4-1BB (CD137, TNFRSF9) is a costimulatory receptor belonging to the tumor necrosis factor receptor superfamily and is primarily expressed on activated T cells and other immune cells. Engagement of 4-1BB plays a critical role in T-cell activation, proliferation, and antitumor immune responses, making it an important target in cancer immunotherapy.

In 4-1BB humanized mouse model (B-h4-1BB Mice), the endogenous mouse Tnfrsf9 gene is replaced with the human TNFRSF9 gene, resulting in exclusive expression of human 4-1BB at both the mRNA and protein levels. Validation studies demonstrate that human 4-1BB is specifically expressed in homozygous B-h4-1BB mice, while mouse 4-1BB expression is absent. This model has been extensively validated for in vivo efficacy and safety assessment of anti-human 4-1BB antibodies, including agonistic antibodies and bispecific formats. Our humanized 4-1BB Mouse Model (B-h4-1BB Mice) support tumor growth inhibition studies, immune activation analysis, tumor-infiltrating lymphocyte (TIL) profiling, and hepatotoxicity risk evaluation associated with 4-1BB agonism.

Key Advantages

• Human-specific 4-1BB expression: Human TNFRSF9 mRNA and protein are exclusively expressed in homozygous 4-1BB humanized mice, with no detectable mouse 4-1BB expression.
• Validated immune activation: Human 4-1BB expression is functionally inducible in T cells following anti-CD3ε stimulation, supporting immune-relevant in vivo studies.
• Robust antitumor efficacy evaluation: Anti-human 4-1BB antibodies significantly inhibit MC38 tumor growth in B-h4-1BB mice across multiple dosing regimens.
• Safety and toxicity assessment capability: High-dose urelumab analog studies reveal liver toxicity and inflammatory responses, enabling translational safety risk evaluation.

Validation

• Human 4-1BB expression: RT-PCR confirms human TNFRSF9 mRNA expression exclusively in homozygous 4-1BB humanized mice. Flow cytometry demonstrates human 4-1BB protein expression on activated T cells, while mouse 4-1BB is detected only in wild-type C57BL/6 mice.
• In vivo antitumor efficacy: Anti-human 4-1BB antibodies significantly inhibit MC38 tumor growth in 4-1BB humanized mice. Multiple antibodies (including urelumab and utomilumab analogs) demonstrate dose-dependent antitumor activity with stable body weight profiles.
• Safety and Hepatotoxicity: High-dose urelumab analog induces elevated ALT/AST levels and liver inflammatory infiltration, supporting translational toxicity assessment.

Application

This model is well suited for in vivo functional, efficacy, and safety studies of anti-human 4-1BB antibodies. The model also enables mechanistic investigation of 4-1BB–mediated immune responses and tumor immunology in preclinical cancer research.

4-1BB

• Exons 2-7 of mouse 4-1BB gene that encode signal peptide and extracellular domain are replaced by human counterparts in 4-1BB humanized 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.

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

Strain specific 4-1BB protein expression analysis in C57BL/6 mice and homozygous 4-1BB humanized 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 and human 4-1BB expression analysis in splenocytes of 4-1BB humanized mice. Splenocytes were collected from wild-type C57BL/6 mice and homozygous 4-1BB humanized 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.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood of 4-1BB humanized mice. Splenocytes, peripheral blood, and lymph nodes were isolated fromfemale C57BL/6JNifdc and of 4-1BB humanized 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.

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood of 4-1BB humanized mice. Splenocytes, peripheral blood, and lymph nodes were isolated fromfemale C57BL/6JNifdc and of 4-1BB humanized mice (female, 9-week-old, n = 3). Single live cells were gated on the T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

Efficacy of anti-human 4-1BB antibodies in 4-1BB humanized mice. (A) Anti-human 4-1BB antibodies inhibited MC38 tumor growth in 4-1BB humanized 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.

Efficacy of anti-human 4-1BB antibodies in 4-1BB humanized mice. (A) Anti-human 4-1BB antibodies inhibited MC38 tumor growth in 4-1BB humanized mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous 4-1BB humanized 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.

Efficacy of anti-human 4-1BB antibodies in 4-1BB humanized mice. (A) Anti-human 4-1BB antibodies inhibited MC38 tumor growth in 4-1BB humanized mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous 4-1BB humanized 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.

Efficacy of anti-human 4-1BB antibodies in 4-1BB humanized mice. (A) Anti-human 4-1BB antibodies inhibited MC38 tumor growth in 4-1BB humanized mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous 4-1BB humanized 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.

Efficacy of anti-human 4-1BB antibodies in 4-1BB humanized mice. (A) Anti-human 4-1BB antibodies inhibited MC38 tumor growth in 4-1BB humanized mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous 4-1BB humanized 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.

Efficacy of anti-human 4-1BB antibodies in 4-1BB humanized mice. (A) Anti-human 4-1BB antibodies inhibited MC38 tumor growth in 4-1BB humanized mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous 4-1BB humanized 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 4-1BB humanized mice. 4-1BB humanized 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 4-1BB humanized 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 4-1BB humanized mice.

Q1: What are 4-1BB humanized mice (B-h4-1BB mice)?

B-h4-1BB mice are a humanized model in which the mouse Tnfrsf9 gene is replaced by the human TNFRSF9 gene. This enables exclusive expression of human 4-1BB (CD137) under endogenous mouse regulatory control.

Q2: Why is humanized 4-1BB (B-h4-1BB) mouse model important for immunotherapy research?

4-1BB receptor is a critical costimulatory immune checkpoint that amplifies T-cell activation, proliferation, and survival. Our humanized 4-1BB (B-h4-1BB) mouse model enables preclinical evaluation of human-specific 4-1BB-targeting therapies, such as agonistic antibodies, by faithfully recapitulating human biology, making it a valuable platform for assessing efficacy and mechanism of action in cancer immunotherapy.

Q3: How is human 4-1BB expression validated?

RT-PCR confirms human TNFRSF9 (4-1BB) mRNA expression exclusively in homozygous B-h4-1BB mice, and flow cytometry verifies exclusive human 4-1BB protein expression on activated immune cells.

Q4: What are the main applications of 4-1BB humanized mice (B-h4-1BB)?

4-1BB humanized (B-h4-1BB) mice are widely used for preclinical evaluation of anti-human 4-1BB immunotherapies, including agonistic antibodies and bispecific antibodies. Key applications include: 

• Assessing in vivo antitumor efficacy
• Profiling tumor-infiltrating lymphocytes
• Evaluating potential on-target, treatment-related toxicities

Q5: What evidence supports the use of 4-1BB humanized mice (B-h4-1BB) in efficacy studies?

Preclinical studies in 4-1BB humanized (B-h4-1BB) mice demonstrate that anti-human 4-1BB antibodies significantly inhibit MC38 tumor growth. Treatment also remodels the tumor immune microenvironment, marked by increased CD8⁺ T cell infiltration and reduced regulatory T cells, supporting the model's value for antitumor efficacy and immunotherapy mechanism-of-action studies.