<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>

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>Conventional cancer treatment based on radiotherapy or chemotherapy affects all dividing cells. By directing the therapy specifically to the tumor cells, normal cells can be spared. Tumor targeting molecules carrying a cytotoxic moiety is then an attractive approach. </p><p>In this thesis, an affibody molecule with high affinity for the protein HER-2, that is strongly associated with aggressive forms of breast cancer, was selected. After radiolabeling with ¹²⁵I, the affibody molecule, in monovalent and bivalent form, was tested <i>in vitro</i> in HER-2 overexpressing tumor cells and in transplanted tumors in mice. </p><p>It was shown that the HER-2 targeting affibody molecule bound its target in a specific manner, both <i>in vitro</i> and <i>in vivo</i>. The small size of the affibody molecule resulted in fast clearance through the kidneys. An impressive tumor-to-blood ratio of 10 eight hours post injection was achieved and the tumors could easily be visualized in a gamma camera. </p><p>The biologic effects of the bivalent affibody molecule and a monovalent affinity maturated version was measured and compared with the effects of the monoclonal antibody trastuzumab. It was found that although all molecules target the same protein, the effects differed greatly.</p><p>The affibody molecule was also labeled with the alpha-emitting radionuclide ²¹¹At. Specific decrease in survival was seen in HER-2 overexpressing cells receiving the ²¹¹At labeled affibody molecule. The sensitivity to the treatment differed between cell lines, probably as a result of differences between the cell lines in internalization and nuclear size. The ²¹¹At labeled affibody molecules were also tested <i>in vivo</i>, where stability of the ²¹¹At label was a problem. To circumvent this problem, more stable conjugation chemistry was tested, as well as strategies to prevent uptake of released ²¹¹At by normal organs.</p><p>This thesis describes the selection and optimization of affibody molecules for medical use for the first time.</p>

Conventional cancer treatment based on radiotherapy or chemotherapy affects all dividing cells. By directing the therapy specifically to the tumor cells, normal cells can be spared. Tumor targeting molecules carrying a cytotoxic moiety is then an attractive approach. In this thesis, an affibody molecule with high affinity for the protein HER-2, that is strongly associated with aggressive forms of breast cancer, was selected. After radiolabeling with 125I, the affibody molecule, in monovalent and bivalent form, was tested in vitro in HER-2 overexpressing tumor cells and in transplanted tumors in mice. It was shown that the HER-2 targeting affibody molecule bound its target in a specific manner, both in vitro and in vivo. The small size of the affibody molecule resulted in fast clearance through the kidneys. An impressive tumor-to-blood ratio of 10 eight hours post injection was achieved and the tumors could easily be visualized in a gamma camera. The biologic effects of the bivalent affibody molecule and a monovalent affinity maturated version was measured and compared with the effects of the monoclonal antibody trastuzumab. It was found that although all molecules target the same protein, the effects differed greatly. The affibody molecule was also labeled with the alpha-emitting radionuclide 211At. Specific decrease in survival was seen in HER-2 overexpressing cells receiving the 211At labeled affibody molecule. The sensitivity to the treatment differed between cell lines, probably as a result of differences between the cell lines in internalization and nuclear size. The 211At labeled affibody molecules were also tested in vivo, where stability of the 211At label was a problem. To circumvent this problem, more stable conjugation chemistry was tested, as well as strategies to prevent uptake of released 211At by normal organs. This thesis describes the selection and optimization of affibody molecules for medical use for the first time.

