tumour targeting using radiolabelled affibody molecules735588/fulltext01.pdf · dissertation...

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2014

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1013

Tumour Targeting usingRadiolabelled Affibody Molecules

Influence of Labelling Chemistry

MOHAMED ALTAI

ISSN 1651-6206ISBN 978-91-554-8983-0urn:nbn:se:uu:diva-229090

Dissertation presented at Uppsala University to be publicly examined in Rudbecksalen,DagHammarskjölds väg 20, Uppsala, Saturday, 20 September 2014 at 09:00 for the degree ofDoctor of Philosophy (Faculty of Medicine). The examination will be conducted in English.Faculty examiner: Professor Jacques Barbet (Head of GIP ARRONAX, National AcceleratorResearch Centrum in Nantes, France).

AbstractAltai, M. 2014. Tumour Targeting using Radiolabelled Affibody Molecules. Influenceof Labelling Chemistry. Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1013. 77 pp. Uppsala: Acta Universitatis Upsaliensis.ISBN 978-91-554-8983-0.

Affibody molecules are promising candidates for targeted radionuclide-based imaging andtherapy applications. Optimisation of targeting properties would permit the in vivo visualizationof cancer-specific surface receptors with high contrast. In therapy, this may increase the ratio ofradioactivity uptake between tumour and normal tissues. This thesis work is based on 5 originalresearch articles (papers I-V) and focuses on optimisation of targeting properties of anti-HER2affibody molecules by optimising the labelling chemistry.

Paper I and II report the comparative evaluation of the anti-HER2 ZHER2:2395 affibody moleculesite specifically labelled with 111In (suitable for SPECT imaging) and 68Ga (suitable for PETimaging) using the thiol reactive derivatives of DOTA and NODAGA as chelators. Theincorporation of different macrocyclic chelators and labelling with different radionuclidesmodified the biodistribution properties of affibody molecules. This indicates that the labellingstrategy may have a profound effect on the targeting properties of radiotracers and must becarefully optimized.

Paper III reports the study of the mechanism of renal reabsorption of anti-HER2 ZHER2:2395

affibody molecule. An unknown receptor (not HER2) is suspected to be responsible for the highreabsorption of ZHER2:2395 molecules in the kidneys.

Paper IV reports the optimization and development of in vivo targeting properties of 188Re-labelled anti-HER2 affibody molecules. By using an array of peptide based chelators, it wasfound that substitution of one amino acid by another or changing its position can have a dramaticeffect on the biodistribution properties of 188Re-labelled affibody molecules. This permitted theselection of –GGGC chelator whichdemonstrated the lowest retention of radioactivity in kidneyscompared to other variants and showed excellent tumour targeting properties.

Paper V reports the preclinical evaluation of 188Re-ZHER2:V2 as a potential candidate fortargeted radionuclide therapy of HER2-expressing tumours. In vivo experiments in mice alongwith dosimetry assessment in both murine and human models revealed that future humanradiotherapy studies using 188Re-ZHER2:V2 may be feasible.

It would be reasonable to believe that the results of optimisation of anti-HER2 affibodymolecules summarized in this thesis can be of importance for the development of other scaffoldprotein-based targeting agents.

Keywords: HER2, Affibody molecule, Radionuclide molecular maging, Targeted radionuclidetherapy, Labeling chemistry

Mohamed Altai, Department of Oncology, Radiology and Clinical Immunology, BiomedicalRadiation Sciences, Rudbecklaboratoriet, Uppsala University, SE-75185 Uppsala, Sweden.

© Mohamed Altai 2014

ISSN 1651-6206ISBN 978-91-554-8983-0urn:nbn:se:uu:diva-229090 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-229090)

The value of the man resides in his education not his fortune.

To my parents who taught me these wordsand to my beloved brother and sister.

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals:

I Altai M, Perols A, Eriksson Karlström A, Sandström M,

Boschetti F, Anna Orlova A, Tolmachev V. Preclinical evalua-tion of anti-HER2 affibody molecules site-specifically labeled with 111In using a maleimido derivative of NODAGA Nuclear Medicine and Biology, 2012;39:518-29.

