Improved stability and yield of a Fv-toxin fusion protein by computer design and protein engineering of the Fv

The conversion of the anti-mesothelin monoclonal antibody K1 to a single-chain Fv (scFv) that is fused to a truncated form of Pseudomonas exotoxin A (PE) results in a fusion protein (immunotoxin) that is unstable and refolds very inefficiently. We have devised a method that identifies candidate residues in the framework region of K1 Fv that, when mutated, improved the yield and stability of the protein. The method works by initially aligning the framework sequences of K1 V(H) and V(L) with those of other scFvs that are stable and give a good yield as immunotoxins. Then we assigned a character to each residue that indicates its state of exposure based on the known crystal structures of Fabs. This identifies residues that are not compatible with their environment in the folded state of the protein. Next we calculated the frequencies of different amino acids for each position of the Fvs based on the available sequence database. This identifies residues that are not commonly present in the conserved positions. If these residues are compatible with their exposure profile they are left unaltered. Otherwise, they are identified as candidate residues for mutation. We identified two such residues in the V(H) (T82 and A85) and two in the V(L) (H36 and V60) of K1 that did not seem appropriate for their respective positions. By mutating these residues in K1 into those that occur most commonly in the sequence database or in stable scFvs, we significantly improved the stability and yield of the K1 scFv immunotoxins. By making single and combined mutations we assessed the relative contribution of mutations at these four sites towards the stability and yield of K1 scFv immunotoxins. The method we devised is probably general and can be used to improve other scFvs.