Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies

Bispecific single-chain diabodies (scDb) consist of the variableheavy and light chain domains of two antibodies connected bythree linkers. The structure of an scDb in the V_H–V_L orientationis V_HA–linkerA–V_LB–linkerM–V_HB–linkerB–V_LA,with linkers A and B routinely chosen to be 5–6 residuesand linker M 15–20 residues. Here, we applied displayof scDb on filamentous phage to analyse the composition of optimallinker sequences. The three linkers were randomized in lengthand sequence using degenerated triplets coding for only sixhydrophilic or aliphatic amino acids (Thr, Ser, Asp, Asn, Gly,Ala). Antigen-binding clones were then isolated by one to tworounds of selection on the two different antigens recognizedby the bispecific scDb. Using an scDb directed against carcinoembryonicantigen (CEA) and ß-galactosidase (Gal), we foundthat monomeric scDb had a preferred length of 15 or more aminoacid residues for the middle linker M and of 3–6 residuesfor the linkers A and B. No obvious bias towards a preferredlinker sequence was observed. Reduction of the middle linkerbelow 13 residues led to the formation of dimeric scDb, whichmost likely results from interchain pairing between all theV_H and V_L domains. Dimeric scDb were also formed by fragmentspossessing a long linker M and linkers A and B of 0 or 1 residue.We assume that these dimeric scDb are formed by intrachain pairingof the central variable domains and interchain pairing of theflanking variable domains. Thus, the latter molecules representa novel format of bispecific and tetravalent molecules. Thedescribed strategy allows for the isolation of both optimizedand minimal linker sequences for the assembly of monomeric ordimeric single-chain diabodies.