• Tumor necrosis factor alpha (TNFα) is a central inflammatory cytokine that activates immune responses through TNFR1 and TNFR2 signaling pathways.
• TNFR1 and TNFR2 regulate overlapping but distinct biology in inflammation, autoimmunity, cancer, tissue injury, and immune-cell activation.
• TNFA/TNFR2/TNFR1 humanized mice (B-hTNFA/hTNFR2/hTNFR1) are triple target-humanized mice carrying human TNFA, humanized TNFR2, and humanized TNFR1 in a C57BL/6 background.
• TNFA/TNFR2/TNFR1 humanized mice (B-hTNFA/hTNFR2/hTNFR1) enable in vivo evaluation of anti-human TNFα antibodies, anti-human TNFR2 antibodies, TNFR1/TNFR2 pathway modulators, and TNFA-targeted therapies.
• Human TNFα, TNFR1, and TNFR2 expression, immune-cell profiling, LPS-induced cytokine response, MC38 tumor efficacy, and collagen-induced arthritis efficacy have been validated in this model.

Key Advantages

• Human TNFα expression validated by ELISA after LPS stimulation
• Human TNFR1 and human TNFR2 expression validated by flow cytometry in splenocytes
• Normal spleen and blood leukocyte subpopulation profiles compared with wild-type mice
• Supports anti-human TNFα antibody efficacy in LPS response and CIA arthritis models
• Supports anti-human TNFR2 antibody evaluation in MC38 tumor-bearing TNFA/TNFR2/TNFR1 humanized mice (B-hTNFA/hTNFR2/hTNFR1)

Validation

• TNFα Protein Validation:
  Human TNFα was detectable in homozygous TNFA/TNFR2/TNFR1 humanized mice (B-hTNFA/hTNFR2/hTNFR1) after LPS stimulation, while mouse TNFα was detected in wild-type mice.
• TNFR1 Protein Validation:
  Human TNFR1 was detectable in homozygous TNFA/TNFR2/TNFR1 humanized mice (B-hTNFA/hTNFR2/hTNFR1)by flow cytometry, while mouse TNFR1 was detected in wild-type mice.
• TNFR2 Protein Validation:
  Human TNFR2 was detectable in homozygous TNFA/TNFR2/TNFR1 humanized mice (B-hTNFA/hTNFR2/hTNFR1) after anti-CD3ε stimulation, while mouse TNFR2 was detected in wild-type mice.
• Phenotypic Validation:
  Spleen and blood leukocyte subpopulations, hematology, and serum biochemistry were characterized in TNFA/TNFR2/TNFR1 humanized mice (B-hTNFA/hTNFR2/hTNFR1).
• Efficacy Validation:
  Adalimumab and anti-human TNFR2 antibodies demonstrated in vivo activity in LPS response, MC38 tumor, and CIA arthritis models.

Application

• Anti-human TNFα antibody efficacy evaluation
• Anti-human TNFR2 antibody and TNFR2 agonist/antagonist studies
• TNFR1/TNFR2 pathway pharmacology and mechanism-of-action research
• LPS-induced inflammatory cytokine response studies
• MC38 syngeneic tumor efficacy studies
• Collagen-induced arthritis (CIA) and rheumatoid arthritis drug development

TNFA

• The targeted mouse Tnfa whole genomic sequences including 5'UTR, 3'UTR and coding region were replaced by human TNFA whole genomic sequences in TNFA/TNFR2/TNFR1 humanized mice (B-hTNFA/hTNFR2/hTNFR1).

TNFR2

• The exons 2~6 of mouse Tnfr2 gene that  encode the extracellular domain were replaced by human TNFR2 exons 2~6 in TNFA/TNFR2/TNFR1 humanized mice (B-hTNFA/hTNFR2/hTNFR1).
• Expression of the human TNFR2 protein is driven by the native mouse Tnfr2 promoter, while endogenous mouse Tnfr2 transcription and translation are disrupted.

