CD3: An Important Therapeutic Target in Immunotherapy

• Gene Information: CD3 is a protein complex that forms part of the T-cell receptor (TCR) signaling machinery. It consists of four chains encoded by CD3D, CD3E, CD3G, and CD247. Together with the TCR, CD3 transmits antigen recognition signals from the cell surface to the cell interior and is essential for T-cell development and activation.
• Protein Expression: CD3 is expressed on nearly all mature T cells, including CD4⁺ T cells, CD8⁺ T cells, and γδ T cells. It is generally absent from B cells, NK cells, and myeloid cells.
• Signaling Pathway: CD3 mediates TCR signaling through intracellular ITAM motifs. After antigen recognition, CD3 recruits signaling molecules such as ZAP-70, leading to activation of the NFAT, NF-κB, and MAPK/AP-1 pathways.
• Therapeutic Inhibition: CD3 is an important therapeutic target for modulating T-cell activity. Anti-CD3 antibodies, such as Teplizumab, can suppress or regulate T-cell responses and are used in autoimmune diseases. CD3 is also widely used in bispecific T-cell engagers, which redirect T cells to kill tumor cells, as seen with Blinatumomab.

Mouse and human CD3E, CD3D, CD3G analysis in thymus of CD3EDG humanized mice by RT-PCR and sequencing. Total RNA was isolated from thymus tissues of wild-type C57BL/6 mice (+/+) and homozygous CD3EDG humanized mice (H/H). cDNA was synthesized by reverse transcription, followed by PCR amplification using mouse- or human-specific primers targeting CD3E, CD3D, and CD3G.

Mouse and human CD3E expression analysis in splenocytes of CD3EDG humanized mice. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous CD3EDG humanized mice (H/H). Mouse and human CD3E protein expression was analyzed by flow cytometry using species-specific anti-mouse CD3E and anti-human CD3E antibodies.

In vitro T-cell activation analysis in CD3EDG humanized mice. T cells (2×10⁵) were isolated from splenocytes of wild-type C57BL/6 mice and CD3EDG humanized mice (16 weeks old, n = 3). T cells were incubated with anti-mouse CD3ε antibody (Bio X Cell, BE0001-1, clone 145-2C11, 2 μg/mL), anti-human CD3ε antibody, and anti-mouse CD28 antibody for 48 hours. T-cell activation was tested by flow cytometry.

In vitro T-cell activation analysis in CD3EDG humanized mice. T cells (2×10⁵) were isolated from splenocytes of wild-type C57BL/6 mice and CD3EDG humanized mice (16 weeks old, n = 3). T cells were incubated with anti-mouse CD3ε antibody, anti-human CD3ε antibody, and anti-mouse CD28 antibody for 24, 48, and 72 hours. IFN-γ and IL-2 production was subsequently measured by ELISA. Values are expressed as mean ± SEM. ND: not detectable.

In vitro cytotoxicity evaluation of an anti-CD3/BCMA bispecific antibody against human BCMA-expressing MC38 cells using CD3EDG humanized mice. Splenocytes isolated from CD3EDG humanized mice were mixed with human BCMA-expressing MC38 cells (B-hBCMA MC38) in the presence of various concentrations of client-provided CD3/BCMA bispecific antibodies. The killing activity was detected after 48 hours. When the ratio of effector cells to target cells  (E:T) were 20:1 and 40:1, the killing effect of the test sample was detected.

Serum titers of OVA-specific antibodies in CD3EDG humanized mice. CD3EDG humanized mice (6 weeks old, n = 5) were immunized three times with ovalbumin (OVA) at 2-week apart. Blood samples were collected one week after immunization. (A) Quantification of serum antibody isotypes before immunization. (B) Measurement of OVA-specific serum antibody titers after the second and third immunizations.

Flow cytometry analysis of leukocyte subpopulations in immune organs of CD3EDG humanized mice. Splenocytes, lymph nodes, and thymus were collected from wild-type C57BL/6 mice and CD3EDG humanized mice (7 weeks 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.

Flow cytometry analysis of T-cell subpopulations in immune organs of CD3EDG humanized mice. Splenocytes, lymph nodes, and thymus were collected from wild-type C57BL/6 mice and CD3EDG humanized mice (7 weeks old, n = 3). Single live cells were gated on the mouse TCRβ⁺ (mTCRβ⁺) T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

(A) Spleen size in wild-type C57BL/6 and CD3EDG humanized mice (7 weeks old, n = 3). (B) Spleen weights were similar between wild-type C57BL/6 and CD3EDG humanized mice. (C) Total splenocyte counts were comparable between the two groups.

(A) Thymus size in wild-type C57BL/6 and CD3EDG humanized mice (7 weeks old, n = 3). (B) Thymus weights were lower in CD3EDG humanized mice than in wild-type C57BL/6 mice. (C) Total thymocyte counts were lower in CD3EDG humanized mice than in wild-type C57BL/6 mice. (D) Histopathological analysis of the thymus in CD3EDG humanized mice (female, 8 weeks old, n = 3) revealed no significant abnormalities, with clear boundaries between the cortex and medulla and normal cellular morphology.

Growth curve of CD3EDG humanized mice. CD3EDG humanized mice were weaned at 3 weeks of age and grouped by sex (20 males and 20 females). Body weight was measured weekly on the same day for 15 consecutive weeks. The minimum and maximum body weights presented in the table were calculated as mean ± SD.

