Construction of miniantibodies for the in vivo study of human autoimmune diseases in animal models

Abstract

Background: Phage display antibody libraries have been made from the lymphocytes of patients suffering from autoimmune diseases in which the antibodies are known to play a role in the pathogenesis or are important for the diagnosis of the disease. In the case of Celiac Disease, the immune response is directed against the autoantigen tissue transglutaminase. However, despite numerous studies, the role of these antibodies in the pathogenesis of this disease has not been elucidated.

Results: We were able to engineer specific anti-transglutaminase antibody fragments in the form called "miniantibody". These are produced by genetic fusion of anti-tTG scFv to Human, Mouse or Rat Fc domains, making them suitable for in vivo expression. The results obtained here indicate that the miniantibody molecule is efficiently secreted, and that the reactivity to the antigen is retained even after fusion to heterologous Fc domains. Further analysis demonstrate that the molecule is secreted as homodimeric, mimicking original antibody structure. Finally, the in vivo expression in mice leads to detectable serum levels with no apparent gross immune response by the host.

Conclusion: In this work we demonstrated the usefulness of a method for the in vivo expression of miniantibodies specific to transglutaminase, corresponding to the autoimmune specificity of Celiac Disease. This can be proposed as a general method to study the pathogenic role of autoimmune antibodies in autoimmune diseases.

Figure 1

Schematic representation of the cloning vector. The Human IgG1 CH2-CH3 domains gene in the vector pMB-SV5 could be substituted by Human IgA, Mouse and Rat Fc domain genes using restriction sites NheI and SpeI. Different scFvs can be exchanged by using restriction sites BssHII and NheI.

Figure 2

ELISA of supernatants of cultured HEK 293T cells transfected with the series of plasmids pMB-SV5 carrying 2.8 scFv gene (A and B) and 3.7 scFv gene (C and D) fused to CH2-CH3 domains genes from human, mouse and rat. Antigens: human tTG, mouse tTG and BSA. Secondary antibodies: A) and C) biotinylated mAb SV5 and streptavidin conjugated with peroxidase; B) and D) goat anti human, mouse and rat IgG or IgA conjugated with peroxidase.

Figure 3

A, Western blotting of the miniantibody MB-MoG-2.8 with reducing agents (lane 2), treated with glycosidase PNGase F (lane 1), and in not reducing not denaturing conditions (lane 3). B, Immunohistochemistry performed on histological section of mouse muscle tissue with the miniantibodies constructs MB-MoG-2.8 and 3.7. Secondary antibodies: biotinylated mAb SV5 followed by streptavidin conjugated with alkaline phosphatase (western blotting) or horseradish peroxidase (immunohistochemistry).

Figure 4

Complement fixation assay with the miniantibodies constructs MB-HuG-2.8 and 3.7, MB-MoG-2.8 and 3.7, MB-RaG-2.8 and 3.7. The binding of C1q to the miniantibodies is revealed with biotinylated anti-C1q and streptavidin conjugated with alkaline phosphatase. Positive and negative control are represented by the murine anti-His D8 (IgG2a) and CUB7402 (IgG1) mAb, both recognizing the coated tTG.

Figure 5

Inhibitory effect of purified MB-MoG-3.7 (grey bars), MB-MoG-2.8 (black bars) and mAb CUB7402 (hatched bars) on mouse tTG activity. Elisa plates coated with gliadin, a tTG substrate, are incubated with 0.2 mM 5-(biotinamido)pentylamine and 0.25 μg of mouse tTG, with increasing amounts of purified miniantibody or mAb. The incorporation of 5-(biotinamido)pentylamine is revealed by streptavidin conjugated with peroxidase.

Figure 6

ELISA time course of the serum anti-tTG miniantibody average titer in 8 BALB/c mice injected at 0 and 14 days with pMB-MoG-3.7 (panel A) and 2.8 (panel B) DNA. Serum dilution 1:50. Secondary antibodies: biotinylated mAb SV5 and streptavidin conjugated with peroxidase.

Figure 7

In situ PCR on histological section of quadriceps muscle of a mouse injected with pMB-MoG-3.7 construct. PCR was performed after 40 days since injection. The arrows point at cells with positive reaction.