Engineered antibody and neuropeptide mediated radionuclide targeting in prostate cancer
Prostate cancer (PC) is the most common cancer type in men in the western world and to date no definitive stratergy to image PC is avaliable. This thesis explores the possibility of using Prostate Specific Membrane Antigen (PSMA) and Gastrin Releasing Peptide Receptor (GRP-R) as biomarkers for the targeting and imaging of PC. The development of an imaging radiopharmaceutical to image all stages of PC growth would improve diagnosis, staging and personalised treatment, as present imaging modalities for PC rely largely on anatomical changes to allow visualisation and have limited sensitivity for imaging metastatic spread of the disease. PSMA was selected due to its up-regulation in advanced carcinoma and metastatic disease and GRP-R due to its high levels of expression in the early stages of PC. The hypothesis is that PC can be imaged by a suitably designed radioligand directed against an appropriate molecular target, such as PSMA and GRP-R. Both of these targets were believed to be appropriate as both are present preferntially in prostate tissue and they both internalise when bound by their ligand. To target PSMA, phage libraries were screened for scFv against both cell-expressed PSMA and recombinant PSMA and diabodies were also generated from high binding clones. Several promising candidates were produced which selectively bound to LNCaP cells and PSMA protein in both FACS and ELISA. Diabodies showed improved binding over corresponding scFv’s. In vivo analysis of tumour-bearing mice failed to reveal tumour uptake of either the scFv or the diabody. In vitro analysis suggested that the affinity of the antibody fragments were not sufficiently high. [99mTc]-Demobesin 4 (DB 4), a radiolabelled GRP-R binding peptide was synthesised. Radioligand binding assays performed on a range of androgen-independent and androgen-dependent PC cell lines showed high GRP-R expression in the androgen dependent LNCaP line but also in the androgen-independent cell lines PC3 and DU145. GRP-R expression, measured by RT-PCR to determine the amount of GRP-R RNA, was similar to that seen using radioligand binding assays and similar patterns were observed in autoradiographic studies. In vivo studies on mice bearing the PC xenografts showed tumour uptake and localisation of [99mTc]-DB 4 within one hour. A limited correlation was observed between results obtained in vivo and in vitro. In conclusion, the results were partly consistant with the hypothesis, whereby initial aims for the PSMA project were successfully achieved with generation of scFv and diabodies that specifically bound, however they proved unsuitable as potential imaging agents, perhaps owing to low binding affinity. GRP-R was shown to be an effective candidate for radioimmaging PC which has the potential to descrininate [99mTc]-DB4 uptake between androgen-independent/dependent cells. Thus this radiopharmaceutical may prove a useful imaging agent for early prostate cancer but that further studies are required to assess its usefulness in the androgen-independent stages of the disease.
AuthorsKashani, Roxana M. G.
- Theses