TNFR1

• The exons 2~6 of mouse Tnfr1 gene that  encode the extracellular domain were replaced by human TNFR1 exons 2~6 in TNFA/TNFR2/TNFR1 humanized mice (B-hTNFA/hTNFR2/hTNFR1).
• Expression of the human TNFR1 protein is driven by the native mouse Tnfr1 promoter, while endogenous mouse Tnfr1 transcription and translation are disrupted.

• Mouse TNFA was detected exclusively in wild-type C57BL/6 mice.
• Human TNFA was detected exclusively in homozygous TNFA/TNFR2/TNFR1 humanized mice and was not detected in wild-type C57BL/6 mice.

Strain-specific analysis of mouse and human TNFA protein expression in wild-type C57BL/6 mice and TNFA/TNFR2/TNFR1 humanized mice by ELISA. Wild-type C57BL/6 mice (+/+) and homozygous TNFA/TNFR2/TNFR1 humanized mice (H/H; H/H; H/H) were stimulated in vivo with lipopolysaccharide (LPS). Serum samples were subsequently collected, and mouse and human TNFA protein levels were measured by ELISA using species-specific TNFA ELISA kits. Mouse TNFA was detected exclusively in wild-type C57BL/6 mice, whereas human TNFA was detected exclusively in homozygous TNFA/TNFR2/TNFR1 humanized mice and was not detected in wild-type C57BL/6 mice. Values are expressed as mean ± SEM. ND: not detectable.

• Human TNFR1 expression on neutrophils was detected exclusively in TNFA/TNFR2/TNFR1 humanized mice.

Strain-specific analysis of mouse and human TNFR1 protein expression in wild-type C57BL/6 mice and TNFA/TNFR2/TNFR1 humanized mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous TNFA/TNFR2/TNFR1 humanized mice (H/H; H/H; H/H). Mouse and human TNFR1 protein expression was analyzed by flow cytometry using species-specific anti-TNFR1 antibodies. Mouse TNFR1 was detected in wild-type C57BL/6 mice, whereas human TNFR1 was detected exclusively in homozygous TNFA/TNFR2/TNFR1 humanized mice and was not detected in wild-type C57BL/6 mice.

• Human TNFR2 expression on regulatory T cells was detected exclusively in TNFA/TNFR2/TNFR1 humanized mice.

Strain-specific analysis of mouse and human TNFR2 protein expression in wild-type C57BL/6 mice and TNFA/TNFR2/TNFR1 humanized mice by flow cytometry. Wild-type C57BL/6 mice (+/+) and homozygous TNFA/TNFR2/TNFR1 humanized mice (H/H; H/H; H/H) were stimulated in vivo with anti-CD3ε antibody. Splenocytes were subsequently collected, and mouse and human TNFR2 protein expression was analyzed by flow cytometry using species-specific anti-TNFR2 antibodies. Mouse TNFR2 was detected in wild-type C57BL/6 mice, whereas human TNFR2 was detected exclusively in homozygous TNFA/TNFR2/TNFR1 humanized mice and was not detected in wild-type C57BL/6 mice.

• The percentages of T cells, B cells, NK cells, dendritic cells (DCs), neutrophils, monocytes, and macrophages in homozygous TNFA/TNFR2/TNFR1 humanized mice were comparable to those in wild-type C57BL/6N mice.
• Humanization of TNF, TNFR1, and TNFR2 does not affect normal immune cell development or splenic distribution.

Flow cytometry analysis of leukocyte subpopulations in immune organs and peripheral blood of TNFA/TNFR2/TNFR1 humanized mice. Splenocytes, peripheral blood, and lymph nodes were collected from female wild-type C57BL/6N mice and TNFA/TNFR2/TNFR1 humanized mice (9 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.

• The proportions of CD4⁺ T cells, CD8⁺ T cells, and regulatory T cells (Tregs) in homozygous TNFA/TNFR2/TNFR1 humanized mice were comparable to those in wild-type C57BL/6N mice.
• Humanization of TNF, TNFR1, and TNFR2 does not affect normal T cell development, differentiation, or splenic distribution.

