Abstract
Due to their lower production cost compared with monoclonal antibodies, single-chain variable fragments (scFvs) have potential for use in several applications, such as for diagnosis and treatment of a range of diseases, and as sensor elements. However, the usefulness of scFvs is limited by inhomogeneity through the formation of dimers, trimers, and larger oligomers. The scFv protein is assumed to be in equilibrium between the closed and open states formed by assembly or disassembly of VH and VL domains. Therefore, the production of an scFv with equilibrium biased to the closed state would be critical to overcome the problem in inhomogeneity of scFv for industrial or therapeutic applications. In this study, we obtained scFv clones stable against GA-pyridine, an advanced glycation end-product (AGE), by using a combination of a phage display system and random mutagenesis. Executing the bio-panning at 37 °C markedly improved the stability of scFvs. We further evaluated the radius of gyration by small-angle X-ray scattering (SAXS), obtained compact clones, and also visualized open–close dynamics of these scFvs by high-speed atomic force microscopy (HS-AFM), revealing that one of the compact clones was biased to the closed state. Finally, nuclear magnetic resonance (NMR) analysis revealed that peak intensity and line width became homogeneous, supporting that dynamic features and/or formation of oligomers was improved in the thus-obtained clone. These findings should contribute to the future industrial and therapeutic use of scFvs.
Highlights
1.1.IntroductionCurrently, monoclonal monoclonal antibodies antibodies are are widely widely used used for for the the diagnosis diagnosis and and treatment treatment of of various variousCurrently, diseases,and andas assensor sensorelements elements[1,2,3,4]. [1,2,3,4].MonoclonalMonoclonalantibodies antibodiesrequire requirecultivation cultivationof ofmammalian mammaliancells cells diseases, forpreparation, preparation,resulting resultinginin a high production cost
Our results demonstrate that the combination of a phage display system and high-speed atomic force microscopy (HS-AFM) analyses for obtaining a clone that exhibits plus reduced
The mouse antibody library immunized with GA-modified keyhole limpet hemocyanin (KLH) was displayed on the phage and used for bio-panning
Summary
1.1.IntroductionCurrently, monoclonal monoclonal antibodies antibodies are are widely widely used used for for the the diagnosis diagnosis and and treatment treatment of of various variousCurrently, diseases,and andas assensor sensorelements elements[1,2,3,4]. [1,2,3,4].MonoclonalMonoclonalantibodies antibodiesrequire requirecultivation cultivationof ofmammalian mammaliancells cells diseases, forpreparation, preparation,resulting resultinginin a high production cost. Monoclonal monoclonal antibodies antibodies are are widely widely used used for for the the diagnosis diagnosis and and treatment treatment of of various various. Diseases,and andas assensor sensorelements elements[1,2,3,4]. Monoclonalantibodies antibodiesrequire requirecultivation cultivationof ofmammalian mammaliancells cells diseases, forpreparation, preparation,resulting resultinginin a high production cost. Fv fragment antibodies for a high production cost. Fv fragment (scFv)(scFv) antibodies may may aoffer a promising approach for overcoming this These problem. These antibodyare derivatives are offer promising approach for overcoming this problem. Antibody derivatives recombinant recombinant proteinsofconsisting of a heavy-chain variable region and light-chain proteins consisting a heavy-chain variable region (VH) and(VH)
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