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

Abstract

Multivalency is a recognized means of increasing the functional affinity of single-chain Fvs (scFvs) for optimizing tumor uptake. A unique divalent single-chain Fv protein [sc(Fv)2], based on the variable regions of the monoclonal antibody (MAb) CC49, has been generated that differs from other dimeric single-chain constructs in that a linker sequence (L) is encoded between the repeated VL and VH domains (VL-L-VH-L-VL-L-VH). This construct was expressed in soluble form in Escherichia coli and purified by ion-exchange and gel-filtration chromatography. Purity and immunoreactivity were determined by SDS-PAGE, HPLC and competitive RIA. sc(Fv)2 exhibited a relative KA (3.34 × 107 M−1) similar to that of the native IgG (1.14 × 108 M−1) as determined by BIAcore analysis. Pharmacokinetic studies showed rapid blood clearance for sc(Fv)2, with a T1/2 less than 40 min. Whole-body clearance analysis also revealed rapid clearance, suggesting no significant retention in the extravascular space or normal tissues. Biodistribution studies of radiolabeled sc(Fv)2 showed tumor uptake greater than 6% ID/g after 30 min, which remained at this level for 6 hr. High tumor uptake and retention of sc(Fv)2 coupled with rapid blood and whole-body clearance makes this dimeric scFv of MAb CC49 a strong candidate for imaging and therapeutic applications. Int. J. Cancer 81:911–917, 1999. © 1999 Wiley-Liss, Inc.