II Altai M *, Strand J *, Rosik D, Selvaraju RK, Karlström A, Or-lova A, Tolmachev V. Influence of nuclides and chelators on imaging using affibody molecules : comparative evaluation of recombinant affibody molecules site-specifically labeled with 68Ga and 111In via maleimido derivatives of DOTA and NODAGA. Bioconjug Chem, 2013;24:1102-9.

III Altai M, Varasteh Z, Andersson K, Eek A, Boerman O, Orlova A In vivo and in vitro studies on renal uptake of radiolabelled affibody molecules for imaging of HER2 expression in tumors. Cancer Biother Radiopharm 2013;28:187-95.

IV Altai M. Honarvar H, Wållberg H, Strand J, Varasteh Z, Rosestedt M, Orlova A, Dunås F, Sandström M, Löfblom J, Tolmachev V, Ståhl S. Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody mol-ecules with 188Re. Conditionally accepted by the European Journal of Medicinal Chemistry.

V Altai M. Wållberg H, Honarvar H, Strand J, Orlova A, Va-rasteh Z, Sandström M, Löfblom J, Larsson E, Strand SE, Lub-berink M, Ståhl S, Tolmachev V. 188Re-ZHER2:V2, a promising af-fibody-based targeting agent against HER2-expressing tumors: preclinical assessment.Conditionally accepted by Journal of Nuclear medicine.

* indicates equal contribution.

Reprints were made with permission from the respective publishers.

Additional papers (not included in this thesis): I Altai M, Wållberg H, Orlova A, Rosestedt M, Hosseinimehr SJ,

Tolmachev V. Ståhl S, Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences biodistribution of 99mTc-labeled recombinant affibody molecules. Amino Acids, 2012;42:1975-85.

II Altai M, Tolmachev V, Orlova A. Radiolabelled probes targeting tyrosine-kinase receptors for personalized medicine. Curr Pharm Des. 2014;20:2275-92.

III Tolmachev V, Altai M, Sandström M, Perols A, Ericsson Karlström A, Boschetti F, Orlova A. Evaluation of a maleimido derivative of NOTA for site-specific labeling of affibody molecules. Bioconjug Chem, 2011;22:894-902.

IV Hofström C, Altai M, Honarvar H, Strand J, Malmberg J, Hosse-inimehr SJ, Orlova A, Gräslund T, Tolmachev V, HAHAHA, HEHEHE, HIHIHI or HKHKHK: influence of histidine containing tags position and composition on biodistribution of [99mTc(CO)3]+- labelled affibody molecules. J Med Chem, 2013;56:4966-74.

V Mitran B, Altai M, Hofström C, Honarvar H, Sandström M, Orlova A, Tolmachev V, Gräslund T. Evaluation of 99mTc-ZIGF1R:4551-GGGC affibody Molecule, a New Construct for Imaging the Insulin-like Growth Factor Type 1 Receptor Expression. Conditionally accepted by Amino Acids.

VI Wållberg H, Orlova A, Altai M, Widström C, Hosseinimehr SJ, Malmberg J, Ståhl S, Tolmachev V. Molecular design and optimiza-tion of 99mTc-labeled recombinant affibody molecules improves their biodistribution and imaging. J Nucl Med, 2010;52:461–469.

VII Hofström C, Orlova A, Altai M, Wångsell F, Gräslund T, Tolma-chev V. The use of a HEHEHE-purification tag improves biodistri-bution of affibody molecules site-specifically labeled with 99mTc, 111In and 125I compared to a hexahistidine-tag. J Med Chem, 2011;54:3817-3826.

VIII Lindberg H*, Hofström C*, Altai M, Honorvar H, Wållberg H, Or-lova A, Ståhl S, Gräslund T, Tolmachev V. Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for 99mTc-labellingat the C-terminus Tumour Biol. 2012;33:641-51.

IX Rosik D, Orlova A, Malmberg J, Altai M, Varasteh Z, Sandström M, Eriksson Karlström A, Tolmachev V. Direct in vivo comparison of 2-helix and 3-helix affibody molecules. Eur J Nucl Med Mol Imag-ing. 2012;39:693-702.

X Malmberg J, Perols A, Varasteh Z, Altai M, Sandström M, Garske U, Tolmachev V, Orlova A, Eriksson Karlström A. Comparative evaluation of synthetic anti-HER2 affibody molecules site-specifically labelled with (111)In using N-terminal DOTA, NOTA and NODAGA chelators in mice bearing prostate cancer xenografts. Eur J Nucl Med Mol Imaging. 2012;39:481-92.