Metastatic and primary bone cancers are devastating diseases of paediatric and adult humans because of the morbidity associated with bone pain. Controlling bone pain from multiple metastatic sites can be difficult in end-stage cancers using conventional therapies. Bone-seeking radiopharmaceuticals have been successful in this area as radiation can be delivered with moderate selectivity to the target. Unfortunately, targeted radiotherapy using radiopharmaceuticals have been relegated to palliative therapy as myelosuppression largely limits the radiation dose to sub-therapeutic levels. Efforts to overcome this therapeutic limitation include autologous bone marrow transplants in combination with chemotherapy-radiosensitization and the development of new radiopharmaceuticals. Development work using laboratory rodent models has been complicated by dosimetric limitations because of size and inherent problems with human xenografted tumour models in rodents. To address this need we studied naturally occurring canine osteosarcoma as a translational model for human bone cancer. Central to our hypothesis was that naturally occurring canine osteosarcoma would serve as an investigational model for comparing the pharmacokinetics (biodistribution), dosimetry, toxicity, and therapeutic effect of 153Sm-EDTMP, 188Re-HEDP, 186Re-HEDP, and a novel ligand, polyethyleneiminomethyl phosphonic acid (PEI-MP). Data collected from existing radiopharmaceuticals was then compared to PEI-MP labelled with 99mTc, 153Sm, and 186Re. This innovative and unique study allowed for the evaluation of various radiopharmaceuticals in a naturally occurring animal model of bone cancer, documenting the pharmacokinetics and dosimetry of a novel radiolabelled-ligand (PEI-MP). Benefits resulting from the successful completion of the study would allow more rapid transfer of rodent preclinical data into a naturally occurring cancer model more resembling to the human diseases and would thus more likely identify problems with pharmacokinetics and toxicity before proceeding to expensive clinical trials. The expected outcomes of the study were originally formulated based on limited previous published data in dogs. For instance, no data exists describing the pharmacokinetics or toxicity of 188Re-HEDP and 186Re-HEDP in the dog. The study was conducted in two phases. The first phase deals with the evaluation of 153Sm-EDTMP, 188Re-HEDP, and 186Re-HEDP in the dog model. Phase-two was the development of a novel ligand (PEI-MP) in the dog model of osteosarcoma, which has the characteristics of an ideal ligand. Pharmacokinetic results for 153Sm-EDTMP in normal dogs (n=4) for blood were similar to published reports for dogs and human. When compared statistically to human data the majority of results were the same, lending credence to the hypothesis that dogs could serve as models for human radiopharmaceutical research. Normal dogs and the osteosarcoma dog did differ from human pharmacokinetics in the urine elimination phase (t½-â). This can most likely be explained by the tumour burden in the human research populations or due to the fact that most humans were aged and likely to have some renal disease. Certainly, the trend in dogs with osteosarcoma was to have a prolonged urine elimination phase (t½-â) compared to normal dogs which supported the hypothesis that the biodistribution and pharmacokinetics results from dogs were similar to human published data. Statistical comparisons were also made between normal dogs receiving 188Re-HEDP and 153Sm-EDTMP. The prolonged urine elimination phase (t½-â) and increased blood retention of 188Re-HEDP was most likely a reflection of prolonged bone washout and soft issue retention. This could be attributed to the differences between the antiresorptive capability of bisphosphonate ligands e.g., EDTMP (lexidronam) with a greater than 100-fold antiresorptive capability than HEDP (etidronate). Additional observational biodistribution studies using macro- and micro-autoradiography techniques were also performed in canine tissue and Sprague-Dawley rats. Results from the studies showed heterogeneous uptake within tumours in dogs. In rats, localization of 153Sm-EDTMP in red marrow areas would lead to a high radiation dose to blood producing elements. In addition, high uptake was documented at the metaphyseal growth plate confirming the likelihood of a delay or cessation of growth if 153Sm-EDTMP were used in growing children. Phase-one of the clinical trial in dogs with naturally occurring osteosarcoma identified only mild toxicity at the dosage rate of 37 MBq/kg (1 mCi/kg) for both 153Sm-EDTMP and188Re-HEDP. In addition, a pilot trial was conducted in dogs receiving 153Sm-EDTMP which also received a carboplatin infusion at the time of the radiopharmaceutical administration followed by another 3 cycles of carboplatin at 3 weekly intervals. No differences in toxicity were noted between the carboplatin group and dogs receiving only 153Sm-EDTMP. As a part of the 188Re-HEDP clinical trial, 3 dogs with osteosarcomas received weekly dose of 188Re-HEDP at 37MBq/kg for 4 weeks in which only mild toxicity was noted. Unfortunately, there was no cessation in growth of the tumours, with all dogs showing progression. The median survival time for both radiopharmaceuticals was 4 months, significantly shorter than the 10-month median survival time for amputation and chemotherapy. Interestingly six dogs that had 99mTc-MDP and 153Sm-EDTMP showed scans of tumours that had consistently lower 99mTc-MDP uptake ratios (normal bone compared to cancerous bone) compared to solely 153Sm-EDTMP. In contrast, this was not evident for uptake ratios between 188Re-HEDP and 99mTc-MDP scans. Once again, this finding highlights the differences between the antiresorptive capabilities of the bisphosphonates ligands. Interestingly, another three dogs were scanned with 99mTc-MDP , 153Sm-EDTMP, and 99mTc-PEI-MP (10-30 kDa) showed a variation in uptake between scans of the same tumours. More importantly, the uptake ratios of 