• CD3EDG consists of the CD3ε, CD3δ, and CD3γ chains of the TCR/CD3 complex, which transduces antigen-recognition signals and mediates T cell activation.
• CD19 is a B cell lineage marker and co-receptor widely used in B cell malignancy, autoimmune disease, CAR-T, and T cell engager therapeutic research.
• BCMA (TNFRSF17, CD269) is expressed in plasma cells and multiple myeloma cells, making BCMA a major target for antibody, CAR-T, and bispecific antibody development.
• B-hCD3EDG/hCD19/hBCMA mice are triple-target humanized mice carrying humanized CD3E/CD3D/CD3G, human CD19, and human BCMA targets in a C57BL/6 background.
• This model supports preclinical evaluation of CD3-based T cell engagers, CD19-targeted therapeutics, BCMA-targeted therapeutics, and multi-target B cell or plasma cell immunotherapies.

Key Advantages

• Human CD3E, CD19, and BCMA target expression supports multi-target immunotherapy evaluation
• Characterized immune-cell profiles and target expression enable CD3-, CD19-, and BCMA-directed therapeutic studies
• Useful for CD3 T cell engager, CD19 bispecific, BCMA bispecific, and B cell/plasma cell immunotherapy research

Validation

• Molecular Validation: Human BCMA mRNA was detected in spleen from B-hCD3EDG/hCD19/hBCMA mice by RT-PCR, while mouse Bcma mRNA was detected in wild-type C57BL/6 mice.
• CD3E Protein Validation: Human CD3E was detected on T cells from spleen and blood of B-hCD3EDG/hCD19/hBCMA mice by flow cytometry.
• CD19 Protein Validation: Human CD19 was detected on B cells from spleen and blood of B-hCD3EDG/hCD19/hBCMA mice by flow cytometry.
• Phenotypic Validation: Leukocyte and T cell subpopulation frequencies in spleen, blood, and lymph nodes were comparable between B-hCD3EDG/hCD19/hBCMA mice and wild-type C57BL/6 mice.

Application

• CD3 T cell engager efficacy and pharmacology studies
• CD19-targeted bispecific antibody and B cell therapy studies
• BCMA-targeted antibody, CAR-T, and bispecific antibody research
• Multi-target CD3/CD19/BCMA immunotherapy evaluation
• B cell, plasma cell, and multiple myeloma therapeutic development
• Immune-cell profiling and translational immuno-oncology research

B-hCD3EDG/hCD19/hBCMA mice combine humanized CD3EDG, CD19, and BCMA targets in a C57BL/6 background. The model was generated by introducing human CD3E/CD3D/CD3G sequences, human CD19 sequence, and human TNFRSF17/BCMA sequence into the corresponding mouse loci.
This targeting design enables species-specific detection of human CD3E on T cells, human CD19 on B cells, and human BCMA in spleen while preserving an immunocompetent mouse background for in vivo pharmacology. B-hCD3EDG/hCD19/hBCMA mice provide a translational platform for evaluating CD3-engaging CD19- or BCMA-targeted immunotherapies.

Strain-specific analysis of BCMA mRNA expression was performed in wild-type C57BL/6 mice and homozygous B-hCD3EDG/hCD19/hBCMA mice by RT-PCR.
Spleen RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hCD3EDG/hCD19/hBCMA mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse and human primers. Mouse Bcma mRNA were detectable in wild-type mice. Human BCMA mRNA were detectable in B-hCD3EDG/hCD19/hBCMA mice but not in wild-type mice.

CD3E protein expression was analyzed in wild-type C57BL/6 mice and homozygous B-hCD3EDG/hCD19/hBCMA mice by flow cytometry.
Splenocytes (A) and blood cells (B) were collected from wild-type C57BL/6 mice and homozygous B-hCD3EDG/hCD19/hBCMA mice (female, 8-week-old, n=1). 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 only detectable in wild-type C57BL/6 mice. Human CD3E was exclusively detectable in homozygous B-hCD3EDG/hCD19/hBCMA mice, but not in wild-type C57BL/6 mice.

CD19 protein expression was analyzed in wild-type C57BL/6 mice and homozygous B-hCD3EDG/hCD19/hBCMA mice by flow cytometry.
Splenocytes (A) and blood cells (B) were collected from wild-type C57BL/6 mice and homozygous B-hCD3EDG/hCD19/hBCMA mice (female, 8-week-old, n=1). Protein expression was analyzed with anti-mouse CD19 antibody (Biolegend, 115507) and anti-human CD19 antibody (Biolegend, 392503) by flow cytometry. Mouse CD19 was only detectable in wild-type C57BL/6 mice. Human CD19 was exclusively detectable in homozygous B-hCD3EDG/hCD19/hBCMA mice, but not in wild-type C57BL/6 mice.

Frequency of leukocyte subpopulations in spleen was analyzed by flow cytometry.
Splenocytes were isolated from wild-type C57BL/6 mice (female, n=3, 8-week-old) and homozygous B-hCD3EDG/hCD19/hBCMA 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, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hCD3EDG/hCD19/hBCMA mice were similar to those in wild-type C57BL/6 mice. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in blood was analyzed by flow cytometry.
Blood cells were isolated from wild-type C57BL/6 mice (female, n=3, 8-week-old) and homozygous B-hCD3EDG/hCD19/hBCMA mice (female, n=3, 8-week-old). A. Flow cytometry analysis of the blood 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, CD4+ T cells, CD8+ T cells and Tregs in B-hCD3EDG/hCD19/hBCMA mice were similar to those in wild-type C57BL/6 mice. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in lymph nodes was analyzed by flow cytometry.
Lymph nodes cells were isolated from wild-type C57BL/6 mice (female, n=3, 8-week-old) and homozygous B-hCD3EDG/hCD19/hBCMA mice (female, n=3, 8-week-old). A. Flow cytometry analysis of the Lymph nodes cells was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells and Tregs in B-hCD3EDG/hCD19/hBCMA mice were similar to those in wild-type C57BL/6 mice. Values are expressed as mean ± SEM.

Q1: What are B-hCD3EDG/hCD19/hBCMA mice?

B-hCD3EDG/hCD19/hBCMA mice are triple gene-humanized mice carrying humanized CD3EDG, human CD19, and human BCMA targets in a C57BL/6 background.

Q2: Why are CD3EDG, CD19, and BCMA important therapeutic targets?

CD3EDG enables T cell engagement through the TCR/CD3 complex, CD19 is a B cell target, and BCMA is a plasma cell and multiple myeloma target used in antibody, CAR-T, and bispecific drug development.

Q3: How was target expression validated in this model?

Human BCMA mRNA was validated in spleen by RT-PCR, while human CD3E and human CD19 protein expression were validated in spleen and blood by flow cytometry.

Q4: Can B-hCD3EDG/hCD19/hBCMA mice be used for immune profiling?

Yes. Spleen, blood, and lymph node leukocyte and T cell subpopulations were characterized by flow cytometry and compared with wild-type C57BL/6 mice.

Q5: What are the main applications of this model?

Applications include CD3 T cell engager studies, CD19-targeted B cell therapy research, BCMA-targeted multiple myeloma therapy studies, bispecific antibody evaluation, and translational immuno-oncology research.