Noncovalent scFv multimers of tumor‐targeting anti‐Lewis^y hu3S193 humanized antibody

Abstract

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.

Multimerization of anti‐(epidermal growth factor receptor) IgG fragments induces an antitumor effect: the case for humanized 528 scFv multimers

“…2). Multimerization of scFvs is thought to be affected by not only the length of the linker, but also the domain order [8,9,26], however, there have been no reports on the influence of domain order on the antitumor effects of scFv multimers. We recently reported a cytotoxic enhancement by converting the VH-VL order to the VL-VH order in a bispecific diabody; structural superiority for cross-linking target cells may have contributed to this enhancement [27].…”

“…For example, the anti-CD19 scFv trimers were predominantly inactive and showed only monovalent binding to the target cell surface [26,28], and in the case of antiLewis y scFv, the trimers dissociated into inactive monomers in a rapid equilibrium [9]. By contrast, the HLG0 trimers in this study showed the highest growth inhibition effects and higher apparent affinity compared with the HLG1 dimers (Figs 2 and 3, Table 1), suggesting that HLG0 forms functional trimers with three active binding sites and that EGFR is an ideal target for therapeutics using specific scFv multimers.…”

“…Further reduction to fewer than five residues or elimination of the linker leads to the formation of more higher order multimers such as scFv trimers, tetramers (known as triabodies, tetrabodies), etc. [7][8][9][10][11]. These increases in molecular size are thought to prolong serum half-lives, which may bring better tumor-to-blood ratios than either IgG or scFv monomers because of the rapid tumor uptake and clearance of scFv multimers [12].…”

“…Here, we prepared scFv multimers using two types of h528 scFvs each with a different domain order, because the domain order of VH and VL in scFv may influence multimerization [8,9]. The h528 scFv multimers were successfully fractionated from dimers to pentamers, and the h528 scFv trimers with the VH-VL order and no linker showed the highest in vitro and in vivo antitumor effects, comparable with those of Cetuximab and Panitumumab.…”

Antigen binding and stability properties of non‐covalently linked anti‐CD22 single‐chain Fv dimers

“…Further shortening the linker peptide below three residues was shown to result in the formation of trimeric, tetrameric, or larger aggregate species [9][10][11][12]. More recently, the formation of V H -V L oriented scFv antibodies with <3 residue linkers to predominantly dimeric molecules has been described [13,14]. Several studies have shown that in comparison with monovalent counterparts, multivalent scFv fragments exhibited increased functional antigen binding affinity [12,15], favorable in vivo retention within tumor tissue [7,16,17], and markedly slower clearance from the bloodstream [6,18].…”