Multivalent Fvs: characterization of single-chain Fv oligomers and preparation of a bispecific Fv

Abstract

Single-chain Fv proteins are known to aggregate and form multimeric species. We report here that these molecules represent a new class of molecular assembly, which we have termed multivalent Fvs. Each binding site in a multivalent Fv comprises the variable light-chain (VL) domain from a single-chain Fv, and the variable heavy-chain (VH) domain from a second single-chain Fv. Each single-chain Fv in a multivalent Fv is part of two binding sites. We have characterized the multivalent forms of the 4-4-20, CC49 and B6.2 sFvs. The degree of multivalent Fv formation is linker-dependent. Multivalent Fvs cannot form in the absence of an intact linker. Multivalent Fvs can be stabilized by their antigen. The conversion between different forms of the multivalent Fvs can be catalyzed by disassociating agents such as 0.5 M guanidine hydrochloride with 20% ethanol. Multivalent Fvs have significantly different stabilities depending on the specific variable domains from which they are constructed. Two models have been proposed for the structure of a multivalent Fv. We have tested each model by attempting to produce a heterodimer from the anti-fluorescein 4-4-20 and anti-tumor CC49 variable regions. We successfully produced a 4-4-20/CC49 heterodimer that comprises two mixed sFvs. The first mixed sFv is composed of the 4-4-20 VL domain, a 12 residue linker and the CC49 Vh domain. The second mixed sFv is composed of a CC49 VL domain, a 12 residue linker and the 4-4-20 VH domain. The 4-4-20/CC49 heterodimer bound both fluorescein and the tumor-associated glycoprotein-72 antigen. These results support a VH/VL 'rearrangement' model in which each variable domain of a multivalent Fv binding site comes from a different polypeptide chain.

The Functional Architecture of an Acetylcholine Receptor-mimicking Antibody

“…Therefore, our data strongly suggest that the periplasmic space of the bacteria contained the expected scFv form of M␣2-3. We also noted that neither inversion of the order of the variable domains (VH was followed by the 14-amino acids linker and VL) nor an increase of the length of the linker peptide from 14 to 18 residues (GSTSGSGKPGSGEGSTKG), 2 as suggested in other instances (27), modified the specificity and affinity of the recombinant hybrid (data not shown). Therefore, the N terminus residues from VL or VH domains of M␣2-3 as well as the linker length play little role in the functional site of the antibody, in agreement with previous data obtained with other scFvs.…”

The 2.0-Å resolution crystal structure of a trimeric antibody fragment with noncognate VH–VLdomain pairs shows a rearrangement of VH CDR3

“…Because the V H and V L domains are directly fused to each other, it is not sterically possible for the V H and V L domains in a single polypeptide to pair with each other. Consequently, the polypeptides form oligomers in which the V H -V L pairing is achieved intermolecularly (16)(17)(18)(19). This type of multivalent antibody fragment has been referred to as a diabody (15,17), although dimeric, trimeric, and higher-order multimers can be isolated (16)(17)(18)(19).…”

Thermal stabilization of a single‐chain Fv antibody fragment by introduction of a disulphide bond

“…Although both the scFv and ds-Fv existed predominantly in the monomeric form, trace amounts of dimer were removed by HPLC prior to surface plasmon resonance analysis. Dimerization is frequently seen with scFv m~ lecules [16] and has an especially significant effect on the bil ding properties of this antibody because of the multivalent naure of its carbohydrate antigen. Both the association and dissociation phases were fitted well by the BIAevaluation 2.0 on :-component interaction model.…”