The cloning, characterisation and engineering of an IGF-I-BINDING single chain Fv

摘要

This thesis describes the construction and characterisation of an insulin-like growth factor (IGF-I)-binding single chain Fv (scFv) and the utilisation of this scFv as a model protein for the study of the application of DNA shuffling and ribosome display to antibody engineering. The variable domain genes were isolated from the hybridoma cell line producing the monoclonalantibody and successfully joined by PCR for the construction of the scFv, named anti-GPE.Sequencing of the gene revealed an unusually short heavy chain CDR2 region. The cloned scFv was expressed in E. coli and purified. Expression levels were low and the protein has poor solubility, most likely due to a reduction in folding efficiency caused by the abbreviated CDR2. The purified monomeric form of the protein was analysed for binding to IGF-I using surface plasmon resonance on the BIAcore 1000 with the specificity of the IgG version of the antibody for the three N-terminal residues of IGF-I - Gly-Pro-Glu - reproduced. The scFv's calculated dissociation constant of 3.68 µM is a low affinity for an antibody and is approximately 36-fold weaker than was calculated for the Fabversion of the antibody, but it is concluded that the calculated affinity for the scFv was an apparent affinity that may be an underestimation of true affinity due to the presence of non-functional or misfolded scFv species within the gel-filtration purified monomer peaks. A mutant version of anti-GPE with residues inserted in the CDR2 to restore it to normal length produced a protein with improved expression and solubility characteristics while retaining IGF-I-binding. It was concluded that the shortCDR2 was due to deletions generated during the somatic mutation process and a model for this isdescribed.A ribosome display method using a rabbit reticulocyte lysate as a source of ribosomes was developed for specific selection of anti-GPE against IGF-I. Error prone PCR was used to produce a random point mutated library of anti-GPE (EPGPE). This was taken through several cycles of display and selection but selection for non-specifically binding scFvs was commonly observed. This was probably due to poor folding of ribosome-displayed proteins in the system used, possibly caused by the presence of DTT in the lysate and/or the low capacity of the anti-GPE framework to tolerate mutation while retaining stability. It is assumed misfolds, exposing hydrophobic regions, would have a tendency to non-specifically interact with the selection surface. Of the 64 EPGPE clones screened from four rounds of display and selection, many were shown to have poor or non-specific binding, but one scFv was characterised that was affinity matured 2.6-fold over anti-GPE wild type affinity for IGF-I.A DNA shuffling method was developed to produce libraries of chimaeric scFvs between anti-GPEand NC10 (anti-neuraminidase scFv) with the objective of isolating functional IGF-I-bindingchimaeras. The NC10 scFv had its CDRs replaced with the anti-GPE CDRs prior to the shuffling toincrease the likelihood of isolating IGF-I binders. Ribosome display was used for selection from the chimaera libraries. Selection strategies included elution of specific binders by GPE peptide and a GPE 10-mer peptide. Selection was also performed using IGF-I immobilised on a BIAcore sensorchip as aselection surface. Again, much non-specific selection was observed as seen for display of EPGPE, for what was expected to be the same reasons. Selected scFvs were genuinely chimaeric but with poor expression and solubility and mostly non-specific in their binding. One characterised selected chimaera, made up of three segments of each of the parental scFvs, was shown to bind specifically to IGF-I by BIAcore. Steps to improve the efficiency of the ribosome display system have been identifiedand are discussed.