<p>Targeted radionuclide therapy (TRT) holds great promise for the treatment of cancer. In TRT, radioactive nuclides are delivered specifically to tumours by molecules that recognise and bind to structures overexpressed by, or specific to, cancer cells. Human epidermal growth factor receptor like protein 2 (HER-2) is an oncogene product overexpressed in e.g. urological, breast, or ovarian cancers that have been correlated to poor prognosis and resistance to hormonal therapy. There is also evidence that tumour cells retain their HER-2 overexpression in metastases.</p><p>Trastuzumab and pertuzumab are two humanised monoclonal antibodies targeting different parts of HER-2. This thesis describes the radiolabelling of these antibodies for use in TRT and diagnostics. The thesis also investigates possible methods for modifying uptake and retention of radioactivity delivered with antibodies binding to HER-2. Modification of the cellular retention of ¹²⁵I by using polyhedral boron anion based linker molecules (DABI and NBI) is investigated, and it is shown that linking ¹²⁵I to trastuzumab using DABI increases cellular accumulation of radioactivity by 33%. It is also shown that trastuzumab can be efficiently coupled to the positron emitter ⁷⁶Br by using NBI. Furthermore, it is shown that cellular uptake of ¹²⁵I can be modified by stimulating EGFR (HER-1) with EGF.</p><p>When labelled with the alpha emitter ²¹¹At, trastuzumab could specifically kill cells in vitro. This cell killing effect could be prevented by saturating the receptors of the target cells with non-radiolabelled trastuzumab.</p><p>Pertuzumab was radiolabelled with the low energy beta emitter ¹⁷⁷Lu without losing affinity or immunocompetence. [¹⁷⁷Lu]pertuzumab was specific to HER-2 in vitro and in vivo. This targeting conjugate was shown to increase median time to tumour progression in mice bearing xenografts of the radioresistant SKOV-3 cell line. </p><p>In conclusion, antibodies against HER-2, especially pertuzumab radiolabelled with ¹⁷⁷Lu, show promise as TRT agents.</p>

<p>After the completion of the human genome project the human genome was annotated with the surprisingly small amount of 24 000 (www.ensemble.com) genes. This has focused research on the contribution of splice variants, posttranslational modifications and interactions of proteins at the proteome level and other regulatory elements in the cell to fully understand the complexity of functions in a higher organism. Proteomic oriented projects are currently aiming to investigate all the splice variants and posttranslational modifications of all the proteins present in an organism or cell type and annotate their function and interaction partners. Projects on this scale are at the moment difficult to achieve and new methodologies are needed. </p><p>Proximity ligation assays (PLAs) are based on a novel protein detection strategy that converts the presence of a target molecule in a unique DNA tag through ligation reactions. PLA detection of proteins requires several independent recognition events by affinity reagents that have been converted into proximity probes. Different formats of the proximity ligation strategy have been developed in both heterogeneous and homogeneous format[1-4]. This thesis presents the development of an antibody based proximity ligation approach and the development of a novel proximity ligation based detection strategy named triple specific proximity ligation (3PLA). To extend the range of target molecules we adapted the proximity ligation assay for the use with antibodies by converting matched monoclonal antibody pairs and polyclonal antibody batches into proximity probes and used them for the detection of several cytokines in complex biological fluids. The novel 3PLA requires the simultaneous detection by three independent affinity binders to create one specific DNA based signal. This requirement for triple recognition extends the biological specificity of immunoassays and allows a proximity ligation design with reduced background signal and thus higher sensitivity. We have established proof of principle detection of the biomarkers troponin I and prostate specific antigen (PSA) alone and in complex with 1-alpha-antichymotrypsin (ACT) and detected as little as 100 molecules of vascular endothelial growth factor (VEGF). To further explore the extended biological specificity of 3PLA we adapted the assay for detection of protein complexes formed during NFκB signaling and used this system to profile the mode of action of three small molecular weight inhibitors of the IκB Kinase (IKK). The development of new protein detection methods hold promises for the investigation of complex interactions and mechanism on the proteome level which are not accessible with current technologies. We have developed tools and protocols useful for the development of new proximity ligation strategies and designs. These protocols allow the rapid and low cost custom set up of PLAs without the need for extensive conjugation protocols or purification procedures.</p>

