This work deals with radiochemistry and new approaches to develop novel PET tracers labelled with the radionuclide 11C. Two methods for the synthesis of 11C-labelled acrylamides have been explored. First, [1-11C]-acrylic acid was obtained from a palladium(0)-mediated 11C-carboxylation of acetylene with [11C]carbon monoxide; this could be converted to the corresponding acyl chloride and then combined with benzylamine to form N-benzyl[carbonyl-11C]acrylamide. In the second method, the palladium(0)-mediated carbonylation of vinyl halides with [11C]carbon monoxide was explored. This latter method, yielded labelled acrylamides in a single step with retention of configuration at the C=C double bond, and required less amine compared to the acetylene method. The vinyl halide method was used to synthesize a library of 11C-labelled EGFr-inhibitors in 7-61% decay corrected radiochemical yield via a combinatorial approach. The compounds were designed to target either the active or the inactive form of EGFr, following computational docking studies. The rhodium(I)-mediated carbonylative cross-coupling of an azide and an amine was shown to be a very general reaction and was used to synthesize a library of dual VEGFr-2/PDGFrβ inhibitors that were 11C-labelled at the urea position in 38-78% dc rcy. The angiotensin II AT1 receptor antagonist eprosartan was 11C-labelled at one of the carboxyl groups in one step using a palladium(0)-mediated carboxylation. Autoradiography shows specific binding in rat kidney, lung and adrenal cortex, and organ distribution shows a high accumulation in the intestines, kidneys and liver. Specific binding in frozen sections of human adrenal incidentalomas warrants further investigations of this tracer. Three angiotensin II AT2 ligands were 11C-labelled at the amide group in a palladium(0)-mediated aminocarbonylation in 16-36% dc rcy. One of the compounds was evaluated using in vitro using autoradiography, and in vivo using organ distribution and animal PET. The compound was metabolized fast and excreted via urine. High radioactivity was also found in the liver, meaning that more metabolically stable compounds are desirable for future development.

Background: Glioblastoma is the most common and aggressive type of glioma and associated with poor prognosis. Apart from ionizing radiation and some rare genetic disorders, few aetiological factors have been identified for primary brain tumours. Inverse associations to asthma and low IgG levels for varicella zoster virus have in previous studies indicated that the immune system may play a role in glioma development. Little is known about prognostic factors in glioma. Previous studies have shown an association between age, Karnofsky performance status, O6-methylguanine-DNA methyltransferase (MGMT) hypermethylation, and prognosis. Polymorphisms in different low penetrance genes have in some studies been associated with glioma prognosis. Material and methods: In paper I, we analysed IgG levels for four different viruses, Epstein-Barr virus (EBV), cytomegalovirus (CMV), varicella zoster virus (VZV) and adenovirus (Ad), in prediagnostic blood samples from 197 cases with glioma and 394 controls collected from three large cohorts: the Northern Sweden Health and Disease Study; the Malmö Diet and Cancer Study; and the Diet, Cancer and Health cohort from Copenhagen. ELISA was used to measure IgG levels and for EBV response to both the nuclear antigen (EBNA1) and the viral capsid antigen (VCA) was measured, for VCA using immunoflourescence. IgG levels were divided into quartiles and binary logistic regression was used to compare the quartiles in cases and controls. All odds ratios were adjusted for age, sex, and cohort. In paper II-IV, we studied 176 glioblastoma cases from Sweden and Denmark. We collected treatment and follow-up data on the cases. We genotyped 30 tagging SNPs in EGF, 89 in EGFR, 27 in VEGFR2, and 17 in VEGF. We also studied 1458 SNPs in 136 DNA repair genes. Hazard ratios were calculated using Cox regression; the major allele was set as categorical variable and all HR were adjusted for age, sex, country, and treatment. For the DNA repair gene results, we adjusted the p-values for multiple testing. Significant findings were confirmed in separate datasets. Results and Discussion: We found a trend towards higher IgG VZV levels in controls compared to glioma cases, especially when restricting the analyses to only include glioma cases with at least 2 years between blood sample and diagnosis. This finding might indicate that there is an aetiological and not a disease-related association. This confirms previous findings and support that a strong immune system can detect and inhibit growth of small cancer clusters. In EGF, we found seven SNPs in one haplotype block that were significantly associated with glioblastoma survival. Four of the SNPs were available for confirmation; however, none reached statistical significance. One explanation could be age differences in the different cohorts. In EGFR, four SNPs associated with survival were found; however, as 89 polymorphisms were tested this was the expected outcome by chance. In VEGF and VEGFR2, we found two SNPs associated with glioblastoma survival, but they could not be confirmed in the separate dataset, and due to multiple testing, were considered to be false positives. Among the DNA repair genes, we found nine SNPs in three genes-MSH2, RAD51L1 and RECQL4-associated with glioblastoma survival after confirmation and adjustment for age, sex, country, and treatment. After adjusting for multiple testing, one SNP in MSH2 and one in RECQL4 remained significant. Conclusions: Our studies provide additional knowledge to the aetiological and prognostic factors important for glioma, emphasising the possible importance of immune function mechanisms. We found limited evidence for the role of genetic variants in glioma progression genes, and some for DNA repair variants as prognostic factors for glioblastoma survival.

