Background: 

• CD3 is a key component of the T cell receptor (TCR) complex, composed of γ, δ, ε, and ζ chains that form dimers (e.g., CD3γε, ζζ). Its intracellular domains contain ITAMs essential for signal transduction. When the TCR engages an MHC–peptide complex, CD3 transmits the signal into the cell, initiating cascades that drive T cell activation, proliferation, and effector functions. The CD8 antigen is a cell surface glycoprotein found on most cytotoxic T lymphocytes that mediates efficient cell-cell interactions within the immune system. 
• The CD8 antigen is a cell surface glycoprotein found on most cytotoxic T lymphocytes that mediates efficient cell-cell interactions within the immune system. The CD8 antigen acts as a coreceptor with the T-cell receptor on the T lymphocyte to recognize antigens displayed by an antigen presenting cell in the context of class I MHC molecules. The coreceptor functions as either a homodimer composed of two alpha chains or as a heterodimer composed of one alpha and one beta chain. Both alpha and beta chains share significant homology to immunoglobulin variable light chains.  

Targeting strategy:

• The exons 2-8 of mouse Cd3e gene that encode the full-length protein were replaced by human CD3E exons 2-9 in B-hCD3EDG/hCD8 mice. The exons 1-5 of mouse Cd3d and the exons 1-6 of Cd3g gene that encode the full-length protein were replaced by human CD3D exons 1-5 and CD3G exons 1-7 in B-hCD3EDG/hCD8 mice, respectively. 
• The exons 1-3 and partial exon 4 of mouse Cd8a gene that encode the extracellular domain were replaced by human CD8A exons 1-3 and partial exon 4 in B-hCD3EDG/hCD8 mice. The exons 1-3 and partial exon 4 of mouse Cd8b1 gene that encode the extracellular domain were replaced by human CD8B exons 1-3 and partial exon 4 in B-hCD3EDG/hCD8 mice.  

Validation:

• Mouse CD3E was detectable on T cells of wild-type C57BL/6JNifdc mice, but not on B-hCD3EDG/hCD8 mice. Human CD3E was detectable on T cells of homozygous B-hCD3EDG/hCD8 mice.
• Mouse CD8A was detectable on T, NKT, and TCRβ+CD4- T cells from the spleen and blood of C57BL/6JNifdc mice, but not on B-hCD3EDG/hCD8 mice. Human CD8A and CD8B were detectable on T, NKT, and TCRβ+CD4- T cells from the spleen and blood of homozygous B-hCD3EDG/hCD8 mice.
• Humanization of CD3E,CD3D, CD3G and CD8 does not change the overall frequency or distribution of immune cell types in the spleen, blood, lymph nodes and thymus.

Application: This product is used for the pharmacological and safety evalsuation of hCD3EDG/hCD8 bispecific antibodies.

CD3EDG

• The chimeric human CD3EDG was expressed, while mouse Cd3edg were knocked out in B-hCD3EDG/hCD8 mice.

CD8

• The exons 1-3 and partial exon 4 of mouse Cd8a gene that encode signal peptide and extracellular domain are replaced by human counterparts in B-hCD3EDG/hCD8 mice. The genomic region of mouse Cd8a gene that encodes transmembrane domain and cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric CD8A expression is driven by endogenous mouse CD8a promoter, while mouse Cd8a gene transcription and translation will be disrupted.
• The exons 1-3 and partial exon 4 of mouse Cd8b1 gene that encode signal peptide and extracellular domain are replaced by human counterparts in B-hCD3EDG/hCD8 mice. The genomic region of mouse Cd8b1 gene that encodes transmembrane domain and cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric CD8B1 expression is driven by endogenous mouse CD8b1 promoter, while mouse Cd8b1 gene transcription and translation will be disrupted.

• Mouse CD3E was detected exclusively in wild-type C57BL/6JNifdc mice , but not in B-hCD3EDG/hCD8 mice.
• Human CD3E was detected in homozygous B-hCD3EDG/hCD8 mice.

Strain specific CD3E expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and analyzed by flow cytometry with species-specific anti-mouse CD3ε antibody (Biolegend, 100312) and anti-human CD3ε antibody (BD Pharmingen, 562426). Mouse CD3E was detectable in wild-type mice, but not in B-hCD3EDG/hCD8 mice. Human CD3E was exclusively detectable in homozygous B-hCD3EDG/hCD8 mice.

• Mouse CD3E was detected exclusively in wild-type C57BL/6JNifdc mice , but not in B-hCD3EDG/hCD8 mice.
• Human CD3E was detected in homozygous B-hCD3EDG/hCD8 mice.

Strain specific CD3E expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Blood were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and analyzed by flow cytometry with species-specific anti-mouse CD3ε antibody (Biolegend, 100312) and anti-human CD3ε antibody (BD Pharmingen, 562426). Mouse CD3E was detectable in wild-type mice, but not in B-hCD3EDG/hCD8 mice. Human CD3E was exclusively detectable in homozygous B-hCD3EDG/hCD8 mice.

