Single-chain Fv multimers of the anti-neuraminidase antibody NC10: the residue at position 15 in the VL domain of the scFv-0 (VL−VH) molecule is primarily responsible for formation of a tetramer–trimer equilibrium

Abstract

Single-chain variable fragment of the murine monoclonal antibody NC10 specific to influenza virus N9 neuraminidase, joined directly in the V(L) to V(H) orientation (scFv-0), forms an equilibrium mixture of tetramer and trimer with the tetramer as the preferred multimeric species. In contrast, the V(H)-V(L) isomer was previously shown to exist exclusively as a trimer. Computer-generated trimeric and tetrameric scFv models, based on the refined crystal structure for NC10 Fv domain, were constructed and used to evaluate factors influencing the transition between V(L)-V(H) trimer and tetramer. These model structures indicated that steric restrictions between loops spanning amino acid residues L55-L59 and L13-L17 from the two adjacent V(L) domains within the V(L)-V(H) trimer were responsible for four scFv-0 molecules assembling to form a tetramer. In particular, leucine at position L15 and glutamate at position L57 appeared to interfere significantly with each other. To minimize this steric interference, the site-directed mutagenesis technique was used to construct several NC10 scFv-0 clones with mutations at these positions. Size-exclusion chromatographic analyses revealed that several of these mutations resulted in the production of NC10 scFv-0 proteins with significantly altered tetramer-trimer equilibrium ratios. In particular, introduction of a polar residue, such as asparagine or threonine, at position L15 generated a highly stable NC10 scFv-0 trimer.

Multivalency: the hallmark of antibodies used for optimization of tumor targeting by design

“…A scFv molecule with a linker of 3-12 residues cannot fold into a functional Fv domain and, instead, associates with a second scFv molecule to form a divalent dimer, that is a diabody, $55 kDa. (30) Reducing the linker length below three residues can force scFv association into trivalent trimers triabodies, $80 kDa (31) or tetravalent tetrabodies, $110 kDa (32) depending on composition and Vdomains orientation. More complicated divalent single chain antibody (VH1-VL2-VH2-VL1) ( Fig.…”

Structural Characterization of a Therapeutic Anti-Methamphetamine Antibody Fragment: Oligomerization and Binding of Active Metabolites

“…Dolezal et. al [22] observed that the anti-neuraminidase scFv exists as trimers and tetramers, however the model they proposed involves swapping of domains in a cyclic manner either to form trimers or tetramers in contrast to the assembly of intact scFv6H4 molecules observed in the present case. Interestingly, we have observed spontaneous conversion of some monomer to multimeric forms in vivo also, but we could not determine a mechanism [12].…”

“…Although homo-oligomerization of scFvs into dimers, trimers and sometimes tetramers is well known [21,22], the common mechanism for formation is an association of a variable heavy region of one chain with the variable light region of another, unlike the associations that we see in the scFv6H4 apo structure. The formation of an oligomeric state is often dependent upon the length of the linker between domains, with shorter linkers favoring higher order oligomers [23].…”

Stabilization of the Single-Chain Fragment Variable by an Interdomain Disulfide Bond and Its Effect on Antibody Affinity

“…The VH domain of one chain was suggested to pair with the VL domain of another chain and vice versa . Besides the external factors, the interface stability affected protein aggregation behavior deeply, such as the linker between two domains [23,24] and the residues at the interface [3,5]. In this paper, we focused on scFv stabilization by an interdomain disulfide bond.…”