4-1BB: A key TNFR superfamily target for enhancing T cell activation in cancer immunotherapy.

• Gene Information: 4-1BB (tumor necrosis factor receptor superfamily member 9) is encoded on human chromosome 1 and functions as a costimulatory receptor. It belongs to the TNFR superfamily and plays a crucial role in sustaining immune cell activation and survival.
• Protein Expression: 4-1BB is highly expressed on activated CD8+ T cells, CD4+ T cells, and NK cells, while maintaining minimal expression on resting immune cells. This activation-induced expression profile makes it a highly specific target for antigen-experienced lymphocytes in the tumor microenvironment (TME).
• Signaling Pathway: Upon binding to its primary endogenous ligand, 4-1BBL, 4-1BB recruits TRAF adapter proteins. This leads to the activation of NF-κB and MAPK pathways, which collectively drive the enhanced proliferation, cytokine production, and prolonged survival functions of effector T cells.
• Therapeutic Agonism: 4-1BB is a key target for therapeutic agonistic monoclonal antibodies designed to specifically enhance tumor-reactive T cells. 4-1BB-targeted therapies are actively being developed for various solid tumors, often as combination therapies with immune checkpoint inhibitors.

4-1BB

• Exons 2-7 of mouse 4-1BB gene that encode signal peptide and extracellular domain are replaced by human counterparts in B-h4-1BB mice.
• The genomic region of mouse 4-1BB 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 4-1BB expression is driven by endogenous mouse 4-1BB promoter, while mouse 4-1BB gene transcription and translation will be disrupted.

• Mouse 4-1BB mRNA was detectable in wild-type C57BL/6 mice.
• Human 4-1BB mRNA was detectable only in homozygous B-h4-1BB mice but not in wild-type mice.

Strain specific analysis of 4-1BB mRNA expression in wild-type C57BL/6 mice and homozygous B-h4-1BB mice by RT-PCR. Spleen RNA was isolated from wild-type C57BL/6 mice (+/+) and homozygous B-h4-1BB mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human 4-1BB primers.

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

Strain specific 4-1BB protein expression analysis in C57BL/6 mice and homozygous B-h4-1BB mice by flow cytometry. Splenocytes were collected from wild type C57BL/6 mice (+/+) and homozygous B-h4-1BB mice (H/H)  that stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-4-1BB antibodies.

• Mouse 4-1BB was only detected on  CD4+ T cells, CD8+ T cells and Treg cells  in wild-type C57BL/6 mice.
• Human 4-1BB was detected on CD4+ T cells, CD8+ T cells and Treg cells in B-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 and homozygous B-h4-1BB mice that stimulated with anti-CD3ε in vivo. 4-1BB expression on T cells, CD4+ T cells, CD8+ T cells and Tregs was analyzed by flow cytometry using species-specific anti-4-1BB antibodies.

• Anti-human 4-1BB antibodies were efficacious in controlling tumor growth in B-h4-1BB mice.
• B-h4-1BB mice provide a powerful preclinical model for in vivo evaluation of anti-human 4-1BB antibodies.

Efficacy of anti-human 4-1BB antibodies in B-h4-1BB mice. (A) Anti-human 4-1BB antibodies inhibited MC38 tumor growth in B-h4-1BB mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-h4-1BB mice (female, 6-7 week-old, n=5). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with two anti-human 4-1BB antibodies with doses and schedules indicated in panel. (B) Body weight changes during treatment. Values are expressed as mean ± SEM.

• Anti-human 4-1BB antibodies were efficacious in controlling tumor growth in B-h4-1BB mice.
• B-h4-1BB mice provide a powerful preclinical model for in vivo evaluation of anti-human 4-1BB antibodies.

Efficacy of anti-human 4-1BB antibodies in B-h4-1BB mice. (A) Anti-human 4-1BB antibodies inhibited MC38 tumor growth in B-h4-1BB mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-h4-1BB mice (male, 7-8 week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with an anti-human 4-1BB antibody with doses and schedules indicated in panel. (B) Body weight changes during treatment. Values are expressed as mean ± SEM.