XI Rosik D, Thibblin A, Antoni G, Honarvar H, Strand J, Selvaraju RK, Altai M, Orlova A, Eriksson Karlström A, Tolmachev V. Triglutam-yl spacer improves biodistribution of synthetic affibody molecules radiofluorinated at N-terminus using 18F-4-fluorobenzaldehyde via oxime formation. Bioconjug Chem. 2014;25:82-92.

XII Orlova A, Malm M, Rosestedt M, Varasteh Z, Andersson K, Selva-raju RK, Altai M, Honarvar H, Strand J, Ståhl S, Tolmachev V, Löfblom J. Imaging of HER3-expressing xenografts in mice using a (99m)Tc(CO)3-HEHEHE-ZHER3:08699 affibody molecule. Eur J Nucl Med Mol Imaging. 2014;41:1450-9.

XIII Varasteh Z, Rosenström U, Velikyan I, Mitran B, Altai M, Honar-var H, Sörensen J, Rosestedt M, Lindeberg G, Larhed M, Tolmachev V, Orlova A. The effect of PEG-based spacer length on binding and pharmacokinetic properties of a 68Ga-labeled NOTA-conjugated an-tagonistic analog of bombesin. Molecules, 2014;19:10455-72.

XIV Honarvar H, Garousi J, Gunneriusson E, Höidén-Guthenberg I, Altai M, Widström C, Tolmachev V, Frejd FY. Imaging of CAIX-expressing xenografts in vivo using 99mTc-HEHEHE-ZCAIX:1 affibody molecule. manuscript.

Contents

Introduction ................................................................................................... 13 Receptor tyrosine kinases as potential therapeutic targets ....................... 14 Anti-cancer therapies targeting RTKs ...................................................... 14

Immunotherapy .................................................................................... 14 Tyrosine kinase inhibitors .................................................................... 15

Resistance to molecular targeted therapies and current solutions ............ 16

Personalising cancer treatment: current status .............................................. 18 Biomarkers in cancer ................................................................................ 18 Molecular profiling of cancer and patient stratification ........................... 19 Response monitoring using anatomical imaging ...................................... 20

Radionuclide molecular imaging for personalising medicine ....................... 21 Radionuclide-based imaging techniques .................................................. 21 Imaging metabolism and proliferation ..................................................... 21 Imaging of receptor tyrosine kinases ........................................................ 22 Imaging probes targeting RTK: current status ......................................... 24

Affibody molecules ....................................................................................... 27 Background .............................................................................................. 27 Labelling chemistry .................................................................................. 29

Macrocyclic chelators .......................................................................... 30

Optimisation of the targeting properties of affibody molecules for targeted radionuclide therapy ...................................................................................... 33

Optimisation of peptide-based chelators .................................................. 35 Kidney reabsorption of radiolabelled affibody molecules: Understanding the mechanism ................................................................. 37

The present investigation .............................................................................. 40 Evaluation of anti-HER2 affibody molecules site-specifically labelled with 111In using a maleimido derivative of NODAGA (Paper I) .............. 40

Aim ...................................................................................................... 40 Method ................................................................................................. 40 Results and discussion ......................................................................... 40 Brief conclusion ................................................................................... 42

Influence of 68Ga and 111In in combination with DOTA and NODAGA chelators on imaging properties of affibody molecules: a comparative evaluation (Paper II) ................................................................................. 43

Aim ...................................................................................................... 43 Methods ............................................................................................... 43 Results and discussion ......................................................................... 43 Brief conclusion ................................................................................... 45

In vivo and in vitro studies on renal uptake of radiolabelled affibody molecules: understanding the mechanism (Paper III) .............................. 46


Selection of an optimal cysteine-containing peptide-based chelator for labelling of affibody molecules with 188Re (Paper IV) ............................. 49


188Re-ZHER2:V2, a promising affibody-based targeting agent against HER2-expressing tumors: preclinical assessment (Paper V). .................. 53


Concluding remarks ...................................................................................... 57

Acknowledgements ....................................................................................... 62

References ..................................................................................................... 65

