Engineering antibody therapeutics: Approaches to neutralizing bacterial toxins.

摘要

Work during the past ten years has set the stage for the Golden Era of protein engineering, during which hundreds of designer proteins will be developed for therapeutic, industrial and technological uses. Most importantly, we now understand the basic science underlying many systems well enough to effectively engineer proteins to produce a desired effect. Antibodies are a common target for engineering efforts because their high affinity and specificity for a binding partner renders them one of the most useful classes of molecules for biotechnology and biomedical applications. Engineering can increase antibody affinity, specificity and stability, reduce immunogenicity and optimize pharmacokinetic parameters.;This work focuses on antibodies that bind and neutralize the bacterial toxins of Bacillus anthracis (protective antigen, PA) and Bordetella pertussis (pertussis toxin, PT), and these antibodies have potential for use as both diagnostic and therapeutic reagents. Efforts have been concentrated in two areas: (1) improving and evaluating antibody neutralization in vivo and in vitro by manipulating biophysical parameters, and (2) identifying the molecular determinants of binding to predict antibody-antigen interactions.;The role of affinity has been addressed by generating a panel of an anti-anthrax toxin scFv which range in affinity from >500 nM to 0.25 nM. The generation of multimeric (more than one binding site per molecule) and higher molecular weight antibodies has been explored to evaluate the effects of avidity and biodistribution in vivo. Neutralization of an anthrax toxin challenge was shown to correlate with antibody affinity both in an in vitro assay and an in vivo rat model. Importantly, an affinity matured scFv is more protective in vivo than lower affinity scFvs, and a higher molecular weight version compares favorably to the parental monoclonal antibody. Similarly, increased molecular weight of an antibody conferred increased protection. Using anti-PT antibodies, low stability has been shown to significantly compromise neutralizing ability in vitro, while high stability appears to be able to compensate for less favorable binding kinetics. This work comprises the first systematic study of the effects of engineering anti-toxin antibodies, and has demonstrated that protein engineering techniques can be used to develop highly potent anti-toxin preparations.