CD3EDG/CD20/CD2: A multi-protein panel targeting T and B-cell coreceptors.

• Gene Information: This target combination clusters CD3EDG on chromosome 11q23, CD2 on 1p13, and MS4A1 (CD20) on 11q12, creating a multi-chromosomal genomic profile for dual T/B-cell targeting.
• Protein Expression: CD3 complex and CD2 are highly expressed on mature T cells and thymocytes, whereas CD20 expression is strictly restricted to B-cell lineages from pre-B stages to mature lymphocytes.
• Signaling Pathway: CD3/CD2 orchestrate T-cell receptor activation, ITAM phosphorylation, and downstream flux, while CD20 independently acts as a calcium channel modulator regulating B-cell activation, survival, and cell-cycle progression.
• Therapeutic Inhibition: This targeted panel enables the evaluation of multi-specific T-cell engagers (BiTEs) that bind CD3/CD2 to redirect cytotoxic T cells, as well as therapeutic antibodies driving anti-CD20 mediated B-cell depletion.

CD3EDG

• The human CD3EDG was expressed, while mouse Cd3edg were knocked out in B-hCD3EDG/hCD20/hCD2 mice.

CD20

• The exons 2-7 of mouse Cd20 gene that encode the whole molecule (ATG to STOP codon) were replaced by human counterparts in B-hCD3EDG/hCD20/hCD2 mice.
• The promoter and 5’UTR and 3’UTR region of the mouse gene were retained. The human CD20 expression was driven by endogenous mouse Cd20 promoter, while mouse Cd20 gene transcription and translation will be disrupted.

CD2

• The exons 1-4 of mouse Cd2 gene that encode signal peptide and extracellular domain were replaced by human counterparts in B-hCD3EDG/hCD20/hCD2 mice.
• The genomic region of mouse Cd2 gene that encodes transmembrane domain and cytoplasmic portion was retained. The promoter, 5’UTR and 3’UTR region of the mouse gene were also retained. The chimeric CD2 expression was driven by endogenous mouse Cd2 promoter, while mouse Cd2 gene transcription and translation will be disrupted.

B-hCD3EDG/hCD20/hCD2 mice (113866) were obtained by mating B-hCD3EDG/hCD20 mice (121703) and B-hCD2 mice (111132).

• Mouse CD3E was detectable on T cells in spleen of wild-type C57BL/6JNifdc mice.
• Human CD3E was detectable on T cells in spleen of homozygous B-hCD3EDG/hCD20/hCD2 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific CD3E expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hCD20/hCD2 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/hCD20/hCD2 mice. Protein expression was analyzed with anti-mouse CD3E antibody (Biolegend, 100312) and anti-human CD3E antibody (BD, 562426) by flow cytometry.

• Mouse CD20 was detectable on B cells in spleen of wild-type C57BL/6JNifdc mice.
• Human CD20 was detectable on B cells in spleen of homozygous B-hCD3EDG/hCD20/hCD2 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific CD20 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hCD20/hCD2 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/hCD20/hCD2 mice. Protein expression was analyzed with anti-mouse CD20 antibody (Biolegend, 152108) and anti-human CD20 antibody (Biolegend, 302305) by flow cytometry.

• Mouse CD2 was detectable on T cells and B cells in spleen of wild-type C57BL/6JNifdc mice.
• Human CD2 was detectable on T cells and B cells in spleen of homozygous B-hCD3EDG/hCD20/hCD2 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific CD2 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hCD20/hCD2 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice and homozygous B-hCD3EDG/hCD20/hCD2 mice. Protein expression was analyzed with anti-mouse CD2 antibody (Biolegend, 100107) and anti-human CD2 antibody (Biolegend, 300213) by flow cytometry.

• No abnormalities were observed.

Average weight of the main organs of B-hCD3EDG/hCD20/hCD2 mice. Values are expressed as mean ± SD.

• No obvious abnormalities were observed in any organs examined (brain, heart, lung, liver, spleen, kidney, bone marrow, stomach, small intestine, colon, lymph node, uterus, and ovary).

Histopathological analysis of organs in B-hCD3EDG/hCD20/hCD2 mice. Major organs from B-hCD3EDG/hCD20/hCD2 mice were collected at 8 weeks of age and analyzed by H&E staining (male, n=3; female, n=3).

• No obvious abnormalities were observed in any organs examined (brain, heart, lung, liver, spleen, kidney, bone marrow, stomach, small intestine, colon, lymph node, and testis).

Histopathological analysis of organs in B-hCD3EDG/hCD20/hCD2 mice. Major organs from B-hCD3EDG/hCD20/hCD2 mice were collected at 8 weeks of age and analyzed by H&E staining (male, n=3; female, n=3).