Optimization of conditions for formation and analysis of anti-CD19 FVS191 single-chain Fv homodimer (scFv')2.
In this report, we present the production of a dimeric form of anti-CD19 scFv, the FVS191cys (scFv')2. Anti-CD19 scFv FVS191cys was constructed by engineering a cysteine residue at the C terminus of the V1, domain of scFv FVS191. FVS191cys (scFv')2 was formed through a disulfide bond between two FVS191cys molecules. To optimize the yield of FVS191cys (scFv')2, the effects of oxidation time, buffer pH, and temperature on the formation of dimeric scFv were analyzed. Our study indicates that the formation of FVS191cys (scFv')2 is oxidation time- and buffer pH-dependent; a high pH buffer facilitates the formation of disulfide-linked (scFv')2. The maximum yield of FVS191cys (scFv')2 can be achieved when FVS191cys is air-oxidized at 4 degrees C, in buffer with a pH of 8.5-9. The biological activity of FVS191cys (scFv')2 was analyzed by ELISA and an internalization assay. FVS191cys (scFv')2 has a CD19 binding ability similar to that of its parental mAb B43 and is internalized by CD19 positive Nalm 6 cells. This study indicates that FVS191cys (scFv')2 is a potential candidate for tumor diagnosis or therapy.
Wang
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D
University of Minnesota Cancer Center, Department of Laboratory Medicine/Pathology, University of Minnesota, Minneapolis 55455, USA.
Berven
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E
Li
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Q
Uckun
F
F
Kersey
J H
JH
D000818:Animals; D018941:Antigens, CD19 / Q000276:immunology*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000378:metabolism; D004705:Endocytosis; D005818:Genetic Engineering; D006863:Hydrogen-Ion Concentration; D007128:Immunoglobulin Fragments / Q000378:metabolism; D007135:Immunoglobulin Variable Region; D057127:Single-Chain Antibodies; D013696:Temperature; D013997:Time Factors
D018941:Antigens, CD19; D004220:Disulfides; C105673:FVS191Cys-(scFv')2; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D057127:Single-Chain Antibodies