Squamous cell carcinoma of the head and neck (SCCHN), the 6th most common cancer worldwide, has a low 5-year survival. Disease as well as treatment often causes patients severe functional and aesthetic problems. In order to improve treatment and diagnosis at earlier stages of tumour development it is important to learn more about the molecular mechanisms behind the disease. p63, an important regulator of epithelial formation, has been suggested to play a role in the development of SCCHN. Six different isoforms of p63 have been found and shown to have various functions. The aim of the studies in this thesis was to learn more about the role of p63 and proteins connected to p63 in SCCHN. Expression of p63, Cox-2, EGFR, beta-catenin, PP2A and p53 isoforms was mapped in tumours and normal tumour adjacent tissue from patients with SCCHN using western blot or RT-PCR. Results showed no significant difference between tumours and normal tumour adjacent tissue concerning expression of EGFR and beta-catenin. Cox-2 and PP2A showed significantly higher expression in tumours while p63 was more expressed in normal tumour adjacent tissue. However, expression of all these proteins in normal tumour adjacent tissue differed from tissue from disease-free non-smoking individuals. Smoking in itself did not affect expression of these proteins. The p53 isoforms p53, p53beta, p53gamma, ∆133p53, ∆133p53beta and ∆133p53gamma were expressed at RNA level in samples both from tumours and normal tumour adjacent tissue, though most of them at fairly low levels. The functional properties of the different p63 isoforms have not been fully mapped. By establishing stable cell lines over-expressing the different p63 isoforms we investigated their specific effect on tumour cells from SCCHN. Only the ∆Np63 isoforms could be stably over-expressed, whereas no clones over-expressing TAp63 could be established. Using microarray technique, cell lines stably expressing the ∆Np63 isoforms were studied and CD44, Keratins 4, 6, 14, 19 and Cox-2 were found to be regulated by p63. In conclusion, the present project adds new data to the field of p63 and SCCHN. For example, we have shown that clinically normal tumour adjacent tissue is altered compared to normal oral mucosa in non tumour patients, and that smoking does not change expression of p63, Cox-2, EGFR, beta-catenin or PP2A in oral mucosa. Novel p53 isoforms are expressed in SCCHN, and even though levels are very low they should not be overlooked. Furthermore, CD44, keratins 4, 6, 14, 19 and Cox-2 were identified as p63 targets in SCCHN.

