2; G4: male, 5-week-old , n=2) , analyzed by IHC with anti-EGFR (abcam, ab32198). EGFR was detectable in both C57BL/6 mice and homozygous B-hCD3EDG/hEGFR mice because of antibody crossover. The arrow indicates tissue cells with positive EGFR staining (brown). “+” indicates that the tissue is positive, and “-” indicates that the tissue is negative. "unknown" means it can not be determined.