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A single-chain Fv (scFv) fragment derived from the murine antibody 4G7, specific for human lymphocyte CD19, was engineered for stability and expression in Escherichia coli in view of future use as a therapeutic protein. We compared two orthogonal knowledge-based procedures. In one approach, we designed a mutant with 14 single amino-acid substitutions predicted to correct destabilizing residues in the 4G7-wt sequence to create 4G7-mut. In the second variant, the murine CDRs were grafted to the human acceptor framework huVkappa3-huV(H)3, with 11 additional point mutations introduced to obtain a better match between CDR graft and acceptor framework, to arrive at 4G7-graft. Compared to 4G7-wt, 4G7-mut showed greater thermodynamic stability in guanidinium chloride-induced equilibrium denaturation experiments and somewhat greater stability in human serum. The loop graft maintained the comparatively high stability of the murine loop donor, but did not improve it further. Our analysis indicates that this is due to subtle strain introduced between CDRs and framework, mitigating the otherwise highly favorable properties of the human acceptor framework. This slight strain in the loop graft is also reflected in the binding affinities for CD19 on leukemic cells of 8.4 nM for 4G7-wt, 16.4 nM for 4G7-mut and 30.0 nM for 4G7-graft. This comparison of knowledge-based mutation and loop-grafting-based approaches will be important, when moving molecules forward to therapeutic applications.

protein engineering, design and selection

Stabilization and humanization of a single-chain Fv antibody fragment specific for human lymphocyte antigen CD19 by designed point mutations and CDR-grafting onto a human framework