Complete blood count (CBC) of CD3EDG humanized mice. Values are expressed as mean ± SEM.

Blood biochemical parameters of CD3EDG humanized mice are shown. Values are expressed as mean ± SEM.

Establishment of a MC38 model and in vivo efficacy study of anti-hCD3ε antibody and anti-mPD-1 antibody in CD3EDG humanized mice. Murine colon cancer MC38 cells were implanted subcutaneously into homozygous CD3EDG humanized mice (female, 8 weeks old, n = 5). When the average tumor volume reached approximately 100±50 mm³, mice were randomized and subsequently administered with anti-mPD-1 antibody or anti-CD3ε antibody via intraperitoneal injection.

Efficacy of anti-hCD3ε antibody and anti-mPD-1 antibody in CD3EDG humanized mice. (A) Tumor growth curves. (B) Body weight changes during treatment. As shown in panel A, anti-mPD-1 antibody significantly inhibited tumor growth in CD3EDG humanized mice, indicating that their T cells function normally.  However, the tumor growth was faster after anti-hCD3ε antibody treatment, which may be caused by activation induced cell death (AICD). Values are expressed as mean ± SEM.

The overage of this tumor model is 60%.

T cells and B cells activation analysis in CD3EDG humanized mice. Lymphocytes were isolated from peripheral blood and tumor at the endpoint of the in vivo experiment. The proportions of T cells and B cells in total CD45+ cells were shown. In the anti-hCD3ε antibody treatment group, the proportion of T cells was significantly decreased due to the activation induced cell death (AICD) effect caused by anti-hCD3ε antibody treatment.

Establishment of a human CD20-expressing MC38 model and in vivo efficacy study of Glofitamab in CD3EDG humanized mice. Human CD20-expressing MC38 mouse colon carcinoma cells (B-hCD20 MC38) were subcutaneously implanted into homozygous CD3EDG humanized mice (female, 7 weeks old, n = 6). Mice were grouped once tumor volume reached approximately 100 mm³, at which time they were injected intravenously with anti-human CD3/CD20 bispecific antibodies (Glofitamab analog, Commercial).

Efficacy of anti-human CD3/CD20 bispecific antibodies (Glofitamab, Commercial) in CD3EDG humanized mice. (A) Tumor growth curves. (B) Body weight changes during treatment. As shown in panel A, anti-human CD3/CD20 bispecific antibodies inhibited B-hCD20 MC38 tumor growth in CD3EDG humanized mice. Values are expressed as mean ± SEM.

The overage of this tumor model is 60%.

Establishment of a B-hDLL3 B16-F10 model and in vivo efficacy study of Tarlatamab in CD3EDG humanized mice. B-hDLL3 B16-F10 mouse melanoma cells were subcutaneously implanted into homozygous CD3EDG humanized mice (female, 7–9-week-old, n=6). Mice were grouped when tumor volume reached approximately 100 ± 20 mm³, at which time they were injected intraperitoneally with anti-human CD3/DLL3 bispecific antibodies (Tarlatamab analog, in-house).

Efficacy of anti-human CD3/DLL3 bispecific antibodies (Tarlatamab, in-house) in CD3EDG humanized mice. (A) Tumor growth curves. (B) Body weight changes during treatment. As shown in panel A, Anti-human CD3/DLL3 bispecific antibodies inhibited B-hDLL3 B16-F10 tumor growth in CD3EDG humanized mice. Values are expressed as mean ± SEM.

The overage of this tumor model is 100%.

Establishment of a B-hSTEAP1 MC38 model and in vivo efficacy study of Xaluritamig in CD3EDG humanized mice. B-hSTEAP1 MC38 mouse colon carcinoma cells were subcutaneously implanted into homozygous CD3EDG humanized mice (female, 10-week-old, n=6). Mice were grouped once tumor volume reached approximately 100 mm³, at which time they were injected intraperitoneally with anti-human CD3/STEAP1 bispecific antibodies (Xaluritamig analog, Commercial).

Efficacy of anti-human CD3/STEAP1 bispecific antibodies (Xaluritamig, Commercial) in CD3EDG humanized mice. (A) Tumor growth curves. (B) Body weight changes during treatment. As shown in panel A, anti-human CD3/STEAP1 bispecific antibodies inhibited B-hSTEAP1 MC38 tumor growth in CD3EDG humanized mice. Values are expressed as mean ± SEM.

The overage of this tumor model is 60%.

Q1: What are B-hCD3EDG mice?

A1: B-hCD3EDG mice are humanized models that express human CD3E, CD3D, and CD3G proteins. They provide a more predictive in vivo system for evaluating therapies targeting human CD3, which is critical for T cell activation and immunotherapy research.

Q2: Why use B-hCD3EDG mice instead of wild-type mice?

A2: Wild-type mice express murine CD3 chains that human antibodies may not recognize. B-hCD3EDG mice allow reliable testing of monoclonal antibodies, bispecifics, and T cell engagers directed at human CD3.

Q3: Do B-hCD3EDG mice have normal immune function?

A3: Yes. They maintain normal T, B, and NK cell development, with comparable immune cell distribution and T cell activation responses to wild-type mice.

Q4: What research areas benefit from B-hCD3EDG mice?

A4: These mice are valuable for immuno-oncology, antibody validation, T cell activation studies, and preclinical safety testing of CD3-directed therapies.