• Mouse CD8A was detected on TCRβ+ T cells of wild-type C57BL/6JNifdc mice, but not on B-hCD3EDG/hCD8 mice.
• Human CD8A and CD8B was detected on  TCRβ+ T cells of in B-hCD3EDG/hCD8 mice.

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and then analyzed by flow cytometry with species-specific anti-CD8 antibodies (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392). Mouse CD8A was detectable in spleen T cells of wild-type mice, but not in the B-hCD3EDG/hCD8 mice. Human CD8A and CD8B were exclusively detectable in spleen  TCRβ+ T cells of B-hCD3EDG/hCD8 mice.

• Mouse CD8A was detected on TCRβ+CD4- T cells of wild-type C57BL/6JNifdc mice, but not on B-hCD3EDG/hCD8 mice.
• Human CD8A and CD8B was detected on  TCRβ+CD4-  T cells of in B-hCD3EDG/hCD8 mice.

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and then analyzed by flow cytometry with species-specific anti-CD8 antibodies (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392). Mouse CD8A was detectable in spleen T cells of wild-type mice, but not in the B-hCD3EDG/hCD8 mice. Human CD8A and CD8B were exclusively detectable in spleen TCRβ+CD4- T cells of B-hCD3EDG/hCD8 mice.

• Mouse CD8A was detected on NKT cells of wild-type C57BL/6JNifdc mice, but not on B-hCD3EDG/hCD8 mice.
• Human CD8A and CD8B was detected on  NKT cells of B-hCD3EDG/hCD8 mice.

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and then analyzed by flow cytometry with species-specific anti-CD8 antibodies (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392). Mouse CD8A was detectable in spleen T cells of wild-type mice, but not in the B-hCD3EDG/hCD8 mice. Human CD8A and CD8B were exclusively detectable in spleen NKT cells of B-hCD3EDG/hCD8 mice.

• Mouse CD8A was detected on TCRβ+ T cells of wild-type C57BL/6JNifdc mice, but not on B-hCD3EDG/hCD8 mice.
• Human CD8A and CD8B was detected on  TCRβ+ T cells of B-hCD3EDG/hCD8 mice.

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and then analyzed by flow cytometry with species-specific anti-CD8 antibodies (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392). Mouse CD8A was detectable in spleen T cells of wild-type mice, but not in the B-hCD3EDG/hCD8 mice. Human CD8A and CD8B were exclusively detectable in blood TCRβ+ T cells of B-hCD3EDG/hCD8 mice.

• Mouse CD8A was detected on TCRβ+CD4- T cells of wild-type C57BL/6JNifdc mice, but not on B-hCD3EDG/hCD8 mice.
• Human CD8A and CD8B was detected on  TCRβ+CD4- T cells of B-hCD3EDG/hCD8 mice.

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and then analyzed by flow cytometry with species-specific anti-CD8 antibodies (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392). Mouse CD8A was detectable in spleen T cells of wild-type mice, but not in the B-hCD3EDG/hCD8 mice. Human CD8A and CD8B were exclusively detectable in blood TCRβ+CD4- T cells of B-hCD3EDG/hCD8 mice.

• Mouse CD8A was detected on NKT cells of wild-type C57BL/6JNifdc mice, but not on B-hCD3EDG/hCD8 mice.
• Human CD8A and CD8B was detected on  NKT cells of B-hCD3EDG/hCD8 mice.

Strain specific CD8 expression analysis in homozygous B-hCD3EDG/hCD8 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc mice (WT) and homozygous B-hCD3EDG/hCD8 mice (HO), and then analyzed by flow cytometry with species-specific anti-CD8 antibodies (anti-human CD8A, Biolegend, 300908; anti-mouse CD8A, Biolegend, 100708; anti-human CD8B, BD, 742392). Mouse CD8A was detectable in spleen T cells of wild-type mice, but not in the B-hCD3EDG/hCD8 mice. Human CD8A and CD8B were exclusively detectable in blood NKT cells of B-hCD3EDG/hCD8 mice.

• The frequencies of T cells, B cells, NK cells, DCs, neutrophils, monocytes, and macrophages in homozygous B-hCD3EDG/hCD8 mice were similar to those in C57BL/6JNifdc mice
• Humanization of CD3EDG and CD8 does not affect normal immune cell development or splenic distribution.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, lymph nodes, and thymus were isolated from C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (male, 9-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• The proportions of CD4⁺ T cells, CD8⁺ T cells, and Tregs in homozygous B-hCD3EDG/hCD8 mice were comparable to those in C57BL/6JNifdc mice
• Humanization of CD3EDG and CD8 does not affect normal T cell development, differentiation, or splenic distribution.

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, lymph nodes, and thymus were isolated from C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (male, 9-week-old, n = 3). Single live cells were gated on the TCRβ⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• B-hCD3EDG/hCD8 mice have normal T cell immunogenic function induced by the OVA.

