BINDING CHARACTERISTICS AND TUMOR TARGETING OF A COVALENTLY LINKED DIVALENT CC49 SINGLE-CHAIN ANTIBODY Guy W. BERESFORD 1 , Gabriela PAVLINKOVA 1 , Barbara J.M. BOOTH 1 , Surinder K. BATRA 2 and David COLCHER 1 * 1 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA 2 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA Multivalency is a recognized means of increasing the func- tional 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 constructsin that a linker sequence (L) is encoded between 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-exchange and gel-filtration chromatography. Purity and immunoreactivity were deter- mined by SDS-PAGE, HPLC and competitive RIA. sc(Fv) 2 exhibited a relative K A (3.34  10 7 M 1 ) similar to that of the native IgG (1.14  10 8 M 1 ) as determined by BIAcore analy- sis. Pharmacokinetic studies showed rapid blood clearance for sc(Fv) 2 , with a T 1/2 lessthan 40 min. W hole-body clearance analysis also revealed rapid clearance, suggesting no signifi- cant 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. H igh tumor uptake and retention of sc(Fv) 2 coupled with rapid blood and whole-body clearance makes thisdimeric scFv of MAb CC49 a strong candidate for imaging and therapeutic applications. Int. J. Cancer 81:911–917, 1999.  1999 Wiley-Liss, Inc. Several anti-carcinoma single-chain Fvs (scFvs) have been evaluated for specific tumor targeting in vivo to antigens such as TAG-72, CEA and the c-erbB-2 receptor. In comparison with the much larger Fab', F(ab' ) 2 and IgG forms of monoclonal antibodies (MAb), scFvs have lower retention times in non-target tissues and exhibit more rapid blood clearance and better tumor penetration (Adams et al., 1993; Colcher et al., 1990; Milenic et al., 1991; Yokota et al., 1993). However, due to rapid clearance from the blood pool (with half-lives less than 15 min) of these 25- to 30-kDa proteins, the absolute amount of scFv uptake by a tumor is limited. Valency is an effective means of increasing the functional affinity (Karush, 1970) of an antibody to the surface of a polymeric antigen. Multivalent scFvs exhibit increased functional affinity compared to monovalent forms (Pack et al., 1995). Consequently, multivalent radiolabeled scFvs should show improved performance in imaging and immunotherapy. The effect of valency is not restricted to IgG-based molecules; it has been well documented in other systems. The hepatic lectin binds to the trimeric ligand with a 100- to 1,000-fold higher functional affinity than the monovalent ligand (Verrey and Drickamer, 1993). The functional affinity of the dimeric Helix pomatia A hemagglutin to its ligand on human A erythrocytes is 10,000-fold higher than the intrinsic affinity of monomeric hemagglutin (Hammarstro ¨m, 1973). Consequently, different strategies are being explored for the formation of scFv dimers and multimers to optimize tumor uptake and clearance properties. The simplest approaches to production of multimeric scFvs are based on the spontaneous formation of non-covalently linked dimers, such as 50-kDa diabodies (Holliger et al., 1993), or trimers as triabodies and trimers having no additional linker sequence between the V L and V H chains (Iliades et al., 1997; Kortt et al., 1997). Introduction of a cysteine at the C terminus of the scFv has been utilized to covalently link 2 scFvs together via site-specific dimerization (Adams et al., 1993). Hu et al. (1996) have produced an antibody fragment called a minibody by fusion of T84.66 anti-CEA scFV to the human IgG 1 CH 3 domain. To promote multimer formation, other investigators have fused scFvs to protein domains capable of multimerization, e.g., amphipathic helices (Pack et al., 1995), leucine zipper proteins (Kostelny et al., 1992) and the -constant region (McGregor et al., 1994). Although several groups have successfully expressed different forms of multivalent scFvs, few have evaluated the targeting or therapeutic efficacy of these constructs in an in vivo model. Biodistribution studies of Adams et al. (1993) using a cysteine-linked divalent scFv showed a 2-fold improvement of in vivo tumor targeting compared with the corresponding monomeric form. Wu et al. (1996) showed improved targeting by a non-covalently linked dimeric form of T84.66 scFv, with tumor uptakes reaching the range of 5% to 15% ID/g compared with the monomeric form (1% to 5% ID/g). In this report, we have used a novel approach to the generation of a divalent scFv for the MAb CC49, which reacts with a unique disaccharide, Sialyl-Tn, present on the tumor-associated mucin TAG-72, expressed by a majority of human adenocarcinomas. The unique Sialyl-Tn epitope has provided a potential target for cancer immunotherapy. The monomeric construct of this CC49 scFv consists of the V L and V H variable regions of MAb CC49 (Muraro et al., 1988) joined with a 25–amino acid helical linker (205C), resulting in a (V L -L-V H ) structure. When expressed, this construct forms monomeric (scFv) and a non-covalently linked dimeric scFv, (scFv) 2 . We have developed a construct in which 2 repeating chains of V L and V H are covalently linked in tandem, using the same helical linker between each region (V L -L-V H -L-V L -L-V H ). This dimeric scFv was designated sc(Fv) 2 to distinguish it from the non-covalently linked dimer derived from the monomeric construct (scFv) 2 . The dimeric structure of sc(Fv) 2 does not rely on non- covalent interactions as found in diabodies and constructs using carboxy-terminal multimerizing complexes, nor does the sc(Fv) 2 molecule require additional chemical processing, such as that used in the formation of disulfide-linked complexes. This novel divalent scFv of MAb CC49 was analyzed for binding characteristics, pharmacokinetics and biodistribution in athymic mice bearing xenografts of the LS-174T human colon carcinoma cell line. MATERIAL AND METHODS Construction of the expression vector The CC49 scFv was constructed through a collaborative effort with the National Cancer Institute’s Laboratory of Tumor Immunol- ogy and Biology and the Dow Chemical Company (Midland, MI). The CC49 scFv (V L -L-V H ) and dimer sc(Fv) 2 (V L -L-V H -L- V L -L-V H ) constructs, in which the V L region sequence and the V H region sequence were combined via a linker sequence designated 205C, were assembled as described by Pavlinkova et al. (1999a). The nucleotide sequences of the CC49 scFv and sc(Fv) 2 constructs were confirmed by sequence analysis. These constructs were cloned into the pRW83 vector, which contained a chloram- Grant sponsor: United States Department of Energy; Grant number: DE-FG02-95ER62024. *Correspondence to: University of Nebraska Medical Center, 983135 Nebraska Medical Center, Omaha, NE 68198-3135, USA. Fax: (402) 559-8112. E-mail: dcolcher@unmc.edu Received 14 September 1998; Revised 14 December 1998 Int. J. Cancer: 81, 911–917 (1999)  1999 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer