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 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.