<p>Osteoporosis is a disease that leads to decreased bone mineral density (BMD), an altered bone micro-architecture and fragile bones. The disease is highly heritable and numerous genes are thought to be involved, making it difficult to identify the causative genetic elements.</p><p>Animal models, mainly intercrosses between laboratory strains of mice, have been succesfully used to map genes affecting these traits, but may not mirror the multifactorial genetic etiology of highly complex traits such as osteoporosis.</p><p>Over the course of tens of thousand years humans have kept domestic animals whose phenotypic repertoires have been tailored to meet our needs. Wild-type red junglefowl (RJ) and domestic White Leghorn (WL) chicken differ for several bone traits. </p><p>In this thesis Quantitative Trait Loci (QTL) mapping was used to trace the inheritance of bone traits in two separate intercrosses between RJ and WL. In these studies we identified several QTL that contributed to differences in BMD, bone size and biomechanical strength of bone. In a comparison of QTL identified in the two intercrosses it was observed that nine QTL had overlapping genomic positions, implicating these loci as important to bone phenotypic variation in chicken.</p><p>In two separate studies, microarray technology was used to compare global gene expression in bone tissue from RJ and WL. In these studies, differential expression was observed for 779 and 560 genes, respectively. Many differentially expressed genes were co-localized with QTL, which implicates them as QTL-candidates. </p><p>Results presented in this thesis link several genomic regions and genes to variation in bone traits. Increased knowledge about these identified genes and regions will contribute to a better understanding of the mechanisms underlying inter-individual differences in bone metabolism, both in chicken and man.</p>

After the completion of the human genome project the human genome was annotated with the surprisingly small amount of 24 000 (www.ensemble.com) genes. This has focused research on the contribution of splice variants, posttranslational modifications and interactions of proteins at the proteome level and other regulatory elements in the cell to fully understand the complexity of functions in a higher organism. Proteomic oriented projects are currently aiming to investigate all the splice variants and posttranslational modifications of all the proteins present in an organism or cell type and annotate their function and interaction partners. Projects on this scale are at the moment difficult to achieve and new methodologies are needed. Proximity ligation assays (PLAs) are based on a novel protein detection strategy that converts the presence of a target molecule in a unique DNA tag through ligation reactions. PLA detection of proteins requires several independent recognition events by affinity reagents that have been converted into proximity probes. Different formats of the proximity ligation strategy have been developed in both heterogeneous and homogeneous format[1-4]. This thesis presents the development of an antibody based proximity ligation approach and the development of a novel proximity ligation based detection strategy named triple specific proximity ligation (3PLA). To extend the range of target molecules we adapted the proximity ligation assay for the use with antibodies by converting matched monoclonal antibody pairs and polyclonal antibody batches into proximity probes and used them for the detection of several cytokines in complex biological fluids. The novel 3PLA requires the simultaneous detection by three independent affinity binders to create one specific DNA based signal. This requirement for triple recognition extends the biological specificity of immunoassays and allows a proximity ligation design with reduced background signal and thus higher sensitivity. We have established proof of principle detection of the biomarkers troponin I and prostate specific antigen (PSA) alone and in complex with 1-alpha-antichymotrypsin (ACT) and detected as little as 100 molecules of vascular endothelial growth factor (VEGF). To further explore the extended biological specificity of 3PLA we adapted the assay for detection of protein complexes formed during NFκB signaling and used this system to profile the mode of action of three small molecular weight inhibitors of the IκB Kinase (IKK). The development of new protein detection methods hold promises for the investigation of complex interactions and mechanism on the proteome level which are not accessible with current technologies. We have developed tools and protocols useful for the development of new proximity ligation strategies and designs. These protocols allow the rapid and low cost custom set up of PLAs without the need for extensive conjugation protocols or purification procedures.

