B-hTNFRSF9 (4-1BB) mice

Basic Information

Strain Name
C57BL/6-Tnfrsf9tm1(TNFRSF9)Bcgen/Bcgen
Stock Number
110004
Common Name
B-hCD137 (4-1BB) Mice
Source/Investigator
Bcgen (Beijing Biocytogen Co., Ltd)
Related Genes
Tnfrsf9 (tumor necrosis factor receptor superfamily, member 9)
Species
C57BL/6
Appearance
Black
Genotypes
Homozygous

Description

TNFRSF9 (Tumor necrosis factor receptor super family, member9), also called CD137 and 4-1BB, is a co-stimulatory molecule and is mainly expressed on the surface of T, NK and mononuclear cells. CD137 is activated by itsligand CD137L or activating anti-CD137 antibodies, resulting in T cell activation, proliferation and cytokine production. CD137L is expressed on antigen presenting cells such as dendritic cells and macrophages and activated B cells. In vivo studies show that CD137 activation increases anti-tumor immune responses, providing a new target for immunotherapy

Targeting Strategy

Details

Phenotype

Protein Expression Analysis

Strain specific 4-1BB protein expression analysis in wild-type (+/+) or homozygous B-h4-1BB (H/H) mice by flow cytometry.

Splenocytes were collected from +/+ and homozygous mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-4-1BB antibodies. Mouse 4-1BB was detectable in +/+ mice, while human 4-1BB was exclusively detectable in B-h4-1BB H/H mice.

High dose of urelumab resulted in liver toxicity in B-h4-1BB mice

Anti-h4-1BB antibody was injected into homozygous h4-1BB mice. Blood was collected on Day 14 & 21 and analyzed by blood biochemical test.

A. On Day 14, compared to the control group G1, the concentration of ALT was significantly increased after treated with high dose (20 mg/kg) of urelumab (in house) (G2). On Day 21, compared to the control group G1, the concentration of ALT was more significantly increased after treated with high dose (20mg/kg) of urelumab (in house) (G2). There was no significant difference in concentrations of ALT between group G1 and group G3 (1 mg/kg). B. There was no significant difference in body weight among the groups.

High dose of urelumab therapy resulted in increased lymphocyte infiltration in liver in B-h4-1BB mice

Chronic inflammation (arrow) was observed in liver of B-h4-1BB mice which treated with high dose (20mg/kg) of urelumab (in house) for 21 days. While there was no microscopic changes in groups G2 and G4 which respectively treated with low dose (1mg/kg) of urelumab (in house) and high dose (20mg/kg ) of Isotype.

Application

In vivo efficacy of anti-human 4-1BB antibodies

Antitumor activity 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. As shown in panel A, anti-human 4-1BB antibodies were efficacious in controlling tumor growth in B-h4-1BB mice, demonstrating that the B-h4-1BB mice provide a powerful preclinical model for in vivo evaluation of anti-human 4-1BB antibodies. Values are expressed as mean ± SEM.

In vivo efficacy of anti-human 4-1BB antibody

Antitumor activity 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 an anti-human 4-1BB antibody with doses and schedules indicated in panel. (B) Body weight changes during treatment. As shown in panel A, anti-human 4-1BB antibodies were efficacious in controlling tumor growth in B-h4-1BB mice, demonstrating that the B-h4-1BB mice provide a powerful preclinical model for in vivo evaluation of anti-human 4-1BB antibodies. Values are expressed as mean ± SEM.

Antitumor activity 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. As shown in panel A, anti-human 4-1BB antibodies were efficacious in controlling tumor growth in B-h4-1BB mice, demonstrating that the B-h4-1BB mice provide a powerful preclinical model for in vivo evaluation of anti-human 4-1BB antibodies. Values are expressed as mean ± SEM.

Antitumor activity 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. As shown in panel A, anti-human 4-1BB antibodies were efficacious in controlling tumor growth in B-h4-1BB mice, demonstrating that the B-h4-1BB mice provide a powerful preclinical model for in vivo evaluation of anti-human 4-1BB antibodies. Values are expressed as mean ± SEM.

Murine colon cancer MC38 cells were subcutaneously implanted into C57BL/6 mice. Mice were divided into control and treatment groups (n=5), when the tumor size was approximately 150±50 mm3. m4-1BB antibody showed substantially inhibitory effects in the C57BL/6 mice. (A) Tumor average volume ± SEM, (B) Mice average weight ± SEM.

Antitumor activity 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. As shown in panel A, anti-human 4-1BB antibodies were efficacious in controlling tumor growth in B-h4-1BB mice, demonstrating that the B-h4-1BB mice provide a powerful preclinical model for in vivo evaluation of anti-human 4-1BB antibodies. Values are expressed as mean ± SEM.

Tumor infiltrating lymphocytes (TILs) analysis

Tumor cells were harvested at the endpoint of experiment, then processed and analyzed by flow cytometry.

Ratio of tumor infiltrating lymphocytes (TILs)

Flow cytometry analysis of tumor infiltrating lymphocytes (TILs).

Tumor cells were harvested at the endpoint of experiment (n=3). Flow cytometry analysis of the tumor infiltrating lymphocytes (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.

References

1. PLoS One. 2014 Jan 21;9(1): e86337. doi: 10.1371/journal.pone.0086337. eCollection 2014.
2. Front Oncol. 2015 Jun 8;5:117. doi: 10.3389/fonc.2015.00117. eCollection 2015.
3. Clin Transl Immunology. 2014 Aug 1;3(8): e22. doi: 10.1038/cti.2014.18. eCollection 2014 Aug.
4. J Immunol. 2002 Jun 1;168(11):5483-90.

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