CD3EDG: Signaling in T Cells for Mechanism and Therapeutic Potential

• Gene Information: The CD3E, CD3D, and CD3G genes, located in a cluster on chromosome 11q23, encode the three invariant polypeptide chains that assemble into the CD3εδ and CD3εγ heterodimers of the T-cell receptor (TCR) complex.
• Protein Expression: These proteins are exclusively and constitutively expressed on the surface of mature T lymphocytes and thymocytes, serving as definitive lineage markers for the T-cell population.
• Signaling Pathway: Upon TCR engagement by an antigen-MHC complex, the CD3 chains transduce activation signals through their cytoplasmic Immunoreceptor Tyrosine-based Activation Motifs (ITAMs), which recruit ZAP-70 to trigger downstream MAPK, NF-κB, and calcium signaling pathways.
• Therapeutic Inhibition: Therapeutic strategies utilize monoclonal antibodies or bispecific T-cell engagers (TCEs) to either modulate TCR signaling for immunosuppression in transplantation or to bypass MHC restriction by directly linking T cells to tumor cells for targeted cytotoxicity.

CD3EDG

• The chimeric human CD3EDG was expressed, while mouse Cd3edg were knocked out in B-hCD3EDG/HLA-A11.1 plus mice.

HLA-A11.1

• The B2M gene (Exon1 to Exon3) of mouse were replaced by the sequence encompassing the human B2M CDS and HLA-A*1101 gene that included leader sequence, α1 and α2 domains ligated to a fragment of the murine H-2D^b gene containing the α3, transmembrane and cytoplasmic domains in B-hCD3EDG/HLA-A11.1 plus mice. The mouse B2M gene is knocked into exon 2 of the mouse Fcgrt gene and is fused via a linker to the remaining portion of exon 2, a strategy that enables the co-expression of mouse B2M and Fcgrt in B-hCD3EDG/HLA-A11.1 plus mice.

• Mouse  B2M was detected in wild-type (WT) C57BL/6JNifdc mice and B-hCD3EDG/HLA-A11.1 plus mice because the mouse B2M gene is knocked into exon 2 of the mouse Fcgrt gene and is fused via a linker to the remaining portion of exon 2, a strategy that enables the co-expression of mouse B2M and Fcgrt in B-hCD3EDG/HLA-A11.1 plus mice.
• Human B2M was exclusively detected in homozygous (HO) B-hCD3EDG/HLA-A11.1 plus mice.

Mouse and human B2M expression analysis in splenocytes and blood. Splenocytes and blood were collected from wild-type (WT) C57BL/6JNifdc mice, homozygous (HO) B-hCD3EDG/HLA-A11.1 plus mice. B2M expression was analyzed by flow cytometry using species-specific anti-mouse B2M antibody (BD Biosciences, 744802) and anti-human TNFR2 antibody (Biolegend, 395712).

• Mouse H-2D^b was detected in wild-type (WT) C57BL/6JNifdc mice, but not in B-hCD3EDG/HLA-A11.1 plus mice.
• Human HLA-A was exclusively detected in homozygous (HO) B-hCD3EDG/HLA-A11.1 plus Mice.

Mouse and human HLA-A expression analysis in splenocytes. Splenocytes and blood were collected from wild-type (WT) C57BL/6JNifdc mice, homozygous (HO) B-hCD3EDG/HLA-A11.1 plus Mice. HLA-A expression was analyzed by flow cytometry using species-specific anti-H-2D^b antibody (Biolegend, 111513) and anti-HLA antibody (Biolegend, 311406).

Mouse and human CD3E expression analysis in splenocytes and blood. Splenocytes and blood cells were collected from wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-hCD3EDG/HLA-A11.1 plus mice (male, 9-week-old, n=3). Protein expression was analyzed with anti-mouse CD3ε antibody (Biolegend, 100312) and anti-humanCD3 antibody (BD, 556612) by flow cytometry.

• The percentages of T cells, B cells, NK cells, DCs, neutrophils, monocytes, and macrophages in homozygous B-hCD3EDG/HLA-A11.1 plus mice were similar to those in C57BL/6JNifdc mice.
• Humanization of CD3EDG, and HLA-A 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 mice and B-hCD3EDG/HLA-A11.1 plus mice female, 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/HLA-A11.1 plus mice were comparable to those in C57BL/6JNifdc  mice.
• Humanization of CD3EDG, and HLA-A 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 mice and B-hCD3EDG/HLA-A11.1 plus mice (female, 9-week-old, n = 3). Single live cells were gated on the CD3⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

Establishment of a B-HLA-A11.1/hKRAS*G12V MC38 model and in vivo efficacy study of an Ab1-TCE. B-HLA-A11.1/hKRAS*G12V MC38 cells were implanted subcutaneously into homozygous B-hCD3EDG/HLA-A11.1 plus mice (female, 12-weeks-old, n=6).

Antitumor activity of Ab1-TCE against syngeneic tumors. (A) Tumor growth curves. (B) Body weight changes during treatment. (C) Tumor cells growth of individual mouse. As shown in panel A, HLA-A11.1/hKRASG12 bispecific antibodies (provided by the client) was efficacious in controlling tumor growth in B-hCD3EDG/HLA-A11.1 plus mice. These results demonstrate that B-hCD3EDG/HLA-A11.1 plus mice provide a powerful preclinical model for in vivo evaluation of HLA-A11.1/hKRASG12V targeted TCR mimic antibodies. Values are expressed as mean ± SEM. The overage of this tumor model is 40%.