Abbreviations

ADAPT ABD Derived Affinity Proteins. B Bladder Bmax The maximum amount of drug which can bind specifi-

cally to the receptor DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid DTPA Diethylene triamine pentaacetic acid DU-145 Human prostate cancer cell line EGFR Epidermal growth factor receptor Eβ-max Maximum energy of beta particles Eγ Gamma energy HER2 Human epidermal growth factor receptor 2 IC50 Half maximal inhibitory concentration IGF-1R Insulin-like growth factor-1 receptor K Kidneys KD Dissociation constant keV Kilo electron-Volt maGGG- Mercaptoacetyl-glycyl-glycyl-glycyl maSSS- Mercaptoacetyl-seryl-seryl-seryl maEEE- Mercaptoacetyl-glutamyl-glutamyl-glutamyl mBC Metastatic breast cancer MMA Maleimidoethylmonoamide MOBY phantom Digital mouse whole body phantom NMRI Naval Medical Research Institute NODAGA 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid NOTA 1,4,7-triazacyclononane-N,N',N''-triacetic acid OK cells Opossum Kidney Epithelial cell line OLINDA/EXM Organ Level INternal Dose Assessment / EXponential

Modelling PDGFR Platelet-derived growth factor receptor p.i. Post injection RECIST Response Evaluation Criteria In Solid Tumours RIT Radioimmunotherapy RMI Radionuclide molecular imaging SKOV-3 Human ovarian carcinoma cell line TCO Trans-Cyclooctene

T½ Half-life VEGFR Vascular endothelial growth factor receptor ZHER2:V2 Anti-HER2 affibody molecule variant 2 -GGGC Glycyl-glycyl-glycyl-cysteinyl -GGSC Glycyl-glycyl-seryl-cysteinyl -GGEC Glycyl-glycyl-glutamyl-cysteinyl -GGKC Glycyl-glycyl-glutamyl-cysteinyl %ID/g Percentage of injected dose per gram

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Introduction

Cancer is a heterogeneous group of diseases that have histologically and clinically distinct features. Malignant tumour cells have a number of abnor-mal biological properties, i.e., the hallmarks of cancer. These include sus-tained proliferative signalling, resistance to programmed cell death (apopto-sis), induction of angiogenesis, and activation of invasion and metastasis [1]. The alteration of normal growth-promoting signals is mainly attributed to the genomic instability of cancer cells. Aberrantly expressed receptors with ty-rosine kinase domains are an important class of oncogene products involved in cancer development.

Receptor tyrosine kinases (RTKs) are a broad superfamily of receptors that fall into twenty subfamilies; the most commonly studied of these are class I (EGF receptor family or ErbB family), class II (Insulin receptor fami-ly) and class V (VEGF receptors family) [2]. Mainly, these families of re-ceptors control necessary functions for newly proliferating cells through membrane-nuclear communication [1]. Dysregulation of RTKs is associated with many neoplastic properties of cells. In many cases, the RTK structure is comprised of three components: an intracellular region, a single transmem-brane hydrophobic domain and an extracellular region [3,4]. The extracellu-lar region is the ligand binding and dimerisation domain of this system. The hydrophobic transmembrane-spanning domain is important for regulating the dimerisation and activation of the receptors. Finally, the intracellular domain is mainly associated with an intrinsic tyrosine kinase system devoted to maintaining cellular protein activation and nuclear communication [4].

In general, ligand binding results in receptor dimerisation followed by transphosphorylation and activation of the kinase domain. The activated kinase domain triggers phosphorylation of tyrosines on the adjacent RTK. Phosphorylated tyrosines establish a base for assembly and activation of a large number of downstream signalling molecules. These signalling mole-cules control proliferation, apoptosis, differentiation, vasculature recruitment and motility, among other functions [5,6].

