AN IMPROVED LINKER FOR SINGLE-CHAIN FV WITH REDUCED AGGREGATION AND ENHANCED PROTEOLYTIC STABILITY

The effects of linker length on binding affinity and degree of aggrega tion have been examined in the antifluorescein 4-4-20 and anticarcinom a CC49 single-chain Fvs. Longer linkers in the antifluorescein sFvs ha ve higher affinities for fluorescein and aggregate less. A proteolytic ally susceptible site between Lys8 and Ser9, in the previously reporte d 212 linker has been identified. A new linker sequence, 218 (GSTSGSGK PGSGEGSTKG) was designed in which a proline was placed at the C-termin al side of the proteolytic clip site in the 212 linker. The CC49 sFv c ontaining the 218 linker showed reduced aggregation and was found to b e more stable to proteolysis in vitro, when compared to the CC49/212 s Fv. The CC49 sFv with the longer 218 linker had higher affinity than C C49/212 sFv. An aggregated CC49/212 sFv sample had higher affinity tha n CC49/218 sFv. The CC49/218 and CC49/212 sFvs had similar blood clear ances in mice, while the aggregated CC49/212 sFv remained in circulati on significantly longer. In mice bearing LS-174T human colon carcinoma xenografts, the CC49/218 sFv showed higher tumor uptake than the CC49 /212 sFv and lower tumor uptake than the aggregated CC49/212 sFv. The higher tumor uptake of the CC49/218 is most likely a result of its hig her resistance to proteolysis. The higher affinity and higher tumor up take of the aggregated CC49/212 sFv are most likely due to the repetit ive nature of the TAG-72 antigen and the higher avidity of multivalent aggregates. When the sFvs were radiolabeled with a lutetium-chelate t he CC49/218 sFv showed a lower accumulation in the liver and spleen co mpared to the aggregated CC49/212 sFv.