• Anti-human 4-1BB antibodies were efficacious in controlling tumor growth in B-h4-1BB mice.
• B-h4-1BB mice provide a powerful preclinical model for in vivo evaluation of anti-human 4-1BB antibodies.

Efficacy of anti-human 4-1BB antibodies in B-h4-1BB mice. (A) Anti-human 4-1BB antibodies inhibited MC38 tumor growth in B-h4-1BB mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-h4-1BB mice (female, 6-7 week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with anti-human 4-1BB antibody Urelumab  (in house)  with doses and schedules indicated in panel. (B) Body weight changes during treatment. Values are expressed as mean ± SEM.

• Anti-human 4-1BB antibodies were efficacious in controlling tumor growth in B-h4-1BB mice.
• B-h4-1BB mice provide a powerful preclinical model for in vivo evaluation of anti-human 4-1BB antibodies.

Efficacy of anti-human 4-1BB antibodies in B-h4-1BB mice. (A) Anti-human 4-1BB antibodies inhibited MC38 tumor growth in B-h4-1BB mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-h4-1BB mice (female, 6-7 week-old, n=5). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with anti-human 4-1BB antibody utomilumab (in house) with doses and schedules indicated in panel. (B) Body weight changes during treatment. Values are expressed as mean ± SEM.

• Anti-human 4-1BB antibodies were efficacious in controlling tumor growth in B-h4-1BB mice.
• B-h4-1BB mice provide a powerful preclinical model for in vivo evaluation of anti-human 4-1BB antibodies.

Efficacy of anti-human 4-1BB antibodies in B-h4-1BB mice. (A) Anti-human 4-1BB antibodies inhibited MC38 tumor growth in B-h4-1BB mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-h4-1BB mice (female, 6-7 week-old, n=8). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with two anti-human 4-1BB antibodies with doses and schedules indicated in panel. (B) Body weight changes during treatment.  Values are expressed as mean ± SEM.

• Anti-human 4-1BB antibodies were efficacious in controlling tumor growth in B-h4-1BB mice.
• B-h4-1BB mice provide a powerful preclinical model for in vivo evaluation of anti-human 4-1BB antibodies.

Efficacy of anti-human 4-1BB antibodies in B-h4-1BB mice. (A) Anti-human 4-1BB antibodies inhibited MC38 tumor growth in B-h4-1BB mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-h4-1BB mice (female, 6-7 week-old, n=5). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with anti-human 4-1BB antibody utomilumab (in house) or Urelumab  (in house) with doses and schedules indicated in panel. (B) Body weight changes during treatment. Values are expressed as mean ± SEM.

Analysis of tumor infiltrates lymphocytes(TILs) by FACS. Tumor cells were harvested at the endpoint of experiment (n=3). Flow cytometry analysis of the TILs were performed to assess cell number and proportion changes compared to the group with no anti-h4-1BB treated. CD45+ cells and CD3+ T cells were significantly increased when treated with anti-h4-1BB, while Treg cells were significantly reduced in the two groups treated with anti-h4-1BB compared to the control. Values are expressed as mean ± SEM.(*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)

High-dose Urelumab  analog (in-house) caused chronic inflammatory response in the liver of B-h4-1 BB mice. B-h4-1BB mice were divided into 3 groups and were treated with hIgG4 isotype antibody and Urelumab  analog respectively. Blood were collected on days 14 and 21 after administration to measure the concentrations of ALT (B) and AST (B). On day 21, the mice were euthanized and liver tissues were taken for H&E staining (A). The results showed that in the high-dose group (G3, 20 mg/kg), 21 days after administration, there was a significant infiltration of inflammatory cells in the liver of B-h4-1 BB mice (A), and the ALT levels in the serum of B-h4-1 BB mice significantly increased on days 14 and 21 after administration, the AST level also showed an increasing trend 21 days after drug treatment (B). It is indicated that the high-dose Urelumab  analog can cause significant liver toxicity in B-h4-1BB mice.