Binding characteristics and tumor targeting of a covalently linked divalent CC49 single-chain antibody

Multivalency is a recognized means of increasing the functional affinity of single-chain Fvs (scFvs) for optimizing tumor uptake. A unique divalent si ngle-chain Fv protein [sc(Fv)(2)], based on the variable regions of the mon oclonal antibody (MAb) CC49, has been generated that differs from other dim eric single-chain constructs in that a linker sequence (L) is encoded betwe en the repeated V-L and V-H domains (V-L-L-V-H-L-V-L-L-V-H). This construct was expressed in soluble form in Escherichia coli and purified by ion-exch ange and gel-filtration chromatography, purity and immunoreactivity were de termined by SDS-PAGE, HPLC and competitive RIA, sc(Fv)(2) exhibited a relat ive K-A (3.34 x 10(7) M-1) similar to that of the native IgG (1.14 x 10(8) M-1) as determined by BIAcore analysis. Pharmacokinetic studies showed rapi d blood clearance for sc(Fv)(2), with a T-1/2 less than 40 min. Whole-body clearance analysis also revealed rapid clearance, suggesting no significant retention in the extravascular space or normal tissues. Biodistribution st udies of radiolabeled sc(Fv)(2) showed tumor uptake greater than 6% ID/g af ter 30 min, which remained at this level for 6 hr, High tumor uptake and re tention of sc(Fv)(2) coupled with rapid blood and whole-body clearance make s this dimeric scFv of MAb CC49 a strong candidate for imaging and therapeu tic applications, (C) 1999 Wiley-Liss, Inc.