May 31, 2018
Shiga University of Medical Science
Kyoto University
Keio University
Japan Agency for Medical Research and Development
[Key Points]
Successfully developed a therapeutic antibody that eliminates abnormal aggregates in amyotrophic lateral sclerosis.
The self-degrading intrabody is a system that produces therapeutic antibodies for ALS within cells. It specifically binds to and promotes the degradation of the abnormal structure of TDP-43 involved in the disease's onset, but does not react with normally functioning TDP-43 .
The self-degrading intrabody possesses two signals that promote degradation through the two major degradation pathways, the proteasome and autophagy, and is rapidly degraded after being produced within the cell.
The self-degrading intrabody reduced abnormal aggregates of TDP-43 in cultured cells and significantly suppressed cell death.
The self-degrading intrabody also markedly reduced abnormal TDP-43 aggregates in the brains of fetal mice. Furthermore, fetal mice that produced the intrabody in their brains were born and developed normally.
[ Summary ]
A research group led by Professor Makoto Urushitani and Special Research Student (now Hospital Assistant Professor) Yoshitaka Tamaki of the Department of Neurology, Shiga University of Medical Science, in a joint study with Professor Ryosuke Takahashi of the Department of Neurology, Graduate School of Medicine, Kyoto University, and Associate Professor Yoshiaki Furukawa of the Faculty of Science and Technology, Keio University, has successfully developed a new therapeutic antibody that eliminates abnormal aggregates of TDP-43, the causative protein of amyotrophic lateral sclerosis (ALS). ALS is a severe, intractable neurological disease in which muscles throughout the body atrophy and lose strength, with motor neurons throughout the body disappearing as the disease progresses. Although the development of treatments to slow its progression is gradually advancing, there is still no curative therapy. The cause was long unknown, but it has been discovered that an RNA-binding protein called TDP-43, which normally resides in the cell nucleus, disappears from the nuclei of motor neurons in ALS patients and forms abnormal aggregates in the cytoplasm. Furthermore, it was found that these aggregates cause various toxic events leading to neuronal cell death. Consequently, the possibility that eliminating these abnormal TDP-43 aggregates could directly lead to a curative treatment for ALS has garnered attention.
In 2012, Professor Urushitani's group at Shiga University of Medical Science developed the monoclonal antibody 3B12A, which recognizes only TDP-43 with an abnormal structure and does not bind to normal TDP-43 in the nucleus. In this study, to turn this antibody into a gene therapy drug that removes abnormal proteins within cells, the researchers cloned the variable region genes from the heavy and light chains of the 3B12A antibody molecule that bind to the antigen. They then created an artificial gene that connects the two to produce a single-chain variable fragment (scFv). Furthermore, to efficiently degrade aggregates via autophagy, they added a protein degradation signal gene called a chaperone-mediated autophagy (CMA) signal to the scFv, creating a vector gene (3B12A scFv-CMA) that expresses a self-degrading intrabody. As a result, the self-degrading intrabody bound only to abnormal TDP-43 in cultured cells, reduced aggregates, and significantly suppressed cell death caused by these aggregates. Interestingly, the binding of 3B12A scFv-CMA to abnormal aggregates also induced a molecular chaperone called heat shock protein 70 (HSP70), which was observed to have a refolding effect, reducing abnormal TDP-43 aggregates by untangling them. The aggregate-reducing effect of 3B12A scFv-CMA was observed not only in cultured cells but also in the brains of fetal mice, where the gene was introduced using a technique called in utero electroporation. A significant inhibitory effect on TDP-43 aggregates was confirmed, with no apparent adverse effects on brain development. This self-degrading intrabody is rapidly degraded in cells where no target aggregates are present, reducing concerns about adverse events from antibody accumulation, making it extremely promising as a molecular targeted therapy. While further confirmation of its effects in ALS model mice that replicate abnormal TDP-43 aggregates in motor neurons and safety confirmation experiments in primates such as monkeys are necessary, this achievement opens the way for a curative treatment for the intractable disease ALS through a unique approach: using an antibody endowed with self-degrading capabilities via the two degradation systems of autophagy and the proteasome to eliminate intracellular aggregates.
This research was published online in the British scientific journal *Scientific Reports* on April 16, 2018, at 10:00 a.m. (UK time; 6:00 p.m. JST).
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