Kuo-Shyan Lin

Oncology practice is rapidly shifting toward personalize cancer diagnosis and treatment by utilizing targeted therapies or probes against the specific molecular drivers of tumors in individual patients. Molecular imaging has been playing a major role in personalized cancer management, including detection, staging, and treatment response assessment. However, tumor heterogeneity limits the utility of monospecific radiotracers in prostate cancer diagnosis and therapy. Utilizing heterodimeric radiotracers to target different proteins overexpressed in tumor has been shown to be a promising strategy to overcome these limitations and improve lesion detection. Previous studies evinced that prostate-specific membrane antigen (PSMA) and fibroblast activation protein (FAP) are overexpressed in prostate cancer, and their expressions are associated with poor prognosis. Therefore, both PSMA and FAP are promising biomarkers of prostate cancer and many PSMA- and FAP-targeting radioligands have been developed for imaging and therapy. Despite many effective PSMA-targeting radiotherapeutic agents being developed, patients with low to no PSMA expression are not eligible for these PSMA-targeted radioligand therapies and have very limited treatment options. Since FAP and PSMA are concomitantly expressed in prostate cancer, the use of bispecific PSMA/FAP-targeting radioligands is expected to increase lesion detection sensitivity in this patient cohort.We hypothesize that the bispecific PSMA/FAP-targeting radiotracers will have comparable or even higher tumor uptake than the monospecific radiotracers. In this thesis, we synthesized several bispecific PSMA/FAP-targeting radiotracers, evaluated their binding affinity in vitro using cell-based competitive binding assays, imaging potential and biodistribution in tumor-bearing mice, and compared their data with the monospecific PSMA- and FAP- targeting radiotracers. Here, we also describe the work of developing pyridine-based FAP-targeted pharmacophore which we hypothesized to be more hydrophilic than the quinoline-based pharmacophore, resulting in higher tumor-to-background contrast ratio of the derived radiotracers. Lastly, we synthesized two bispecific PSMA/FAP-targeting tracers containing the pyridine-based FAP-targeted pharmacophore. Overall, this research demonstrates the feasibility of designing PSMA/FAP-targeting radiotracers and how linker selection, length, pharmacophore selection and modification, and hydrophilicity of the compounds affect the binding affinity, tumor uptake, tumor-to-background contrast ratio of the bispecific PSMA/FAP-targeting radiotracers. This can give insights on parameters to consider in designing PSMA/FAP bispecific radiotracers.

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Prostate-specific membrane antigen (PSMA) is a transmembrane protein with an extracellular catalytic site that hydrolyzes N-acetylaspartate glutamate. It is associated with many solid cancers, and most notably prostate cancer as per its namesake. Moreover, PSMA expression correlates with prostate specific antigen expression and Gleason score which means the detected PSMA expression can be utilized to diagnose and stage prostate cancer patients. PSMA expression also has prognostic and predicative values which can further inform prostate cancer management and precision medicine. Its large extracellular domain makes it an ideal drug target for antibody and small molecule development. Antibodies are larger and have longer blood residence time and are expensive to develop, while small molecules have faster clearance and penetrance making them the ideal vector for further uses. Many lysine-urea-glutamate-based PSMA inhibitors targeting the folate enzymatic site such as [¹⁸F]DCFPyL and [⁹⁹ᵐTc]Tc- EDDA/HYNIC-iPSMA have been developed and evaluated in clinical and pre-clinical settings. However, a common problem for glutamate-based tracers is their high non-specific kidney and salivary gland uptake which can be problematic in detecting metastatic lesions and can cause side-effects like xerostomia when translating imaging-based radiopharmaceuticals into therapeutic ones.Hence, there remains a need to further optimize and increase the specificity and affinity of PSMA-targeting radiopharmaceuticals. We present a peptide-chemistry based synthesis of novel PSMA-targeting radiopharmaceuticals which is further evaluated for their in vitro and in vivo characterization based on parameters including binding affinity, hydrophilicity, labelling yield and purity, stability, biodistribution and tracer uptake, and tumour-to-background ratio inLNCaP cell and tumour xenograft models. Here, we report several successful candidates labelled with technetium-99m and fluorine-18 for SPECT and PET imaging, respectively, with ideal pharmacodynamic and pharmacokinetic properties for further pre-clinical evaluation.

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