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Receptor tyrosine kinases as potential therapeutic targets As our understanding of the role of RTKs in both normal physiology and malignancies increases, their importance as clinically relevant targets be-comes clearer [7]. Molecular recognition of aberrantly expressed receptors and hyperactive intracellular signalling proteins forms the basis of targeted therapy [7]. Ideally, the diseased tissue should be selectively targeted while healthy tissues and organs are spared, thus reducing toxicity. Generally, tar-geting strategies can be divided into the following main categories: • The blocking of binding and dimerisation domains of RTKs (e.g., mono-clonal antibodies trastuzumab, cetuximab, and panitumumab); • The inhibition of intracellular receptor tyrosine kinase sites by tyrosine kinase inhibitors (TKIs) (e.g., lapatinib, sorafenib and gefitinib); • The inhibition of intracellular signalling cascades (e.g., vemurafenib target-ing the B-Raf/MEK/ERK pathway). • The inhibition of the chaperone machinery (e.g., HSP90) of malignant cells, preventing correct folding of RTKs and their delivery to cellular mem-branes. • The use of molecular recognition of aberrantly expressed RTKs for target-ed delivery of cytotoxic agents (e.g., toxins or radionuclides) to malignant cells.

So far, agents targeting the extracellular and intracellular domains of RTKs constitute the majority of approved anti-RTK therapies. Several prom-ising compounds aimed at abrogating signalling in tumour cells by altering signalling via the MAPK pathway (Ras/Raf/MEK/ERK pathway) are under extensive development [8,9]. Recently, the FDA approved the B-Raf inhibi-tors Zelboraf (vemurafenib) and Tafinlar (dabrafenib) and the MEK inhibitor Mekinist (trametinib) for the treatment of patients with advanced metastatic or unresectable melanomas. The clinical utility of kinase inhibitors of intra-cellular signalling proteins needs to be further investigated both preclinically and clinically before full approval of their efficacy [8,9].

Anti-cancer therapies targeting RTKs Immunotherapy Targeting the extracellular domain of RTKs with monoclonal antibodies is considered as an important cancer treatment strategy. Monoclonal antibodies selectively target the extracellular domain of aberrantly expressed RTKs, thus permitting specificity in comparison to conventional therapies e.g., chemotherapy. The mechanism of action of monoclonal antibodies (mAbs) involves the triggering of receptor internalisation (and subsequent degrada-

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tion), the inhibition of dimerisation, the activation of antibody-dependent cellular cytotoxicity (ADCC) and the occupancy of the ligand binding pock-et [10].

The anti-HER2, trastuzumab, was the first approved (1998) mAb directed against RTKs. Later, a number of anti-RTK mAbs, such as cetuximab and pantimumab (anti-EGFR, 2004 and 2006, respectively), bevacisumab (anti-VEGF, 2004) and ramucurimab (anti-VEGFR2, 2014), were introduced into clinical practice (Fig. 1). Today, more mAbs are in late phase clinical trials. Examples include the anti-IGF-1R mAbs, figitumumab and ganitumab [11-15].

EGFRHER2HER2 IGF-1R VEGFR

-Trastuzumab-Pertuzumab

- Lapatinib- Neratinib- Afatinib

HSP90

- Cetuximab- Panitumumab

PDGFR

- Erlotinib- Gefitinib- Lapatinib- Neratinib- Afatinib

HSP90 inhibitors e.g. 17-AAG

- Sorafenib- Sunitinib- Pazopanib- Cediranib

- Imatinib- Dasatinib - Sorafenib- Sunitinib

NVP-AEW541

Ramucirumab

- Fugitumumab- Ganitumab

IMC-3G3

Figure 1. Anti-cancer therapies based on the targeting of RTKs.

Tyrosine kinase inhibitors An alternative strategy for the treatment of RTK-driven tumours is to target the intracellular tyrosine kinase domains. Tyrosine kinases transfer a γ-phosphate group from adenosine triphosphate (ATPs) to a specific tyrosine-residue in target proteins. Tyrosine kinase inhibitors are small molecules capable of penetrating the cell membrane and blocking the catalytic activity of TKs, hence arresting receptor-oncogene communication signals [16].

Imatinib (PDGFR, Kit, Abl1-2) was the first approved tyrosine kinase in-hibitor for the treatment of chronic myeloid leukemia. Later, imatinib proved to be efficient in patients with gastrointestinal stromal tumours (GIST) with

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mutant c-Kit [17,18]. The commercial success of imatinib attracted consid-erable interest in developing new TKIs. Lapatinib (EGFR; HER2) was ap-proved for the treatment of certain types of advanced or metastatic breast cancer [19]. Today, there are 13 FDA-approved TKIs and more are in the final stages of approval [20].

