Single-chain variable fragments (scFvs) of anti-Lewis y hu3S193 humanized antibody were constructed by joining the V H and V L domains with either +2 residues, +1 residue, or by directly linking the domains. In addition two constructs were synthesized in which one or two C-terminal residues of the V H domain were removed (-1 residue, -2 residue) and then joined directly to the V L domain. An scFv construct in the reverse orientation with the V L joined directly to the V H domain was also synthesized. Upon transformation into Escherichia coli all scFv constructs expressed active protein. Binding activity, multimeric status, and multivalent properties were assessed by flow cytometry, size exclusion chromatography, and biosensor analysis. The results for hu3S193 scFvs are consistent with the paradigm that scFvs with a linker of +3 residues or more associate to form a non-covalent dimer, and those with a shorter linker or directly linked associate predominantly to form a non-covalent trimer and tetramer that are in equilibrium. While the association of V domains to form either a dimer or trimer/tetramer is governed by the length of the linker, the stability of the trimer/tetramer in the equilibrium mixture is dependent on the affinity of the interaction of the individual V domains to associate to form the larger Fv module.Keywords: scFv multimers; diabody; triabody; trimer; tetrabody; anti-Lewis y antibody; hu3S193Single-chain variable fragments (scFvs) were designed to link the variable heavy-chain domain (V H ) and variable light-chain domain (V L ) via a flexible peptide linker to stabilize and enhance their interaction with each other to form an antigen-binding site (Huston et al. 1991;Malby et al. 1993). Peptide linkers >12 amino-acid residues provide sufficient spatial flexibility for the V H and V L domains to associate predominantly as scFv monomers with only small amounts of higher molecular mass multimers evident (Griffiths et al. 1993;Kortt et al. 1994;Whitlow et al. 1994). Decreasing the linker length to <10 residues prevents the V domain from interacting with its attached partner and promotes the interaction of complementary V H /V L pairs from two scFv molecules to associate to form noncovalent multivalent multimers Iliades et al. 1997;Kortt et al. 1997;Pei et al. 1997;Atwell et al. 1999;Le Gall et al. 1999;Dolezal et al. 2000). The formation of scFv dimers, trimers, and tetramers by noncovalent association of tethered V domains is dependent on the length of the linker joining the domains and the orientation of the V domains in the scFv construct . In general, with linkers of 3 to 10 residues, scFv dimers (diabodies) are predominantly formed whereas trimers (triabodies) and tetramers (tetrabodies) are formed with linkers of 2 residues or less.
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