Development of Polymer Peptide Conjugates for Enhanced Pancreatic Cancer Imaging

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the US with very poor prognosis. All clinically available biomarkers and diagnostic tools either fail to detect early stage PDAC or suffer from low specificity and sensitivity. There is an urgent need for diagnostic agents with greater efficacy for PDAC detection and staging. Nanomaterials such as N-(2-Hydroxypropyl) methacrylamide (HPMA) copolymers can effectively target tumors, and offer novel opportunities for the development of effective diagnostic agents for cancer. However, a major problem of many nanomaterials-based diagnostics is their opsonization and sequestration by the mononuclear phagocyte system (MPS), leading to substantial accumulation in MPS tissues such as the liver and spleen. This MPS accumulation in non-target tissues can hinder identification of resident or nearby metastatic malignant lesions thereby decreasing the diagnostic efficacy. In this thesis, we focused on developing new approaches in which radiolabeled HPMA copolymers can be synthetically modified to significantly reduce the MPS accumulation and/or to increase the tumor accumulation, thereby enhancing the diagnostic and/or radiotherapeutic efficacy of the agents. First, we evaluated the effectiveness of incorporating metabolic active linkers into HPMA copolymers to decrease the radioactivity retention in the liver and spleen in pancreatic tumor xenograft mice. We demonstrated that 177Lu-labeled HPMA copolymers conjugated with cathepsin B/S cleavable peptide linkers exhibited significant long-term reduction in hepatic and splenic radioactivity accumulation as compared to the non-cleavable control. Next we conjugated a 109 kDa HPMA copolymer with three different cathepsin S cleavable linkers and evaluated the structure-activity relationship with regard to the lengths of the linking groups on the in vitro and in vivo efficacy of 177Lu-labeled cathepsin S cleavable HPMA copolymers. Biodistribution results showed that the 177Lu-labeled HPMA copolymer with the shortest length linker had a significant enhancement in the tumor-to-non-target ratios, which was also confirmed by SPECT/CT imaging. Finally, the potential of active tumor targeting for PDAC was evaluated using a plectin-1 targeted peptide (PTP) conjugated HPMA copolymer on pancreatic cancer cells in vitro. Unfortunately, we found that incorporation of the PTP into the HPMA copolymer diminished the binding of the peptide, possibly due to steric hindrance. Overall, our 177Lu-labeled cathepsin cleavable HPMA copolymer showed decreased MPS tissue accumulation and significant improvement in tumor-to-non-target organ ratio, and was successfully applied to SPECT/CT imaging of pancreatic tumors in a xenograft mouse model

Engineered antibody and neuropeptide mediated radionuclide targeting in prostate cancer

PhDProstate 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

Department of Radiology-Annual Executive Summary Report-July 1, 2005 to June 30, 2006

98 page Department of Radiology Annual Executive Summary Report, July 1, 2005 to June 30, 2006, Thomas Jefferson University Hospital, Philadelphia, Pennsylvania, United States

Novel Antibody-Targeted Liposomes for the Treatment of Solid Tumors

In the last decades, several advances have been made in cancer treatment leading to improved patient survival, although certain types of cancer remain very difficult to treat. Surgical resection and radiation therapy are highly effective against primary lesions. Disseminated disease however remains the main cause of cancer related mortality. Chemotherapy has been effective against various cancer types, but is often marred by adverse effects and the development of drug resistance, which eventually leads to withdrawal of patients from chemotherapeutic treatments. Nanomedicine can be used to encapsulate chemotherapeutics and thus protect the healthy tissue from adverse effects. In addition, the specificity of nanomedicine can be increased with a range of targeting modalities, such as antibodies or antibody fragments. The goal of this thesis was to bring these two concepts together to improve drug delivery by developing nanomedicine specifically targeted against certain cancer antigens. As one of the earliest nanomedicine, liposomes have been used as drug-delivery systems and have shown to eradicate conventional side effects of chemotherapeutics. Whereas so-called nanobodies, or single-domain antibodies, are the smallest functional antibody constructs, which are known for escaping detection in the body and do not trigger an immune response. The combination of nanobodies and liposomes creates a platform, which is long-circulating, highly specific and stealth-like in vivo, and ideal for loading with chemotherapeutics. In this thesis, we have shown that targeting with nanobodies offers many advantages compared to conventional targeting ligands and can be used as novel imaging tools against prostate cancer. Moreover, these nanobodies could be used in combination with drug-loaded liposomes where they were effective in inhibiting tumor growth. Furthermore, we have investigated the role of the tumor microenvironment and morphology in the uptake of liposomes in vivo and the use of radioactive labeled liposomes has shown that tumor heterogeneity is a major limiting factor in tumor uptake. Fortunately, the use of mild hyperthermia, as a pretreatment to increase the permeability of the tumor vasculature, increased liposomal uptake in the tumor. Moreover, thermosensitive liposomes can be used which, upon a hyperthermia trigger, release their chemotherapeutic contents at the target site. Collectively, the results of this thesis describe and provide insights into the pitfalls and possibilities of targeted nanomedicine