Osteoporosis is a disease that leads to decreased bone mineral density (BMD), an altered bone micro-architecture and fragile bones. The disease is highly heritable and numerous genes are thought to be involved, making it difficult to identify the causative genetic elements. Animal models, mainly intercrosses between laboratory strains of mice, have been succesfully used to map genes affecting these traits, but may not mirror the multifactorial genetic etiology of highly complex traits such as osteoporosis. Over the course of tens of thousand years humans have kept domestic animals whose phenotypic repertoires have been tailored to meet our needs. Wild-type red junglefowl (RJ) and domestic White Leghorn (WL) chicken differ for several bone traits. In this thesis Quantitative Trait Loci (QTL) mapping was used to trace the inheritance of bone traits in two separate intercrosses between RJ and WL. In these studies we identified several QTL that contributed to differences in BMD, bone size and biomechanical strength of bone. In a comparison of QTL identified in the two intercrosses it was observed that nine QTL had overlapping genomic positions, implicating these loci as important to bone phenotypic variation in chicken. In two separate studies, microarray technology was used to compare global gene expression in bone tissue from RJ and WL. In these studies, differential expression was observed for 779 and 560 genes, respectively. Many differentially expressed genes were co-localized with QTL, which implicates them as QTL-candidates. Results presented in this thesis link several genomic regions and genes to variation in bone traits. Increased knowledge about these identified genes and regions will contribute to a better understanding of the mechanisms underlying inter-individual differences in bone metabolism, both in chicken and man.

Targeted radionuclide therapy (TRT) holds great promise for the treatment of cancer. In TRT, radioactive nuclides are delivered specifically to tumours by molecules that recognise and bind to structures overexpressed by, or specific to, cancer cells. Human epidermal growth factor receptor like protein 2 (HER-2) is an oncogene product overexpressed in e.g. urological, breast, or ovarian cancers that have been correlated to poor prognosis and resistance to hormonal therapy. There is also evidence that tumour cells retain their HER-2 overexpression in metastases. Trastuzumab and pertuzumab are two humanised monoclonal antibodies targeting different parts of HER-2. This thesis describes the radiolabelling of these antibodies for use in TRT and diagnostics. The thesis also investigates possible methods for modifying uptake and retention of radioactivity delivered with antibodies binding to HER-2. Modification of the cellular retention of 125I by using polyhedral boron anion based linker molecules (DABI and NBI) is investigated, and it is shown that linking 125I to trastuzumab using DABI increases cellular accumulation of radioactivity by 33%. It is also shown that trastuzumab can be efficiently coupled to the positron emitter 76Br by using NBI. Furthermore, it is shown that cellular uptake of 125I can be modified by stimulating EGFR (HER-1) with EGF. When labelled with the alpha emitter 211At, trastuzumab could specifically kill cells in vitro. This cell killing effect could be prevented by saturating the receptors of the target cells with non-radiolabelled trastuzumab. Pertuzumab was radiolabelled with the low energy beta emitter 177Lu without losing affinity or immunocompetence. [177Lu]pertuzumab was specific to HER-2 in vitro and in vivo. This targeting conjugate was shown to increase median time to tumour progression in mice bearing xenografts of the radioresistant SKOV-3 cell line. In conclusion, antibodies against HER-2, especially pertuzumab radiolabelled with 177Lu, show promise as TRT agents.

<p>Targeted radiotherapy (TRT) of cancer is a promising approach that enables selective treatment of tumour cells, while sparing normal tissue. The humanized monoclonal antibody A33 (huA33) is a potential targeting agent for TRT of colorectal cancer, since its antigen is expressed in more than 95 % of all colorectal carcinomas. The aim of this thesis was to evaluate the therapeutic potential of the two huA33-based TRT-conjugates, ¹⁷⁷Lu-huA33, and ²¹¹At-huA33.</p><p>The conjugates ¹⁷⁷Lu-huA33, and ²¹¹At-huA33, bound specifically to colorectal cancer cells, both <i>in vitro</i> and <i>in vivo</i>. A dose dependent cytotoxic effect of ²¹¹At-huA33 was also demonstrated <i>in vitro</i>. From a therapeutic perspective, both conjugates had a favourable biodistribution in tumour-bearing nude mice, with high tumour uptake and a low uptake in normal organs (with the exception of an expected thyroid uptake of ²¹¹At). After injection of ²¹¹At-huA33, the blood absorbed a slightly higher dose than the tumour, but for ¹⁷⁷Lu-huA33, the tumour received a 12 times higher dose than blood. Two days after intravenous injection of ¹⁷⁷Lu-huA33 in tumour-bearing mice, the tumours could be clearly visualised by gamma camera imaging, with very low interference from normal tissue radioactivity. In an experimental therapy study, also performed in tumour-bearing mice, there was an excellent therapeutic effect of ¹⁷⁷Lu-huA33. About 50 % of the treated animals were tumour free 140 days after injection of ¹⁷⁷Lu-huA33, while none of the non-radioactive controls survived beyond 20 days after injection of treatment substances.</p><p>In conclusion, this thesis demonstrates that the therapeutic conjugates ¹⁷⁷Lu-huA33, and ²¹¹At-huA33, are promising targeting agents that might help improve therapy of colorectal cancer.</p>