Regulation of growth is of fundamental importance for development of the organism and to maintain health. The induction of cell proliferation and matrix production are influenced by several different signaling systems, most importantly by growth factors. The human HER-family of growth factor ligands and receptors is one of the most studied and, at present, one of the most complex including 4 tyrosine kinase receptors and at least 11 different ligands cooperating in the transfer of signals. The HER-family growth responses are also influenced by other intercellular and extracellular signals, including matrix components, cytokines and hormones mediating e.g. inflammation. HER-1 (EGFR) is one of the best known and most extensively studied growth factor receptors. TGF-alpha is possibly the most potent HER-1 ligand and influences wound healing, epidermal maintenance, gastrointestinal function, lactation, pulmonary function and more. Several studies have shown important regulatory functions for some inflammatory cytokines on TGF-alpha production in white blood cells. HER-1 is widespread in epithelial cells but also in mesenchymal cells such as fibroblasts, osteogenic and chondrogenic cells. Consequently, many tumors arising from these cell types express HER family members and often show TGF-alpha and/or HER activation. Indeed, mammary cancer development has been shown when over expressing both TGF-alpha and HER-2 in mouse mammary cells in vivo. In recent years the first HER-1 and HER-2 inhibitors have come into clinical practice for treatment of breast cancer, lung cancer and gastrointestinal cancers, sometimes with great success. However, more knowledge is needed concerning the inflammatory regulation of HER-family expression including where and how the ligands and receptors cooperate. Therefore we were interested in studying the role of TGF-alpha in normal and abnormal growth. First we showed that the acute inflammatory cytokine IL-6 regulates TGF-alpha expression in U-937-1 monocytoid cells. Secondly, we detected a possible long-term enhancing influence of singledose UVR on HER-1 expression in normal human melanocytes. We continued thirdly by revealing TGF-alpha production concomitant with HER-2 in normal human synovia and release of soluble TGF-alpha into the synovial fluid. Both TGF-alpha and HER-2 production were significantly increased in inflammatory joint conditions, e.g. RA. Fourthly, we demonstrated expression of TGF-alpha, HER-1 and HER-2 in synovial sarcoma cells in culture; the observed HER-2 phosphorylation was dependent on ligand induced HER-1 activation. The presented results indicate that TGF-alpha expression can be enhanced by acute inflammatory cytokine IL-6, possibly contributing to growth stimulatory effects assigned to IL-6 itself. The acute effects of UVR on melanocytes mediate up-regulated steady-state expression of HER-1, constituting a potential target for locally produced TGF-alpha that may induce melanocyte proliferation. TGF-alpha and HER-2 seem to have a role in the maintenance of synovial joint tissues. Upregulation of TGF-alpha and HER-2 in inflammatory joint conditions, e.g. RA, represents a novel mechanism for synovial proliferation contributing to joint deterioration. TGF-alpha, HER1 and HER-2 may have a role in synovial sarcoma proliferation; further investigation is needed to evaluate HER-family inhibitors as a possible treatment alternative in this type of cancer.

The epidermal growth factor receptor (EGFR) family consists of four (EGFR, ErbB2, Erbb3, and ErbB4) receptor tyrosine kinases (RTK) whose signalling is important for physiological and malignant cellular functions such as proliferation, survival, migration, and differentiation. EGFR and ErbB2 in particular are established oncogenes in many solid tumours and are targets for anti-cancer treatment. LRIG1 (leucine-rich repeats and immunoglobulin-like domains-1) is a protein that negatively regulates the EGFR-family, and other RTKs and is a proposed tumour suppressor. This thesis examines the expression of the EGFR-family members and LRIG1 in renal cell carcinoma (RCC) and in prostate cancer (PC). In RCC, up-regulation of EGFR was shown for all RCC types analysed: clear cell (ccRCC), papillary (pRCC), and chromophobe (chRCC). ErbB2 was down-regulated in ccRCC. ErbB3 expression was low in non-neoplastic kidney and not significantly altered in RCC. ErbB4 was strongly down-regulated in the vast majority of RCCs of all types. LRIG1 was down-regulated in ccRCC. No prognostic value was found for any of these factors in RCC. In prostate cancer cells, LRIG1 was shown to be up-regulated by androgen stimulation and suppressed the growth of prostate cancer cells. In prostate cancer, the expression and prognostic value of LRIG1 was investigated in two patient series, one with untreated patients and one with patients who had undergone prostatectomy. In the untreated patient series, LRIG1 correlated with malignancy grade (Gleason score) and poor outcome for patients (both cancer specific and overall survival), being an independent prognostic factor. In contrast, in the series of patients who had undergone prostatectomy, LRIG1 expression correlated with a good outcome (overall survival). Thus in RCC, there were alterations in gene-expression of the EGFR-family members and LRIG1 between kidney cortex and RCC and between the RCC types. Despite few associations with clinical factors, these alterations are likely to be of biological importance. In prostate cancer LRIG1 was up-regulated by androgen stimulation and inhibited cell proliferation. LRIG1 expression had prognostic value in prostate cancer, maybe as a secondary marker of androgen receptor activation or because of growth inhibition of prostate cancer cells. Contradicting findings in untreated patients and patients treated with prostatectomy poses the question of whether the prognostic value of LRIG1 and other markers vary depending on the specific biological and clinical circumstances in the materials studied.

