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

Abstract

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.

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody

“…This is often compensated for by mutations in the framework region to recover the native mouse structure pair. 27,28 In this study, we examined a structure guided approach to reduce the potential risk of immunogenicity of a rodent derived antibody. The antibody selected for humanization was the antimyostatin murine antibody RK35.…”

Dual-Targeting for the Elimination of Cancer Cells with Increased Selectivity

“…Two additional sctbs have been produced and studied, which further illustrate unique functional capabilities offered by dual-targeting, the sctbs 33-ds16-ds19 and HLA-ds16-hu19. The prefix "hu" signifies a murine scFv humanized by CDR-grafting plus additional point mutagenesis [48]. The first of these two sctbs, 33-ds16-ds19, was designed for the treatment of mixed lineage leukemias (MLL), also called bi-phenotypic leukemia.…”

The use of single chain Fv as targeting agents for immunoliposomes: an update on immunoliposomal drugs for cancer treatment

“…It has also been shown that the stability of scFv can be improved when some scFv are expressed as Fab fragments [115]. Other methods, such as point mutation, directed evolution and phage display of scFv constructs, have been used to improve the in vivo stability of scFv [116][117][118].…”

“…In addition, optimization of codon use in specific expression vectors can also increase production yields [157]. Rationalized point mutations designed to improve proper folding and thermal stability should also improve production of scFv [118]. The inclusion of rare codons in the polypeptide linker between the V L and V H has also been shown to improve production yield by slowing down the transcription process and allowing for proper folding of the V L and V H regions [158].…”