Development of Novel Protein-Based MRI Contrast Agents for the Molecular Imaging of Cancer Biomarkers

Temporal and spatial molecular imaging of disease biomarkers using non-invasive MRI with high resolution is largely limited by lack of MRI contrast agents with high sensitivity, high specificity, optimized biodistribution and pharmacokinetics. In this dissertation, I report my Ph. D. work on the development of protein-based MRI contrast agents (ProCAs) specifically targeting different cancer biomarkers, such as grastrin-releasing peptide receptor (GRPR), prostate specific membrane antigen (PSMA), and vascular endothelial growth factor receptor-2 (VEGFR-2). Similar to non-targeted ProCAs, these biomarker-targeted ProCAs exhibit 5 - 10 times higher r1 and r2 relaxivites than that of clinical MRI contrast agents. In addition, these biomarker-targeted ProCAs have high Gd3+ binding affinities and metal selectivities. The highest binding affinity of the three GRPR-targeted contrast reagents obtained by grafting a GRPR ligand binding moiety into ProCA32 for GRPR is 2.7 x 10-9 M. We further demonstrate that GRPR-targeted ProCAs were able to semi-quantitatively evaluate GRPR expression levels in xenograft mice model by MRI. In addition, we have also created a PSMA-targeted ProCA which has a binding affinity to PSMA biomarker of 5.2 x 10-7 M. Further, we developed VEGFR-targeted contrast agent which is able to image VEGFR2 in mice models using T1-weighted and T2-weighted sequences. Moreover, the relaxivities and coordination water numbers of ProCAs can be tuned by protein design of ProCA4. Since disease biomarkers are expressed in various tumors and diseases, our results may have strong preclinical and clinical implications for the diagnosis and therapeutics of cancer and other type of diseases

Endorectal Digital Prostate Tomosynthesis

Several areas of prostate cancer (PCa) management, such as imaging permanent brachytherapy implants or small, aggressive lesions, benefit from high image resolution. Current PCa imaging methods can have inadequate resolution for imaging these areas. Endorectal digital prostate tomosynthesis (endoDPT), an imaging method that combines an external x-ray source and an endorectal x-ray sensor, can produce three-dimensional images of the prostate region that have high image resolution compared to typical methods. This high resolution may improve PCa management and increase positive outcomes in affected men. This dissertation presents the initial development of endoDPT, including system design, image quality assessment, and examples of possible applications to prostate imaging. Experiments using computational phantoms, physical phantoms, and canine prostate specimens were conducted. Initial system design was performed computationally and three methods of endoDPT image reconstruction were developed: shift and add (SAA), backprojection (BP), and filtered BP (FBP). A physical system was developed using an XDR intraoral x-ray sensor and a GE radiography unit. The resolution and radiation dose of endoDPT were measured and compared to a GE CT scanner. Canine prostate specimens that approximated clinical cases of PCa management were imaged and compared using endoDPT, the above CT scanner, and a GE MRI scanner. This study found that the resolution of endoDPT was significantly higher than CT. The radiation dose of endoDPT was significantly lower than CT in the regions of the phantom that were not in the endoDPT field of view (FoV). Inside the endoDPT FoV, the radiation dose ranged from significantly less than to significantly greater than CT. The endoDPT images of the canine prostate specimens demonstrated qualitative improvements in resolution compared to CT and MRI, but endoDPT had difficulty in visualizing larger structures, such as the prostate border. Overall, this study has demonstrated endoDPT has high image resolution compared to typical methods of PCa imaging. Future work will be focused on development of a prototype system that improves scanning efficiency that can be used to optimize endoDPT and perform pre-clinical studies

Molecular Imaging

The present book gives an exceptional overview of molecular imaging. Practical approach represents the red thread through the whole book, covering at the same time detailed background information that goes very deep into molecular as well as cellular level. Ideas how molecular imaging will develop in the near future present a special delicacy. This should be of special interest as the contributors are members of leading research groups from all over the world