No description available.

<p>Proximity ligation is a recently established technique that can provide answers to questions about the concentration, localization, interactions, modifications and functions of proteins. The method enables sensitive protein measurements with a detection limit in the low femtomolar range in complex biological samples. In proximity ligation, the challenge of detecting specific proteins is converted to the analysis of specific DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins, and to form amplifiable tag sequences upon ligation when brought in proximity. Protocols for the conversion of monoclonal or polyclonal antibodies into proximity probes through the attachment of oligonucleotide sequences are described in the thesis. In addition, the thesis describes the adaptation of the proximity ligation technology for detection of microbial pathogens, analysis of interactions between proteins and nucleic acids, and of inhibition of receptor-ligand interactions. </p><p>Nucleic acid amplification allows specific detection of pathogens with single-copy sensitivity. There are many circumstances, however, when analysis of pathogen surface antigens or the antibody response can provide increased diagnostic value. Proximity ligation reactions were used to measure numbers of virus and bacteria by detection of viral or bacterial surface proteins. Detection sensitivities similar to those of nuclear acid-based detection reactions were achieved directly in infected samples for a parvovirus and for an intracellular bacterium. </p><p>Biological processes are orchestrated by interactions of proteins with molecules in their environment, and investigations of interactions between proteins and other biomolecules are thus of great importance. Protocols were established for very specific and sensitive homogeneous-phase analysis of interactions between proteins and specific nucleic acid sequences. Finally, the proximity ligation mechanism was used to monitor interactions between VEGF-A and two of its receptors, VEGFR-1 and VEGFR-2, and to characterize the effects of agents disrupting this interaction.</p>

<p>Improved methods are needed to interrogate the genome and the proteome. Methods with high selectivity, wide dynamic range, and excellent precision, capable of simultaneously analyzing many biomolecules are required to decipher cellular function. This thesis describes a molecular and microfluidic toolbox designed with those criteria in mind. It also presents a tool for graphical representation of nucleic acid sequences.</p><p>Proximity ligation is a novel protein detection method that requires dual and proximate binding of two oligonucleotide-tagged affinity reagents to a protein or protein complex in order to elicit a signal. The responses from such recognition reactions are the formation of specific nucleic acid reporter molecules that are subsequently amplified and quantitatively detected. </p><p>A scalable microfluidic platform suitable for fluorescence detection, cell culture, and actuation is also described. The platform uses rapid injection molding to produce microstructures in thermoplastic materials. By applying a thin layer of silica to the structures, a lid made of silicone rubber coated onto a thermoplastic support can be covalently bonded to generate enclosed channels.</p><p>A method is presented for precise biomolecule counting, termed “amplified single-molecule detection”. The method preserves the discrete nature of biomolecules, converting specific molecular recognition events to fluorescence-labeled micrometer-sized objects that are enumerated in microfluidic channels. </p><p>I also present a novel microarray-based detection method. To attain high selectivity and a wide dynamic range, the method is based on dual recognition with enzymatic discrimination and amplification. Upon target recognition in solution, DNA probes are subjected to thousand-fold amplification in solution, followed by selective detection on arrays and another hundred-fold amplification of reporter molecule created from the first amplification reaction. </p><p>Lastly, I describe a novel graphical representation of nucleic acid sequences using TrueType fonts that can be of value for visual inspection of DNA sequences and for teaching purposes</p>