B6N-hFCRN(Extra) Mice

Catalog Number: C001701

Strain Name: C57BL/6NCya-Fcgrttm2(hFCGRT)/Cya

Genetic Background: C57BL/6NCya

Reproduction: Homozygote x Homozygote

 

Strain Description

Neonatal Fc receptor (FcRn) is a cell surface receptor protein that binds to the Fc region of IgG antibodies. It is structurally similar to MHC class I molecules and is composed of an α-chain and β2-microglobulin (β2M). The α-chain of the FcRn receptor is encoded by the Fcγ receptor and transporter (FCGRT) gene, while β2-microglobulin is encoded by the β-2-microglobulin (B2M) gene. FcRn is expressed widely on epithelial cells, endothelial cells, and hematopoietic cells, and is found in a variety of tissues and organs, including the intestine, placenta, kidney, and liver [1-2].

IgG antibodies are the most abundant immunoglobulins in human serum (about 75%), and play an important role in the immune response by defending against pathogens and toxins. Compared to other immunoglobulins, IgG has a high circulating level, a longer half-life, and the ability to be transferred from mother to offspring. These properties are closely related to its interaction with FcRn. FcRn binds to the Fc region of IgG, preventing IgG molecules from being degraded by lysosomes. This prolongs the in vivo half-life of IgG and is involved in the transport, maintenance, and distribution metabolism of IgG. In addition, the specific transport process of IgG from the mother to the fetus to provide the fetus with short-term passive immunity is also mediated by FcRn [1-2]. In addition to its protective role, IgG autoantibodies are also associated with many pathological conditions. Therefore, novel FcRn blocking therapies are an effective strategy to reduce the circulating levels of pathogenic IgG autoantibodies and to reduce IgG-mediated diseases. In addition, many drugs also utilize FcRn's protective mechanism for IgG by fusing or conjugating with the Fc portion of IgG to prolong its serum half-life and improve its pharmacokinetics. The FCGRT gene encodes the α-chain of the FcRn protein, and its homologous genes are present in most mammals.

This model is a humanized FcRn mouse, in which the sequence encoding the extracellular domain of the endogenous protein in the mouse Fcgrt gene has been replaced by the corresponding sequence in the human FCGRT gene. B6N-hFCRN(Extra) mice are therefore useful for in vivo studies of IgG, screening of IgG antibody drug candidates, and evaluating the pharmacology, efficacy, and pharmacokinetics of drugs. The homozygous mice are viable and fertile.

Strain Strategy

The mouse Fcgrt endogenous extracellular domain (aa.22~297) was replaced with the human FCGRT extracellular domain (aa.24~297). The murine signal peptide(aa.1~21) was remained.


Applications

  • In vivo transport, maintenance, and metabolism studies of IgG;
  • Development and screening of IgG antibody drug candidates;
  • Pharmacology, efficacy, and pharmacokinetics of IgG antibody drugs;
  • Evaluation of Fc-based immunotherapy.

 

Validation Data

1. Expression detection of human FCGRT gene


Figure 1. Expression of the human FCGRT gene (hFCGRT) in the kidneys and livers of wild-type (WT) mice and B6N-hFCRN (Extra) mice. RT-qPCR was used to detect the expression of the human FCGRT gene using species-specific primers. The results showed that B6N-hFCRN (Extra) mice successfully expressed the human FCGRT gene.

2. Expression detection of human FcRn protein


Figure 2. Human FcRn protein expression in the liver and kidney of wild-type mice (WT) and B6N-hFCRN(Extra) mice. The expression of human FcRn protein was detected by Western Blotting using a species-specific FcRn antibody. The results showed that B6N-hFCRN(Extra) mice successfully expressed human FcRn protein.

3. Mouse IgG Expression Assay

Figure 3. Expression of mouse IgG in the serum of B6-hFcRn(Extra) mice and wild-type (WT) mice (7-week-old, homozygous, female, n = 6). The serum of the mice was collected, and the expression level of mouse IgG was detected by enzyme-linked immunosorbent assay (ELISA) using a mouse IgG-specific ELISA kit. The results showed that the content of mIgG in the serum of B6-hFcRn(Extra) mice was lower than that in WT mice.

 

4. In vivo pharmacokinetics (PK) of monoclonal antibody drugs

a. Atezolizumab

Figure 4. Metabolic analysis of Atezolizumab in B6-hFcRn(Extra) mice and wild-type (WT) mice (8-week-old, homozygous, female, n = 3). The human IgG1 subtype antibody Atezolizumab at a concentration of 10 mg/kg was injected into the tail veins of B6-hFcRn(Extra) mice and WT mice respectively. The serum of the mice was collected at different time points, and the concentration of the hIgG1 subtype antibody was analyzed by enzyme-linked immunosorbent assay (ELISA) using a human IgG1-specific ELISA kit. The results showed that, calculated according to the first-order elimination kinetics, the half-life of Atezolizumab in B6-hFcRn(Extra) mice was 66.51 h (2.77 days), and the half-life in WT mice was 65.71 h (2.73 days).

*Atezolizumab is a humanized monoclonal IgG1 antibody. It activates the tumor-specific immune response by blocking the interaction between PD-L1 and PD-1/B7.1 receptors, and can directly inhibit the proliferation of tumor cells, and induce mitochondrial-related apoptosis and autophagy [3].

 

b. Evolocumab

Figure 5. Metabolic analysis of Evolocumab in B6-hFcRn(Extra) mice and wild-type (WT) mice (8-week-old, homozygous, female, n = 3). The human IgG2 subtype antibody Evolocumab at a concentration of 10 mg/kg was injected into the tail veins of B6-hFcRn(Extra) mice and WT mice respectively. The serum of the mice was collected at different time points, and the concentration of the hIgG2 subtype antibody was analyzed by enzyme-linked immunosorbent assay (ELISA) using a human IgG2-specific ELISA kit. The results showed that, calculated according to the first-order elimination kinetics, the half-life of Evolocumab in B6-hFcRn(Extra) mice was 135.62 h (5.65 days), and the half-life in WT mice was 266.94 h (11.12 days).

*Evolocumab is a fully human monoclonal antibody. By specifically binding to and inhibiting proprotein convertase subtilisin/kexin type 9 (PCSK9), it increases the number of low-density lipoprotein (LDL) receptors on the surface of hepatocytes, thereby significantly reducing the level of low-density lipoprotein cholesterol (LDL-C) in the blood. It is suitable for the treatment of primary hypercholesterolemia, familial hypercholesterolemia (including homozygous and heterozygous types), and the control of cardiovascular risks in patients with atherosclerotic cardiovascular diseases [4].

 

References
[1]Challa DK, Velmurugan R, Ober RJ, Sally Ward E. FcRn: from molecular interactions to regulation of IgG pharmacokinetics and functions. Curr Top Microbiol Immunol. 2014;382:249-72.
[2]Patel DD, Bussel JB. Neonatal Fc receptor in human immunity: Function and role in therapeutic intervention. J Allergy Clin Immunol. 2020 Sep;146(3):467-478.
[3]Ilie M, Hofman P. Atezolizumab in advanced non-small cell lung cancer. J Thorac Dis. 2017 Oct;9(10):3603-3606.
[4]Amgen Inc. (2024). REPATHA (evolocumab) injection, for subcutaneous use [PDF document]. U.S. Food and Drug Administration. https://www.accessdata.fda.gov/drugsatfda_docs/label/2024/125522s043lbl.pdf