Flow cytometry analysis of T-cell subpopulations in immune organs and peripheral blood of TNFA/TNFR2/TNFR1 humanized mice. Splenocytes, peripheral blood, and lymph nodes were collected from female wild-type C57BL/6N mice and TNFA/TNFR2/TNFR1 humanized mice (9 weeks 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.

• No significant differences were observed compared with wild-type mice.

Complete blood count (CBC) of TNFA/TNFR2/TNFR1 humanized mice. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Blood biochemical parameters of TNFA/TNFR2/TNFR1 humanized mice are shown. Values are expressed as mean ± SD.

• Anti-human TNFA significantly decreased mouse IL6 and mouse IFNγ expression at four hours after LPS injection, indicating a successful inhibition of the downstream activation caused by hTNFA.

ELISA analysis of human TNFA, mouse IL-6, and mouse IFN-γ levels following LPS stimulation and adalimumab analog treatment in TNFA/TNFR2/TNFR1 humanized mice. Homozygous TNFA/TNFR2/TNFR1 humanized mice (H/H; H/H; H/H) received an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) 2 hours after treatment with PBS or an in-house adalimumab analog administered intravenously (i.v.). Serum samples were collected 1 and 4 hours after LPS injection. Human TNFA, mouse IL-6, and mouse IFN-γ levels were measured by ELISA using species-specific kits (ELISA MAX™ Deluxe Set Human TNF-α, BioLegend, 430204; ELISA MAX™ Deluxe Set Mouse IL-6, BioLegend, 430304; ELISA MAX™ Deluxe Set Mouse IFN-γ, BioLegend, 430804). Following LPS stimulation, human TNFA and mouse IL-6 levels were significantly increased at both 1 and 4 hours, while mouse IFN-γ levels were significantly increased at 4 hours. Pretreatment with the adalimumab analog significantly reduced human TNFA levels at 1 hour and mouse IL-6 and IFN-γ levels at 4 hours after LPS injection, indicating effective inhibition of downstream inflammatory responses mediated by human TNFA. Values are expressed as mean ± SEM. Statistical significance was determined using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

Establishment of a subcutaneous MC38 tumor model and in vivo efficacy evaluation of an anti-human TNFR2 antibody in TNFA/TNFR2/TNFR1 humanized mice. MC38 mouse colon carcinoma cells were subcutaneously implanted into homozygous TNFA/TNFR2/TNFR1 humanized mice (female, 7 weeks old, n = 6). When the average tumor volume reached approximately 110 mm³, mice were randomized into treatment groups and subsequently administered an anti-human TNFR2-specific antibody via intraperitoneal injection.

• Anti-human TNFR2 antibody treatment inhibited tumor growth compared with untreated controls.

In vivo antitumor efficacy of anti-human TNFR2 antibodies in a subcutaneous MC38 tumor model using TNFA/TNFR2/TNFR1 humanized mice. (A) Tumor growth curves. Murine colon carcinoma MC38 cells were subcutaneously implanted into homozygous TNFA/TNFR2/TNFR1 humanized mice (female, 8 weeks old, n = 6). Mice were assigned to treatment groups based on body weight differences and subsequently administered the client-provided anti-human TNFR2 antibody (Ab1) at the doses and schedules indicated in panel A. (B) Body weight changes during treatment. As shown in panel A, treatment with the anti-human TNFR2 antibody inhibited MC38 tumor growth in TNFA/TNFR2/TNFR1 humanized mice in a dose-dependent manner. These results demonstrate the utility of TNFA/TNFR2/TNFR1 humanized mice as a preclinical model for evaluating the in vivo antitumor efficacy of anti-human TNFR2 antibodies. Values are expressed as mean ± SEM.

• Collagen-induced arthritis (CIA) was successfully established in TNFA/TNFR2/TNFR1 humanized mice and wild-type C57BL/6 mice.

