Developing protein conjugation techniques to enhance cell delivery of therapeutic enzymes

The focus of disease research often surrounds therapeutic pathway identification and the subsequent investigation of proteins or compounds that potentially interfere with disease mechanisms. However, finding targets and effective therapeutic domains often overshadows another important aspect of drug delivery and efficacy; the method of domain conjugation. Unfortunately the combination of good therapeutic components and good therapeutic design can often be amiss, with differing skills and groups needed to marry the two together. In recognition of this, there are new techniques emerging that aim to not only address old problems of stable conjugation, but incorporating new knowledge of drug design. This research takes a reverse approach to drug design in order to better comprehend requirements which are often an afterthought; first looking at conjugation technique and then how it can best be exploited. Thereby, exploring how different conjugation methods can be used to complement existing therapeutic and internalisation strategies. Here, two methods of conjugation were investigated through attachment of bacterial toxin domains derived from botulinum A and diphtheria, with the intention of targeting neuroblastoma cells using a variety of specific and non-specific pathways. Both conjugation techniques proved to be stable in vitro and capable of binding a variety of domains consistently. The data here also emphasized the importance of endosomal escape proteins to increase the translocation of catalytic domains into the cytosol. It was demonstrated that the conjugates formed were able to facilitate the binding between targeting and catalytic domains, enabling specific neuroblastoma internalisation. Moreover, that three domains were able to conjugate together using a linking method and could form a co-operative triple-functioning complex.