Production of stable scFv by mutation of the unusual amino acids at the framework region

Anti-CD19-PE38KDEL scFv is a single chain immunotoxin (IT) in which the Fv portion of the anti-CDl9 mAb, HB12A, in a light chain-linker-heavy chain form, is fused to PE38KDEL, a truncated form of Pseudomonas Exotoxin. The scFv-PE38KDEL binds to and is cytotoxic for CD19 positive Burkitts lymphoma cells. However, it is unstable and rapidly looses its activity at 37aC. Alignment of the framework region of \L to known antibody sequences showed 4 unusual amino acids at positions: 3(G), 21(F), 49(H) and 76(G). To investigate whether any of these residues influenced the stability of the scFv, we changed these amino acids to the most common residues found at each position G3V, F21I, H49Y and G76S. Combination changes (F21I + H49Y + G76S) and (G3V + F21I + H49Y + G76S) were also made. The expression and purification patterns of all the mutants were very similar. The stability of these mutated ITs was studied by incubation at 37aC for 4, 8, 16, 24 and 48 h in PBS or in PBS+0.2% HSA followed by cytotoxicity assay on JD38 cells. The results showed that VJ.G76S was the most stable followed by Vi,G3V. Each of Vz,F21I and Vi,H49Y changes had a weak stabilization effect. The combination of weakly stabilizing mutations with strongly stabilizing mutations resulted in stable phenotype. Binding assays showed that the mutants retained antigen specificity. These studies indicate that identifying unusual amino acids in the framework region and mutating these to conserved amino acids can produce more stable scFvs. This approach could be applied to other recombinant antibodies.