[Intellectual Contribution][Advances in Technology]

Single domain intrabodies against WASP inhibit TCR-induced immune responses in a transgenic mouse model

Mitsuru Sato, Chisato Sakuma, Takato Takenouchi, Hiroshi Kitani
Animal Immune and Cell Biology Research Unit
[Abstract]
Intrabody technology provides a novel approach to decipher the molecular mechanisms of protein function in cells. In the present study, we demonstrated that single domain intrabodies composed of only the variable regions (VH or VL) of antibodies retain specific binding activity to the target molecule and efficiently inhibit domain function in a transgenic mouse model.
[Keywords]
intrabody, single domain, functional knockdown, transgenic mouse

[Background]

Gene-knockout and RNA interference (RNAi) technologies are powerful tools for understanding gene function. However, these gene-knockout and silencing techniques cannot be used to analyze domain structures, functions, and post-translationally modified protein functions. Intracellularly expressed antibody fragments (intrabodies) have been used as powerful tools for clinical applications and for basic studies of intracellular proteins. Specific binding of intrabodies to the target domain selectively inhibits the function of intracellular proteins. Among several types of intrabodies developed so far, single domains composed of only the variable regions (VH or VL) of antibodies are the smallest and thus the easiest to design. The present work demonstrates that single domains are sufficient to function as intrabodies.
[Results and Discussion]
  1. We developed transgenic (Tg) mice expressing the VH or VL single domains derived from a monoclonal antibody raised against the N-terminal domain of Wiskott-Aldrich syndrome protein (WASP) (Fig. 1). WASP is predominantly expressed in the cytosol of hematopoietic cells and regulates immune responses, such as the production of interleukin (IL)-2 in T cell receptor (TCR) signaling.
  2. In T cells from anti-WASP VH and VL single domain Tg mice, anti-WASP single domains efficiently bound to the WASP N-terminal domain and inhibit IL-2 production upon TCR stimulation (Fig. 2).
  3. These results strongly suggest that the WASP N-terminal domain plays a pivotal role in IL-2 production in TCR signaling and the VH/VL single domain intrabodies are sufficient to knockdown the domain function of target proteins in the cytosol.
[Future prospects]
  1. The VH/VL single domain intrabodies should be valuable tools for identifying novel protein functions, and transgenic mice that express single domain intrabodies may be useful in functional knockdown models.
  2. Intrabody technology can be used both to elucidate disease mechanisms and to provide novel therapies. In the pharmaceutical industry, intrabodies could be potentially powerful tools particularly for target discovery and validation. Intrabodies offer the possibility of selectively blocking the function of target molecule on a domain or epitope specific basis.

Fig.1. Generation of transgenic mice expressing the VH or VL single domains derived from a monoclonal antibody


Fig.2. Single domain intrabodies against WASP inhibit TCR-induced immune responses in transgenic mice T cells

 

[Reference]

  1. Sato M, Sawahata R, Sakuma C, Takenouchi T, Kitani H (2013) Single domain intrabodies against WASP inhibit TCR-induced immune responses in transgenic mice T cells Scientific Reports 3:3003
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