An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability

The effects of linker length on binding affinity and degreeof aggregation have been examined in the antifluorescein 4-4-20and anticarcinoma CC49 single-chain Fvs. Longer linkers in theantifluorescein sFvs have higher affinities for fluoresceinand aggregate less. A proteolytically susceptible site betweenLys8 and Ser9, in the previously reported 212 linker has beenidentified. A new linker sequence, 218 (GSTSGSGKPGSGEGSTKG)was designed in which a praline was placed at the C-terminalside of the proteolytic clip site in the 212 linker. The CC49sFv containing the 218 linker showed reduced aggregation andwas found to be more stable to proteolysis in vitro, when comparedto the CC49/212 sFv. The CC49 sFv with the longer 218 linkerhad higher affinity than CC49/212 sFv. An aggregated CC49/212sFv sample had higher affinity than CC49/218 sFv. The CC49/218and CC49/212 sFvs had similar blood clearances in mice, whilethe aggregated CC49/212 sFv remained in circulation significantlylonger. In mice bearing LS-174T human colon carcinoma xenografts,the CC49/218 sFv showed higher tumor uptake than the CC49/212sFv and lower tumor uptake than the aggregated CC49/212 sFv.The higher tumor uptake of the CC49/218 is most likely a resultof its higher resistance to proteolysis. The higher affinityand higher tumor uptake of the aggregated CC49/212 sFv are mostlikely due to the repetitive nature of the TAG-72 antigen andthe higher avidity of multivalent aggregates. When the sFvswere radiolabeled with a lutetium-chelate the CC49/218 sFv showeda lower accumulation in the liver and spleen compared to theaggregated CC49/212 sFv.