CD3 and 4-1BB: A New Trends of T cell engager therapeutic

• Gene Information: CD3: Encoded by CD3E, D, G; part of the Ig superfamily. It forms the essential signaling backbone of the T-cell receptor (TCR) complex. 4-1BB: Encoded by TNFRSF9; a member of the TNF receptor superfamily. It acts as a potent inducible costimulatory gene.
• Protein Expression: CD3: Constitutive and universal marker for all mature T cells (CD4+, CD8+). Always present on the cell surface. 4-1BB: Inducible; absent on resting cells. It is expressed only after T-cell activation, marking tumor-reactive or “effector” lymphocytes.
• Signaling Pathway: CD3 (Signal 1): Operates via ITAM phosphorylation and ZAP-70. It triggers the initial "on" switch, Ca2+ flux, and immediate cytotoxicity. 4-1BB (Signal 2): Operates via TRAF1/2 and NF-κB. It drives metabolic fitness, prevents apoptosis (Bcl-xL), and sustains long-term T-cell memory.
• Therapeutic Inhibition: Uses bispecifics (BiTEs) to force T-cell killing of tumor cells regardless of MHC restriction; Cytokine Release Syndrome (CRS) is the primary safety concern. Focuses on boosting T-cell persistence; modern multi-specific formats (CD3×41BB ×TAA) localize activation to the tumor microenvironment.

• Mouse CD3E was detected on T cells populations in wild-type C57BL/6 mice, but not in B-hCD3EDG/h4-1BB mice.
• Human CD3E was detected on T cells populations in B-hCD3EDG/h4-1BB mice, but not in wild-type C57BL/6 mice.

Mouse and human CD3E expression analysis in splenocytes. Splenocytes were collected from wild-type C57BL/6 mice (male, 6-week-old, n = 1) and homozygous B-hCD3EDG/h4-1BB mice (male, 6-week-old, n = 1). CD3E expression on T cells was analyzed by flow cytometry using species-specific anti-CD3E antibodies (anti-human CD3E antibody, BD Horizon, 562426; anti-mouse CD3E antibody, Biolegend, 100312 ).

• Mouse 4-1BB was detected on T cells populations in wild-type C57BL/6 mice, but not in B-hCD3EDG/h4-1BB mice.
• Human 4-1BB was detected on T cells populations in B-hCD3EDG/h4-1BB mice, but not in wild-type C57BL/6 mice.

Mouse and human 4-1BB expression analysis in splenocytes. Splenocytes were collected from wild-type C57BL/6 mice (male, 6-week-old, n = 1) and homozygous B-hCD3EDG/h4-1BB mice (male, 6-week-old, n = 1) that treated with anti-m/hCD3E antibody (7.5 μg/mice, i.p., 24 hours). 4-1BB expression on CD4+ T cells, CD8+ T cells and Tregs were analyzed by flow cytometry using species-specific anti-4-1BB antibodies (anti-human 4-1BB antibody, Invitrogen, 12-1379-42; anti-mouse 4-1BB antibody, Invitrogen, 17-1371-82).

• The frequencies of T cells, B cells, NK cells, Granulocytes, Dendritic cells, Monocytes, and Macrophages in homozygous B-hCD3EDG/h4-1BB mice were similar to those in C57BL/6 mice.
• Humanization of CD3E, CD3D, CD3G, and 4-1BB does not affect normal immune cell development and distribution.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes, lymph nodes, and peripheral blood were isolated from C57BL/6 and B-hCD3EDG/h4-1BB mice (male, 6-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/h4-1BB mice were comparable to those in C57BL/6 mice.
• Humanization of CD3E, CD3D, CD3G, and 4-1BB does not affect normal T cell development, differentiation, or distribution.

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood. Splenocytes, lymph nodes, and peripheral blood were isolated from C57BL/6 and B-hCD3EDG/h4-1BB mice (male, 6-week-old, n = 3). Single live cells were gated on the mTCRβ+ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM

Analysis of blood leukocyte subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hCD3EDG/h4-1BB mice (n=3, 6 week-old). Flow cytometry analysis of the blood leukocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for CD45 population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hCD3EDG/h4-1BB mice were similar to those in the C57BL/6 mice, demonstrating that introduction of CD3EDG and 4-1BB in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in blood. Values are expressed as mean ± SEM.

Analysis of blood T cell subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hCD3EDG/h4-1BB mice (n=3, 6 week-old). Flow cytometry analysis of the leukocytes was performed to assess T cell subsets. A. Representative FACS plots. Single live CD45+ cells were gated for CD3 T cell population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8+ T cells, CD4+ T cells and Treg cells in homozygous B-hCD3EDG/h4-1BB mice were similar to those in the C57BL/6 mice, demonstrating that introduction of CD3EDG and 4-1BB in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in blood. Values are expressed as mean ± SEM.

Establishment of a B-hDLL3 B16-F10 model and in vivo efficacy study of an anti-human CD3/4-1BB/DLL3 Tri-specific antibody. B-hDLL3 B16-F10 melanoma cells were implanted subcutaneously into homozygous B-hCD3EDG/h4-1BB mice (female, 7 weeks old, n = 5). When the average tumor volume reached approximately 80 mm³, mice were randomized and subsequently administered the RG6524 analog (in-house) via intraperitoneal injection.

Efficacy of a CD3/4-1BB/DLL3 Tri-specific Antibody in B-hCD3EDG/h4-1BB mice. (A) Tumor growth curves. (B) Body weight changes during treatment. As shown in panel A, RG6524-analog (in-house) was efficacious in controlling tumor growth in B-hCD3EDG/h4-1BB mice in a dose-dependent manner, demonstrating that the B-hCD3EDG/h4-1BB mice provide a powerful preclinical model for in vivo evaluation of anti-human CD3/4-1BB/DLL3 Tri-specific antibodies. Values are expressed as mean ± SEM.

The overage of this tumor model is 60%.

• The proportions of CTL cells in the blood, spleen, and tumor increased in the RG6524-analog treatment group.
• The proportions of Th cells in the blood, spleen, and tumor decreased in the RG6524-analog treatment group.
• The proportions of CTL cells to Tregs in the blood and tumor showed an increasing trend in the RG6524-analog treatment group, while the spleen showed no clear trend.

• The proportions of mCD69+ CTL and Th cells in the tumor were increased in the high dose RG6524-analog treatment group.
• The proportions of TEM CTL and Th cells in the tumor were increased in the RG6524-analog treatment group.
• The proportions of naïve CTL and Th cells in the tumor were decreased in the RG6524-analog treatment group.