Detection of OVA-induced immune responses in B-hCD3EDG/hCD8 mice by IFN-γ ELISpot assay. Male wild-type C57BL/6JNifdc mice and B-hCD3EDG/hCD8 mice at the age of 9–10 weeks were immunized with intraperitoneal injection of 0.3 mg of OVA protein (Simga, A5503-25MG) and 30 μg poly (I:C) (InvivoGen, tlrl-pic). Mice were immunized with OVA two times at 1-week interval. One week after the last immunization, mice were sacrificed. The splenocytes were extracted, stimulated with OVA peptide257–264, or Cell Activation Cocktail (without Brefeldin A), (BioLegend, 42330) as positive control (PC), and then measured for IFN-γ secretion. (A) Representative results showing stimulation of splenocytes harvested from immunized mice with OVA peptide257–264, or positive control in duplicates. (B) Summary of results. These data indicate that B-hCD3EDG/hCD8 mice have normal T cell immunogenic function.

• Analysis of T cell Activation Stimulated with Anti-CD3ε Antibody in vitro.

CD25 and CD69 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hCD8 mice by flow cytometry. T cells were isolated from splenocytes of C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (male, 9-week-old, n=3), and were incubated in the presence of anti-mouse CD3ε antibody (2 ug/ml, BioXcell, BE0001-2), or anti-human CD3ε antibody (2 ug/ml, BioXcell, BE0231) with or without anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 24h. T cell proliferation was tested by flow cytometry. T cell activation in B-hCD3EDG/hCD8 mice was significantly up-regulated by anti-hCD3ε antibody and anti-mCD28 antibody.

• Analysis of T cell Activation Stimulated with Anti-CD3ε Antibody in vitro.

CD25 and CD69 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hCD8 mice by flow cytometry. T cells were isolated from splenocytes of C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (male, 9-week-old, n=3), and were incubated in the presence of anti-mouse CD3ε antibody (2 ug/ml, BioXcell, BE0001-2), or anti-human CD3ε antibody (2 ug/ml, BioXcell, BE0231) with or without anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 48h. T cell proliferation was tested by flow cytometry. T cell activation in B-hCD3EDG/hCD8 mice was significantly up-regulated by anti-hCD3ε antibody and anti-mCD28 antibody.

• Analysis of T cell Activation Stimulated with Anti-CD3ε Antibody in vitro.

CD25 and CD69 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hCD8 mice by flow cytometry. T cells were isolated from splenocytes of C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (male, 9 week-old, n=3), and were incubated in the presence of anti-mouse CD3ε antibody (2 ug/ml, BioXcell, BE0001-2), or anti-human CD3ε antibody (2 ug/ml, BioXcell, BE0231) with or without anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 72h. T cell proliferation was tested by flow cytometry. T cell activation in B-hCD3EDG/hCD8 mice was significantly up-regulated by anti-hCD3ε antibody and anti-mCD28 antibody.

• Analysis of T cell Activation Stimulated with Anti-CD3ε Antibody in vitro.

The proliferation of CD4+ T cells and CD8+ T cells was analyzed by flow cytometry. T cells were isolated from splenocytes of C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (female, 13 week-old, n=3), and were incubated in the presence of anti-mouse CD3ε antibody (2 ug/ml, BioXcell, BE0001-2), or anti-human CD3ε antibody (2 ug/ml, BioXcell, BE0231) with or without anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 24h. T cell proliferation was tested by flow cytometry.

• Analysis of T cell Activation Stimulated with Anti-CD3ε Antibody in vitro.

The proliferation of CD4+ T cells and CD8+ T cells was analyzed by flow cytometry. T cells were isolated from splenocytes of C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (female, 13 week-old, n=3), and were incubated in the presence of anti-mouse CD3ε antibody (2 ug/ml, BioXcell, BE0001-2), or anti-human CD3ε antibody (2 ug/ml, BioXcell, BE0231) with or without anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 48h. T cell proliferation was tested by flow cytometry.

• Analysis of T cell Activation Stimulated with Anti-CD3ε Antibody in vitro.

The proliferation of CD4+ T cells and CD8+ T cells was analyzed by flow cytometry. T cells were isolated from splenocytes of C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (female, 13 week-old, n=3), and were incubated in the presence of anti-mouse CD3ε antibody (2 ug/ml, BioXcell, BE0001-2), or anti-human CD3ε antibody (2 ug/ml, BioXcell, BE0231) with or without anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 72h. T cell proliferation was tested by flow cytometry.

• Analysis of T cell Activation Stimulated with Anti-CD3ε Antibody in vitro.

In vitro cytokine production (IFN-γ and IL-2) in B-hCD3EDG/hCD8 mice. T cells (2×10⁵) were isolated from the splenocytes of C57BL/6JNifdc and B-hCD3EDG/hCD8 mice (male, 9-week-old, n=3), incubated in the presence of anti-mCD3ε antibody (2 ug/ml
，，，BioXcell，，，，BE0001-2) or anti-hCD3ε antibody (2 ug/ml
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，BE0231) with or without anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 24h, 48h and 72h. IFN-γ and IL-2 productions were then tested using ELISA method.