Gliomas are the most common primary brain tumours, and their capacity to invade surrounding normal brain prevents complete removal of the tumour. Malignant glioma has still a poor prognosis. However, with the rapid development of molecular biology our understanding about glioma has increased dramatically. Among known growth factors, EGF and its receptor are frequently amplified and over expressed in malignant glioma. Therefore, it is of interest to find approaches to hamper the activity of EGF/EGFR. The aim of this thesis was to identify and characterize human analogues to a recently identified gene in Drosophilia, kekkon-1, which negatively regulates the activity of Drosophilia EGF receptor. In the first part, we set up a quantitative real-time RT-PCR assay, which showed good linearity, reproducibility and uniformity. We analyzed the expression of the most commonly used reference genes, and showed that 18S was the most reliable endogenous reference gene in this study. In the second part, we cloned, identified, and sequenced a gene family, which we named leucine-rich repeats and immunoglobulin–like domains family (LRIG). The LRIG gene family had three vertebrate paralogs and one homolog in ascidiacea. The proteins encoded by human LRIG genes shared an overall structure with a signal peptide, 15 tandems leucine-rich repeats with N- and C- terminal flanking regions followed by 3 immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tail. Northern blot showed the mRNA sizes to be 5.5 kb for LRIG1, 4.8 kb for LRIG2, and 5.1 kb for LRIG3. LRIG1-3 mRNAs were detected in all human and mouse tissues analyzed, however, at various levels. FISH and BLAST analysis showed that LRIG1 was located at 3p14, LRIG2 at 1q13, and LRIG3 at 12q13. LRIG1 was shown to be down-regulated in several cancer cell lines and proposed to be a tumour suppressor gene. In the third part, we analysed the expression of LRIG gene family in human astrocytic tumours. LRIG1-3 mRNAs were detected in all human glioma cell lines, in primary tumour tissues and control-matched normal brain tissues, at various levels. Subcellular localizations of LRIG1-GFP fusion proteins were visualized in nuclear, perinuclear, and cytoplasmic compartment. According to the predicted protein sequences, short peptides were synthesized and used to raise antibodies in rabbits. The antibodies were used for immunohistochemical analysis of LRIG1-3 in 404 human astrocytic tumours in a tissue micro array. The pattern of immunoreactivity of LRIG1-3 was heterogeneous with staining in nuclear, perinuclear and cytoplasmic compartment of positive tumour cells. Perinuclear staining of LRIG1-3 displayed a significant inverse correlation with WHO grade and especially positive LRIG3 perinuclear and cytoplasmic staining correlated with a low proliferation index. The LRIGs correlated with survival, and LRIG3 perinuclear staining was in addition to tumour grade an independent prognostic factor. The results suggest that LRIGs may play a role in normal tissue, and may be of importance in the pathogenesis and prognosis of tumours. The exact function of LRIG1-3 remains to be established.

Tumor targeting and molecular imaging of protein markers specific for or overexpressed in tumors can add useful information in deciding upon treatment and assessing the response to treatment for a cancer patient. The epidermal growth factor receptor (EGFR) is one such tumor-associated receptor, which expression is abnormal or upregulated in various cancers and associated with a poor patient prognosis. It is therefore considered a good target for imaging and therapy. Monoclonal antibodies and recently also antibody fragments have been investigated for in vivo medical applications, like therapy and imaging. In molecular imaging a small sized targeting agent is favorable to give high contrast and therefore, antibody fragments and lately also small affinity proteins based on a scaffold structure constitute promising alternatives to monoclonal antibodies. Affbody molecules are such affinity proteins that are developed by combinatorial protein engineering of the 58 amino acid residue Z-domain scaffold, derived from protein A. In this thesis, novel Affibody molecules specific for the EGFR have been selected from a combinatorial library using phage display technology. Affibody molecules with moderate high affinity demonstrated specific binding to native EGFR on the EGFR-expressing epithelial carcinoma A431 cell line. Further cellular assays showed that the EGFR-binding Affibody molecules could be labeled with radiohalogens or radiometals with preserved specific binding to EGFR-expressing cells. In vitro, the Affibody molecule demonstrated a high uptake and good retention to EGFR-expressing cells and was found to internalize. Furthermore, successful imaging of tumors in tumor-bearing mice was demonstrated. Low nanomolar or subnanomolar affinities are considered to be desired for successful molecular imaging and a directed evolution to increase the affinity was thus performed. This resulted in an approximately 30-fold improvement in affinity, yielding EGFR-binding Affibody molecules with KD´s in the 5-10 nM range, and successful targeting of A431 tumors in tumor-bearing mice. To find a suitable format and labeling, monomeric and dimeric forms of one affinity matured binder were labeled with 125I and 111In. The radiometal-labeled monomeric construct, 111In-labeled-ZEGFR:1907, was found to provide the best tumor-to-organ ratio due to good tumor localization and tumor retention. The tumor-to-blood ratio, which is often used as a measure of contrast, was 31±8 at 24 h post injection and the tumor was clearly visualized by gamma-camera imaging. Altogether, the EGFR-binding Affibody molecule is considered a promising candidate for further development of tumor imaging tracers for EGFR-expressing tumors and metastases. This could simplify the stratification of patients for treatment and the assessment of the response of treatment in patients. / QC 20100723

