CD3EDG/BAFFR: A T-cell engager antibody capable of depleting B cells

• Gene Information: CD3 consists of multiple genes (CD3D, CD3E, CD3G, CD3Z) on chromosomes 11 and 1, while TNFRSF13C (chromosome 22q13) encodes BAFFR, a critical tumor necrosis factor receptor superfamily member. 
• Protein Expression: CD3 protein expression is strictly restricted to the surface of mature T cells and thymocytes, whereas BAFFR is predominantly expressed on mature B lymphocytes, excluding plasma cells. 
• Signaling Pathway: Antigen binding to the TCR activates CD3-mediated ITAM phosphorylation to trigger downstream ZAP70 and calcium flux, while BAFF binding to BAFFR drives alternative NF-κB activation for cell survival. 
• Therapeutic Inhibition: Bispecific antibodies and T-cell engagers frequently target CD3 to redirect cytotoxicity against tumors, whereas blocking the BAFFR axis effectively treats autoimmune conditions like lupus by depleting pathogenic B cells.

B-hCD3EDG/hBAFFR mice (113941) were obtained by mating B-hCD3EDG mice (110039) and B-hBAFFR mice (111140).

CD3E

• The exons 2-8 of mouse Cd3e gene that encode the full-length protein were replaced by human counterparts in B-hCD3EDG/hBAFFR mice.
• The endogenous mouse promoter, 5′UTR, and 3′UTR regions were retained. Human CD3E expression to be driven by mouse Cd3e promoter, while mouse Cd3e transcription and translation will be disrupted.

CD3D

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

CD3G

• The exons 1-6 of Cd3g gene that encode the full-length protein were replaced by human counterparts in B-hCD3EDG/hBAFFR mice.
• The promoter, 5′UTR, and 3′UTR regions of the mouse gene were replaced by human counterparts. Human CD3G expression to be driven by human CD3G promoter, while mouse Cd3g transcription and translation will be disrupted.

BAFFR

• The exons 1-2 of mouse Baffr gene that encode extracellular domain and transmembrane domain were replaced by human counterparts in B-hCD3EDG/hBAFFR mice. The genomic region of mouse Baffr gene that encodes cytoplasmic portion was retained. The promoter, 5’UTR and 3’UTR region of the mouse gene were also retained. 
• The chimeric BAFFR expression was driven by endogenous mouse Baffr promoter, while mouse Baffr gene transcription and translation will be disrupted. B-hCD3EDG/hBAFFR mice (113941) were obtained by mating B-hCD3EDG mice (110039) and B-hBAFFR mice (111140).

• Mouse CD3E was only 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/hBAFFR mice, but not in wild-type C57BL/6JNifdc mice.

Mouse and human CD3E expression analysis. Splenocytes were collected from wild-type C57BL/6JNifdc mice  and homozygous B-hCD3EDG/hBAFFR mice. Protein expression was analyzed with anti-mouse CD3E antibody (Biolegend, 100312) and anti-human CD3E antibody (BD Horizon™, 562426) by flow cytometry.

• Mouse BAFFR was only detectable on B cells from spleen of wild-type C57BL/6JNifdc mice.
• Human BAFFR was exclusively detectable on B cells from spleen of  homozygous B-hCD3EDG/hBAFFR mice, but not in wild-type C57BL/6JNifdc mice.

Mouse and human BAFFR expression analysis. Splenocytes were collected from wild-type C57BL/6JNifdc mice  and homozygous B-hCD3EDG/hBAFFR mice. Protein expression was analyzed with anti-mouse BAFFR antibody (Biolegend, 134105) and anti-human BAFFR antibody (Biolegend, 316905) by flow cytometry.

• Anti-hCD3/BAFFR antibody Ab1 (provided by the client) can bind T cells and B cells of B-hCD3EDG/hBAFFR mice.

Anti-human CD3/BAFFR antibody binding assessment in B-hCD3EDG/hBAFFR mice. Blood was collected from homozygous B-hCD3EDG/hBAFFR mice and analyzed by flow cytometry with anti-hCD3/BAFFR antibody Ab1 (provided by the client).

• Anti–human CD3/BAFFR antibody Ab1 (provided by the client) can eliminate B cells in B-hCD3EDG/hBAFFR mice.

In vivo B cell depletion of anti-human CD3/BAFFR antibody  in homozygous B-hCD3EDG/hBAFFR mice. Homozygous B-hCD3EDG/hBAFFR mice (three per group) were intravenously injected with CD3/BAFFR antibody Ab1 (provided by the client) at doses of 0.5 mg/kg on Day 0 and 3 mg/kg on Day 6. Blood was collected at 2 days before treatment, and on Day1, Day5, Day8 and Day11. The cell count and the frequency of mCD45+ cells, B cells (mCD19+) and T cells (mTCRβ+) were determined by flow cytometry. (A) The cell count of CD45+ cells, B cells and T cells in blood. (B) The proportion of T and B cells in the mCD45+ cells. Values are expressed as mean ± SEM.