Development of recombinant antibodies for diagnostic applications by protein engineering: Dissertation

Since the advent of hybridoma technology the exquisite specificity of monoclonal antibodies has been exploited for a number of applications in diagnostics, medicine and research. Today, monoclonal antibodies and binding fragments can also be produced by recombinant methods and this offers, in addition to efficient production, the means for convenient and rapid engineering of antibody fragments for different specific applications. In this work antibody engineering was used to increase the available lysine content of an anti-human alpha-fetoprotein (hAFP) Fab fragment and to improve its labelling properties for applications in which sensitive detection is essential. Monoclonal antibody derivatives binding testosterone (TES) with affinity and specificity sufficient for their use as reagents in immunoassays designed for the quantitative measurement of TES in clinical samples were also developed and characterised. Europium (Eu3+) chelates provide a nonradioactive alternative for sensitive labelling of antibodies for diagnostic immunoassays. Lysine residues at antibody surfaces are suitable targets for labelling by an isothiocyanate derivative of the chelate. In this work the labelling efficiency of a recombinant anti-hAFP Fab fragment was improved by increasing its lysine content by protein engineering. Molecular modelling was used to identify three light chain constant domain surface arginine residues which were mutated to lysine residues. The mutations did not influence the affinity of the lysine-enriched Fab fragment, and its labelling efficiency was found to be about 40 percent higher than that of the wild-type Fab fragment. With a low degree of labelling, the affinities of the two Fab fragments were identical and comparable to that of the original monoclonal anti-hAFP IgG. With higher degrees of labelling the affinities of both Fab fragments decreased more than that of the intact IgG since more lysine residues are available for labelling in the additional heavy chain constant domains of the larger molecule. A great number of small, rigid and hydrophobic steroid hormones, with very similar structures and containing only a few functional groups capable of direct interactions with antibodies, have remained as a major challenge for immunodiagnostics. The majority of commercially available diagnostic test kits for steroid hormones utilise polyclonal antibodies, not because of optimal performance and product quality, but because of the apparent lack of monoclonal antibodies fulfilling the clinical requirements. Semirandom mutagenesis of an existing monoclonal antibody combined with different phage display selection strategies was employed in the development of highly specified TES-binding recombinant antibodies. The optimisation of the CDR sequences and the CDR combination resulted in clones having an excellent overall binding profile. It was shown that many variant sequences of the original 3-C4F5 antibody can confer an antibody TES-binding specificity and affinity comparable to those of a rabbit polyclonal anti-testosterone antiserum currently used in a diagnostic immunoassay. To the best knowledge of this author the developed mutant Fab fragments of the 3-C4F5 antibody are the first steroid hormone-binding recombinant antibodies shown to work accurately over the whole physiological concentration range of clinical samples.