Background:

• 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).

Targeting strategy:

• B-hCD3EDG/hGPC3 mice were obtained by mating B-hCD3EDG mice (110039) and B-hGPC3 mice (110873). In B-hCD3EDG/hGPC3 mice, chimeric human CD3EDG was expressed, while mouse Cd3edg were knocked out. The human GPC3 CDS was inserted after the exon 3 of mouse Gpc3 gene in B-hCD3EDG/hGPC3 mice. The insertion disrupts the endogenous murine Gpc3 gene.

Validation:

• Mouse CD3E was detectable on T cells from spleen of wild-type C57BL/6JNifdc mice. Human CD3E was exclusively detectable on T cells from spleen of homozygous B-hCD3EDG/hGPC3 mice but not in wild-type C57BL/6JNifdc mice.
• GPC3 was detectable in liver and kidney of wild-type C57BL/6JNidfc mice and homozygous B-hCD3EDG/hGPC3 mice, the antibody was across-reactive between human and mouse.
• Mouse Cd3d, Cd3g and Gpc3 mRNA were only detectable in wild-type mice. Human CD3D, CD3G and GPC3 mRNA were only detectable in homozygous B-hCD3EDG/hGPC3 mice, but not in wild-type C57BL/6JNidfc mice.
• Humanization of CD3EDG and GPC3 do not change the overall frequency or distribution of immune cell types in spleen, blood and lymph nodes.

Application: This product is used for the pharmacological and safety evaluation of CD3EDG/GPC3 bispecific antibodies.

Gene targeting strategy for B-hCD3EDG/hGPC3 mice. B-hCD3EDG/hGPC3 mice were obtained by mating B-hCD3EDG mice (110039) and B-hGPC3 mice (110873). In B-hCD3EDG/hGPC3 mice, chimeric human CD3EDG was expressed, while mouse Cd3edg were knocked out. The human GPC3 CDS was inserted after the exon 3 of mouse Gpc3 gene in B-hCD3EDG/hGPC3 mice. The insertion disrupts the endogenous murine Gpc3 gene.

Strain specific CD3E expression analysis in homozygous B-hCD3EDG/hGPC3 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hGPC3 mice (H/H; H/H). Protein expression was analyzed with anti-mouse CD3E antibody (Biolegend, 100312) and anti-human CD3E antibody (BD Horizon™, 562426) by flow cytometry. Mouse CD3E was detectable on T cells from spleen of wild-type C57BL/6JNifdc mice. Human CD3E was exclusively detectable on T cells from spleen of homozygous B-hCD3EDG/hGPC3 mice but not in wild-type C57BL/6JNifdc mice.

Species specific analysis of CD3D, CD3G and GPC3 gene expression in wild-type C57BL/6JNidfc mice and homozygous humanized B-hCD3EDG/hGPC3 mice by RT-PCR. Spleen and kidney were collected from wild-type C57BL/6JNidfc mice (+/+) and homozygous B-hCD3EDG/hGPC3 mice (H/H, H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human CD3D, CD3G and GPC3 primers. Mouse Cd3d, Cd3g and Gpc3 mRNA were only detectable in wild-type mice. Human CD3D, CD3G and GPC3 mRNA were detectable only in homozygous B-hCD3EDG/hGPC3 mice, but not in wild-type C57BL/6JNidfc mice.

Strain specific GPC3 expression analysis in homozygous humanized B-hCD3EDG/hGPC3 mice by western blot. Liver and kidney were collected from wild-type C57BL/6JNidfc mice (+/+) and homozygous B-hCD3EDG/hGPC3 mice (H/H; H/H) and analyzed by western blot with anti-GPC3 antibody (Abcam, ab186872). GPC3 was detectable in liver and kidney of wild-type C57BL/6JNidfc mice and homozygous B-hCD3EDG/hGPC3 mice, the antibody was across-reactive between human and mouse.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6JNifdc mice (Female, n=3, 8-week-old) and homozygous B-hCD3EDG/hGPC3 mice (Female, n=3, 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, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hCD3EDG/hGPC3 mice were similar to those in C57BL/6JNifdc mice. The frequency of leukocyte subpopulations in blood and lymph nodes of B-hCD3EDG/hGPC3 mice were also comparable to wild-type C57BL/6JNifdc mice. (Data not shown). Values are expressed as mean ± SEM.