Establishment and clinical evaluation of a collagen-induced arthritis (CIA) model in TNFA/TNFR2/TNFR1 humanized mice. Arthritis was induced in TNFA/TNFR2/TNFR1 humanized mice and wild-type C57BL/6 mice using type II collagen (CII). (A) Body weight changes. (B) Clinical arthritis scores. The results showed significantly increased clinical arthritis scores in TNFA/TNFR2/TNFR1 humanized mice, indicating successful establishment of the collagen-induced arthritis model.

Histopathological analysis of collagen-induced arthritis (CIA) in TNFA/TNFR2/TNFR1 humanized mice and wild-type C57BL/6 mice. (A) Histopathological scores. (B) Hematoxylin and eosin (H&E) staining of joint tissue sections. In the arthritis model groups, histopathological examination revealed subcutaneous mixed inflammatory cell infiltration, periarticular joint space narrowing, articular cartilage destruction, bone tissue damage, and other arthritis-associated lesions in some or all limb joints. These findings indicate the successful establishment of the collagen-induced arthritis model in TNFA/TNFR2/TNFR1 humanized mice and wild-type C57BL/6 mice.

• Adalimumab analog significantly reduced clinical score compared with controls.

In vivo efficacy of anti-human TNFA antibody adalimumab analog in a collagen-induced arthritis (CIA) model using TNFA/TNFR2/TNFR1 humanized mice. Collagen-induced arthritis was established in female TNFA/TNFR2/TNFR1 humanized mice (n = 9–10 per group) by subcutaneous injection of type II collagen (CII) emulsion on days 0 and 21. Arthritis development was monitored, and clinical arthritis scores were assessed daily. At the onset of inflammation (defined as a clinical score > 1 or a persistent score of 1), mice were assigned to treatment groups, with the treatment initiation day designated as day 0. Mice received intraperitoneal injections of an in-house anti-human TNFA antibody (adalimumab analog) at different doses. (A) Body weight changes. (B) Clinical arthritis scores. Body weight and clinical arthritis scores were monitored daily during treatment. Mice were euthanized 2 days after the final treatment, and paws were collected for histopathological evaluation using decalcified hematoxylin and eosin (H&E)-stained joint sections. No significant changes in body weight were observed, while clinical arthritis scores increased in all groups except the control group, confirming successful establishment of the CIA model. Treatment with the adalimumab analog reduced clinical arthritis scores in a dose-dependent manner. These results demonstrate the utility of TNFA/TNFR2/TNFR1 humanized mice as a preclinical CIA model for evaluating the in vivo therapeutic efficacy of anti-human TNFA antibodies.

Histopathological evaluation of anti-human TNFA antibody efficacy in a collagen-induced arthritis (CIA) model using TNFA/TNFR2/TNFR1 humanized mice. Histopathological examination of limb joints was performed at the study endpoint. (A) Hematoxylin and eosin (H&E) staining of joint tissue sections showing inflammatory cell infiltration (a), synovial hyperplasia (b), and bone structural damage (c). (B) Histopathological scores. The histopathological score was higher in group G2 than in group G1, confirming successful establishment of the CIA model. The score was also higher in group G2 than in group G4, indicating the therapeutic efficacy of anti-human TNFA antibody treatment in reducing arthritis-associated joint pathology. These results demonstrate the utility of TNFA/TNFR2/TNFR1 humanized mice as a preclinical CIA model for evaluating the in vivo therapeutic efficacy of anti-human TNFA antibodies.

Q: Do TNFA humanized mice (B-hTNFA) have efficacy data from autoimmune disease models, such as arthritis and psoriasis?

A: TNFA humanized mice (B-hTNFA) only have modeling data for collagen induced arthritis (CIA), and no data for other autoimmune disease models.
In two collagen-induced arthritis experiments, the clinical scores of TNFA humanized mice (B-hTNFA) were lower than those of C57BL/6 and TNFA/TNFR2/TNFR1 humanized mice (B-hTNFA/hTNFR2/hTNFR1), respectively. Therefore, we recommend using TNFA/TNFR2/TNFR1 humanized mice (B-hTNFA/hTNFR2/hTNFR1)  for modeling collagen-induced arthritis.