99mTc-MDP and 153Sm-EDTMP scans showed wide variation with a coefficient of variance of 52% and 39% respectively. However, the range in uptake from the 99mTc-PEI-MP (10-30 kDa) scan was narrow with a coefficient of variance of only 6%. This could be attributed to more consistent uptake ratio of the unique ligand PEI-MP and its hypothesized mechanism of action: enhanced permeability and retention (EPR) in tumour vasculature. This requires further investigation with larger groups. In phase-two, the pharmacokinetic result for the novel ligand PEI-MP was initially studied labelled with 99mTc. Various molecular weights were tested in normal dogs and compared to previously published results in baboons. Results from the dog studies were found to be similar to those from the primate study. As in the primate study, molecular weight and charge played a significant role in 99mTc-PEI-MP pharmacokinetics. Increasing the size of the macromolecules and altering their charge resulted in marked changes in their pharmacokinetics and biodistribution. The PEI-MP molecular weight of 10-30 kDa and 20-30 kDa were the most promising and fulfilled the hypothesized criteria of an ideal radiopharmaceutical. In keeping with the aims of the study, the 20-30kDa polymer was considered more desirable because of its faster clearance. However, because of the limitations imposed by the percentage yield of the different molecular weights of the ligand during filtration, we decided to label the 10-30kDa molecular weight MW-fraction with 153Sm. Unexpectedly, the 153Sm-PEI-MP 10-30 kDa had a prolonged urine elimination phase (t½-â) associated with increased liver uptake when compared to 99mTc-PEI-MP10-30 kDa. To explain this, computer modelling for blood plasma (ECCLES) was done which predicted that there would be some chemical dissociation of the 153Sm from the PEI-MP polymer in blood. This is due to interaction between the radiopharmaceutical and citrate, forming 153Sm-citrate. The ECCLES model for blood plasma also predicted that the anionic MW-fraction, PEI-MP 10-30kDa, would be a poor ligand complexed to 166Ho, 212Pb, 213Pb, and 89Sr, but was expected to be effective when complexed to 186Re or 188Re, based on their close proximity to 99mTc on the periodic table. As a preliminary study 186Re was complexed to 20-30 kDa (n=2) and 30-50 kDa (n=1) MW-fractions and tested in dogs. The results were similar to 99mTc-PEI-MP 10-30 kDa. The biodistribution data and pharmacokinetic data were also used to do comparative dosimetry between radiopharmaceuticals. Not surprisingly, the dosimetry data confirmed the high red marrow dose for 153Sm-EDTMP and the increased soft-tissue dose of the radionuclides complexed to HEDP. The radiation dose to the tumour for all radiopharmaceuticals fell within the range of 26Gy to 44Gy. This is well within the range used to treat canine osteosarcoma using external beam radiotherapy. When compared to external beam radiotherapy, the probable lack of tumour response in our clinical trial relates to the heterogenous distribution of the radiopharmaceutical in the tumour and the inherent resistance of osteosarcoma cells to continuous low-dose radiation delivery (CLDR) inherent in radionuclide -particle decay. The study met the majority of outcomes with the exception of labelling PEI-MP with 153Sm. This was due to the interaction of the 153Sm-PEI-MP complex with citrate ions in blood. Rapid deterioration of the Rhenium-188 generator also led to earlier than expected curtailment of the 188Re-HEDP therapeutic trial although sufficient data was available to be used in a comparative study. / Thesis (PhD)--University of Pretoria, 2013. / Internal Medicine / unrestricted

Telomerase is expressed in the majority (>85%) of tumours but not in differentiated normal tissue. This enzyme catalyses the elongation of telomeres – a process critical for continued cell proliferation. Telomerase is a potential novel target for molecularly-targeted radiotherapy (mRT), due to its nuclear localization and expression profile. The radiolabelling of telomerase inhibitors may accelerate and enhance the cytotoxicity of such molecules, as a result of irradiation of the DNA. An oligonucleotide targeting telomerase RNA (hTR), shown to inhibit enzyme activity in vitro, was selected for study. Complementary and non-targeting control oligonucleotides were conjugated to a metal chelator (DTPA) to allow radiolabelling with indium-111. The radioiodination of MST-312, BIBR-1532 and flavonoid-derived small molecule inhibitors of telomerase was also pursued. The inhibitory activity of the candidate molecules was analysed using the telomeric repeat amplification protocol (TRAP). The internalization of inhibitors was assessed by gamma-counting following cell lysis. The clonogenic assay was employed to measure the effect of modified inhibitors on cell survival. Small molecule telomerase inhibitors were modified for labelling with iodine-123, which led to a modest decrease in inhibitory potency, compared to the parent molecules. Radiolabelled small molecules exhibited poor stability and internalization into cancer cells, so were unsuitable for mRT. Modified oligonucleotides potently inhibited telomerase activity, whereas a non-targeting oligonucleotide exhibited no inhibitory activity. Indium-111 radiolabelled oligonucleotides decreased the clonogenic survival of telomerase-positive breast cancer cells but not telomerase-negative cells, in a sequence-specific manner. Accordingly, complementary radiolabelled oligonucleotides were found to induce the DNA damage marker γH2AX. Oligonucleotides localized to nuclear Cajal bodies, the sites of telomerase assembly, in a proportion of cancer cells. Telomerase inhibitors of different classes were radiolabelled with Auger electron-emitting radionuclides, and delivered to cells. Radiolabelled oligonucleotides targeting telomerase significantly reduced the clonogenicity of cancer cells in vitro. This study represents a novel approach for the mRT of telomerase-positive cancers.