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• The protein encoded by CD3E gene is the CD3-epsilon polypeptide, which together with CD3D, CD3G and CD3Z, and the T-cell receptor alpha/beta and gamma/delta heterodimers, forms the T-cell receptor-CD3 complex. CD3E is critical for normal formation and function of the TCR-CD3 complex and thus is a promising target for immune activity modulation.
• Glypican 3 (GPC3) is a member of the family of glypican heparan sulfate proteoglycans (HSPGs). The GPC3 gene may play a role in controlling cell migration, negatively regulating cell growth and inducing apoptosis. GPC3 is frequently upregulated in hepatocellular carcinoma (HCC).
• B-hCD3E/hGPC3 mice (112603) was obtained by crossing with B-hCD3E mice (110008) and B-hGPC3 mice (110873). The exons 2-6 of mouse Cd3e gene that encode the extracellular domain were replaced by human CD3E exons 2-7 in B-hCD3E/hGPC3 mice. The human GPC3 CDS was inserted after the exon 3 of mouse Gpc3 gene in B-hCD3E/hGPC3 mice. The insertion disrupts the endogenous murine Gpc3 gene.
• Mouse CD3E was detectable on T cells from spleen of wild-type C57BL/6 mice. Human CD3E was exclusively detectable on T cells from spleen of homozygous B-hCD3E/hGPC3 mice but not in wild-type C57BL/6 mice.
• GPC3 was detectable in liver and kidney of wild-type C57BL/6 mice and homozygous B-hCD3E/hGPC3 mice due to the cross-reactivity of antibodies.
• Humanization of CD3E and GPC3 do not change the overall frequency or distribution of immune cell types in spleen, blood and thymus.
• Humanization of CD3E and GPC3 do not change the blood cell composition and morphology, ALT and AST levels or health of liver.
• This product is used for the pharmacological and safety evaluation of CD3/GPC3 bispecific antibodies.

Gene targeting strategy for B-hCD3E/hGPC3 mice.
The exons 2-6 of mouse Cd3e gene that encode the extracellular domain were replaced by human CD3E exons 2-7 in B-hCD3E/hGPC3 mice. The human GPC3 CDS was inserted after the exon 3 of mouse Gpc3 gene in B-hCD3E/hGPC3 mice. The insertion disrupts the endogenous murine Gpc3 gene.

Species specific analysis of GPC3 gene expression in wild-type C57BL/6 mice and homozygous humanized B-hCD3E/hGPC3 mice by RT-PCR. Kidney was collected from wild-type C57BL/6 mice (+/+) and homozygous B-hCD3E/hGPC3 mice (H/H; H/H). Mouse Gpc3 mRNA was detectable only in wild-type C57BL/6 mice. Human GPC3 mRNA was detectable only in homozygous B-hCD3E/hGPC3 mice, but not in wild-type C57BL/6 mice.

Strain specific GPC3 expression analysis in homozygous B-hCD3E/hGPC3 mice by western blot. Liver and kidney were collected from wild type (WT) mice (+/+) and homozygous B-hCD3E/hGPC3 mice (H/H; H/H), and analyzed by western blot with anti-GPC3 antibody. GPC3 was detectable in WT mice (+/+) and homozygous B-hCD3E/hGPC3 mice (H/H; H/H) due to the cross-reactivity of antibodies.

Strain specific CD3E expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hCD3E/hGPC3 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hCD3E/hGPC3 mice (H/H; H/H), and analyzed by flow cytometry. Mouse CD3E was only detectable in wild-type C57BL/6 mice. Human CD3E was exclusively detectable in homozygous B-hCD3E/hGPC3 mice, but not in wild-type C57BL/6 mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry.
Splenocytes were isolated from wild-type C57BL/6 mice and homozygous B-hCD3E/hGPC3 mice (n=4, 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, monocytes, macrophages, granulocytes, CD4+ T cells, CD8+ T cells, and Tregs in B-hCD3E/hGPC3 mice were similar to those in C57BL/6 mice, demonstrating that humanization of CD3E and GPC3 do not change the frequency or distribution of these cell types in spleen. The frequency of leukocyte subpopulations in thymus and blood of B-hCD3E/hGPC3 mice were also comparable to wild-type C57BL/6 mice (Data not shown). Values are expressed as mean ± SEM.