Targeting therapy to the neuromuscular junction: proof of concept.
Bottom Line: The site of pathology in myasthenia gravis (MG) is the neuromuscular junction (NMJ).A single-chain antibody directed against the alpha subunit of the acetylcholine receptor was synthesized (scFv-35) and coupled to decay-accelerating factor (DAF, scFv-35-DAF). scFv-35-DAF was tested in a passive model of experimentally acquired MG.We demonstrate a method to effectively target a therapeutic agent to the NMJ.
Affiliation: Department of Pharmacology and Physiology, George Washington University, Washington, DC, USA.Show MeSH
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Mentions: We utilized the TIB-175 hybridoma cell line (ATCC), which produces monoclonal antibody 35, an antibody known to bind the AChR,23,24 for isolation of RNA and then performed PCR amplification of variable heavy-chain (VH) and variable light-chain (VL) regions. We then performed overlapping PCR to generate VH–VL fragment with a linker (GGGGS)3 to produce the single-chain AChR antibody scFv-35 (Fig. 1). Monoclonal antibody 35 (MAb-35) is an antibody used commonly in EAMG studies.10,24,25 Constructs were produced with an IgG signal peptide. To attach the DAF domains, we utilized the C57Bl/6J for RNA and PCR to clone the 4 extracellular complement control protein modules of 255 amino acids and produced the scFv-35-Daf (Fig. 2). We cloned all constructs into the pIRES2-AcGFP1 vector (Clontech, Mountain View, California). This vector allows expression of 2 separate proteins, 1 green fluorescent protein (GFP) and the other protein of interest, rather than a single fusion protein, which limits concerns regarding the risk of loss of biological activity of the protein of interest.
Affiliation: Department of Pharmacology and Physiology, George Washington University, Washington, DC, USA.