Dysregulation of protein activity, caused by alterations in protein sequence, expression, or localization, is associated with numerous diseases. In order to control the activity of harmful protein entities, affinity ligands such as proteins, oligonucleotides or small molecules can be engineered to specifically interact with them to modulate their function. In this thesis, non-immunoglobulin based affinity proteins known as affibody molecules are used to functionally inhibit proteins important for signaling through pathways that are overactive in different cancers.   In Paper I and Paper II, affibody molecules with high affinity for the receptor tyrosine kinases HER2 or EGFR are expressed in the secretory compartments of model cancer cell lines SKOV3 or A431 using a retrovirus-based gene delivery system. Equipping the affinity proteins with an ER retention tag, the affibody molecules together with their target protein are retained in the secretory compartments as shown by confocal fluorescence imaging. Flow cytometric analysis showed a 60 % or 80 % downregulation of surface located HER2 or EGFR in these cell lines, respectively. A significant decreased in proliferation rate of the cells was also observed, which for EGFR retention could be correlated with inhibition of phosphorylation in the kinase domain. In Paper III, novel affibody molecules interacting with the hormone binding site of the insulin growth factor-1 receptor were generated. One variant had high (1.2 nM) affinity for the receptor and could be used for immunofluorescence analysis and for receptor pull-out from cell lysates. Addition of this affibody molecule to MCF-7 cells had a dose dependent growth inhibitory effect on the cells. In Paper IV, novel affibody molecules against the intracellular oncoproteins H-Ras and Raf-1 were selected and characterized, and they proved to be specific for their target proteins. Mapping experiments showed that the affibody molecules selected against H-Ras interacted at over-lapping epitopes not affecting the interaction between Ras and Raf. In contrast, the predominant variant isolated during selection against Raf-1 could completely inhibit the Ras/Raf interaction in a real-time biospecific interaction analysis.   Taken together, the affibody molecules presented here and the strategies by which they are used to interfere with cancer related proteins and pathways may be valuable tools for further investigation of these systems and may possibly also be used to generate molecules suitable for cancer therapy. / QC 20100818

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

Affibody molecules are small (7 kDa) affinity proteins of non-immunoglobulin origin that have been generated to specifically interact with a large number of clinically important molecular targets. In this thesis, Affibody molecules have been employed as tracers for radionuclide molecular imaging of HER2- and IGF-1R-expressing tumors, paper I-IV, and for surface knock-down of EGFR, paper V. In paper I, a tag with the amino acid sequence HEHEHE was fused to the N-terminus of a HER2-specific Affibody molecule, (ZHER2), and was shown to enable facile IMAC purification and efficient tri-carbonyl 99mTc-labeling. In vivo evaluation of radioactivity uptake in different organs showed an improved biodistribution, including a 10-fold lower radioactivity uptake in liver, compared to the same construct with a H6-tag. In paper II, it was further shown that an N-terminally placed HEHEHE-tag on ZHER2 provided lower unspecific uptake of radioactivity in liver compared to its H6-tagged counterpart even when radiolabeling was at the C-terminus using alternative chemistries to attach 99mTc, 111In or 125I. In paper III, the H6-tag’s composition and position was varied with regards to charge, hydrophobicity and its C- or N-terminal placement on ZHER2. Among the ten variants investigated, it was found that an N-terminal HEHEHE-tag provided the most favorable overall biodistribution profile and that introduction of hydrophobic and positively charged amino acids provoked liver uptake of radioactivity. In paper IV, the HEHEHE-tag was shown to enable IMAC purification and tri-carbonyl 99mTc-labeling of an IGF-1R-specific Affibody molecule and improved its overall biodistribution when compared to the same construct with a H6-tag. In paper V, the aim was to develop an intracellular receptor-entrapment system to reduce the surface levels of EGFR. An EGFR-specific Affibody molecule was expressed as a fusion to different mutants of an intracellular transport protein in SKOV-3 cells, resulting in a collection of cell lines with 50%, 60%, 80% and 96% reduced surface level of EGFR. Analysis of the proliferation rate of these cell lines showed that a modest reduction (15%) in proliferation occurs between 60% and 80% reduction of the surface level of EGFR. / <p>QC 20130129</p>