B6-hCTLA4 Mice

Catalog Number: C001413

Genetic Background: C57BL/6NCya

Reproduction: Homozygote x Homozygote

Strain Description

Cytotoxic T-lymphocyte-associated protein 4 (CTLA4), also known as cluster of differentiation 152 (CD152), is an immunoglobulin superfamily protein encoded by the CTLA4 gene. CTLA4 is expressed by activated T cells and delivers inhibitory signals to T cells ^[1]. The structure of CTLA4 protein contains a V-domain, a transmembrane domain, and a cytoplasmic tail. Different splicing patterns of CTLA4 pre-mRNA lead to the appearance of different isoforms, among which the membrane-bound isoform is linked by disulfide bonds to form homodimers, while the soluble isoform exists as a monomer. CTLA4 is homologous to CD28, which delivers T-cell activation signals. Both molecules compete for binding to the natural B7 family ligands B7-1 and B7-2 on antigen-presenting cells, but CTLA4 has a much higher affinity for binding to B7-1 and B7-2 than CD28. This results in the inhibition of T cell activation, allowing tumor cells to escape from T cell attack ^[2]. The gene is closely associated with the occurrence or progression of insulin-dependent diabetes mellitus, Graves' disease, Hashimoto's thyroiditis, celiac disease, systemic lupus erythematosus, thyroid-associated ophthalmopathy, and other autoimmune diseases ^[3].

CTLA4 is a membrane protein, with its extracellular domain serving as the receptor/ligand binding region and its intracellular domain responsible for signal transduction ^[4]. This strain was generated by gene editing to replace the extracellular domain of Ctla4 in mice with the humanized version, resulting in a model that expresses the extracellular domain of human CTLA4 and the intracellular domain of mouse CTLA4. This model can be used for the research of the development and screening of CTLA4-related inhibitors or antibody drugs, the evaluation of pharmacodynamics and safety, the evaluation of tumor immunotherapy, and the mechanisms of the immune system.

Strain Strategy

The mouse Ctla4 gene was edited using gene editing technology to replace the sequence encoding the extracellular domain of mouse CTLA4 protein (aa.36~161) with the sequence from the human CTLA4 gene encoding the human CTLA4 protein extracellular domain (aa.36~161) while retaining the mouse signal peptide.

Figure 1. Schematic representation of the gene editing strategy for generating B6-hCTLA4 mice.

Validation Data

1. Detection of species-specific CTLA4 protein expression

Figure 2. Expression of CTLA4 in spleen T cells after activation in wild-type mice (C57BL/6N) and B6-hCTLA4 mice. Spleen cells were stimulated at different times and analyzed by flow cytometry using species-specific CTLA4 antibodies to detect the expression of human and mouse CTLA4 in homozygous B-hCTLA4 mice and wild-type mice. The results showed that the proportion of T cells expressing mouse CTLA4 (mCTLA4) in the spleen cells of wild-type mice significantly increased after 48 hours of stimulation. Similarly, the proportion of T cells expressing human CTLA4 (hCTLA4) in the spleen cells of B6-CTLA4 mice also significantly increased.

 

2. In vivo efficacy of anti-human CTLA4-specific antibody drug (Yervoy)

Figure 3. Anti-tumor effect of human CTLA4 antibody drug Yervoy in B-hCTLA4 mice. Colon adenocarcinoma cell line MC38 cells were subcutaneously implanted into B-hCTLA4 mice (female, 6 weeks old) and treated with different doses of Anti-hCTLA4 antibody Yervoy] to verify its anti-tumor effect [5. The results showed that Yervoy dose-dependently inhibited tumor growth in B-hCTLA4 mice, indicating that B6-hCTLA4 mice can be used for in vivo evaluation of targeted CTLA4 antibody drugs.

Tumor volume calculation formula:
V=0.5×long diameter×short diameter^2; unit: mm^3.
Dosing regimen:
Group 1: PBS, 100 µl; Group 2: Yervoy, 0.1 mg/kg; Group 3: Yervoy, 0.3 mg/kg; Group 4: Yervoy, 1.0 mg/kg.

Reference

[1] National Center for Biotechnology Information. CTLA4 cytotoxic T-lymphocyte associated protein 4 [ Homo sapiens (human) ] - Gene - NCBI. Retrieved [2023 May 23], from https://www.ncbi.nlm.nih.gov/gene/1493

[2] Noel PJ, Boise LH, Thompson CB. Regulation of T cell activation by CD28 and CTLA4. Adv Exp Med Biol. 1996;406:209-17.

[3] Gough SC, Walker LS, Sansom DM. CTLA4 gene polymorphism and autoimmunity. Immunol Rev. 2005 Apr;204:102-15.

[4] Ramagopal UA, Liu W, Garrett-Thomson SC, Bonanno JB, Yan Q, Srinivasan M, Wong SC, Bell A, Mankikar S, Rangan VS, Deshpande S, Korman AJ, Almo SC. Structural basis for cancer immunotherapy by the first-in-class checkpoint inhibitor ipilimumab. Proc Natl Acad Sci U S A. 2017 May 23;114(21): E4223-E4232.

[5] Cameron F, Whiteside G, Perry C. Ipilimumab: first global approval. Drugs. 2011 May 28;71(8):1093-104.