Resistance to molecular targeted therapies and current solutions Regardless of the noteworthy success achieved with mAbs and small mole-cule TKIs, it appears that a considerable number of patients are inherently resistant to these drugs and the majority acquire resistance over time. For instance, it has been reported that 70 % of HER-2 positive breast cancer patients do not benefit from trastuzumab [21]. Multicenter trials revealed efficacy of lapatinib in only a subset of breast cancer patients expressing HER2 [19]. Understanding the underlying biological basis of resistance to mAbs and TKIs has been the subject of intensive research but is beyond the scope of this thesis. Briefly, up regulation of cell death inhibitory mecha-nisms, escape from the cell cycle arrest, and switching to alternative mito-genic signalling pathways are among several proposed mechanisms thought to mediate resistance to molecular targeted therapy [22,23]. These mecha-nisms are controlled by several oncogenic products (i.e., receptors or signal-ling proteins). For example, the PTEN (a tumour suppressor) mutation found in 50 % of metastatic breast cancers was proposed, among other mecha-nisms, to confer resistance of HER2-positive mBC to trastuzumab [21]. In addition, secondary resistance to trastuzumab was found to result from in-creased IGF-1R and HER3 signalling. This counteracts the growth inhibition induced by HER2-blockage.

Failure to obtain a positive response with many TK-targeted therapies made it necessary to reconsider new treatment strategies. Cytotoxic payloads or effector molecules were conjugated to tumour-targeting mAbs or their fragments to enhance their anti-tumour effects and improve response dura-tion and overall response rate. For this purpose, mAbs have been armed with radionuclides, cytotoxic drugs and toxins [24,25]. Particularly, a conjugate of the antibody trastuzumab and emtansine toxin (a tubular polymerisation inhibitor) known as T-DM1 has been shown to significantly improve PFS (progression free survival) and OS (overall survival) with less frequent grade 3 or above adverse effects than lapatinib-capecitabine [26,27].

Given the multiplicity of resistance mechanisms, it is likely that an opti-mal therapeutic approach would be highly dependent on the actual mecha-nism of resistance in each individual patient. This highlights the need for novel therapeutic strategies based on detailed molecular profiling of individ-

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ual relapsed tumours. Such understanding would open the door for an indi-vidualised drug administration strategy for distinct patient subgroups to al-low for the personalisation of cancer treatments [28].

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Personalising cancer treatment: current status

Our understanding of cancer biology continues to evolve, and cancer is now detected, characterised and even treated on a molecular level. Clinicians and scientists aim to design cancer study protocols that assess patients’ molecu-lar status, which is important to overcome the pleomorphic nature of cancer [29].

Biomarkers in cancer During the era of targeted therapy, the concept of biomarkers has evolved to facilitate the development of new therapies and to rationalise clinical deci-sion making regarding treatment with a specific drug. A biomarker, in gen-eral, is a parameter that is objectively measured and evaluated to assess pathogenic status, biological processes or pharmacologic response to a ther-apeutic intervention. In cancer, biomarkers act as useful tools for the predic-tion of both survival and treatment outcome. Therefore, biomarkers can be divided into three categories: prognostic (estimates the natural course of the disease and the patients’ overall outcome); predictive (estimates the response or survival of a specific patient on a specific treatment compared with anoth-er treatment) and pharmacodynamic (evaluates the effect of a drug or other intervention) [30,31]. There is a growing interest in the pharmaceutical in-dustry and in clinics for the discovery of biomarkers to be used as surrogate endpoints to allow for more rapid evaluation of the performance of a new drug in clinical trials.

The expression level of an oncogene or a receptor, either in the cancer cell or stromal tissue, might act as a predictive biomarker. Single or multiple hyperactive intracellular signalling proteins can also act as predictive bi-omarkers. The successful development and use of kinase inhibitors for can-cer therapy has been shown to be very much dependent on predictive bi-omarkers for patient selection [32]. This has led to the approval of several pioneering anti-cancer drugs (e.g., trastuzumab for HER2-expressing tu-mours and imatinib for tumours expressing BCR-ABL1). An important lesson accounting for the relevance of predictive biomarkers comes from the development of the anti-IGF-1R mAb, figitumumab. Figitumumab has demonstrated promising clinical outcomes in several phase I/II studies. Sur-prisingly, figitumumab has failed to achieve similar results in large random-

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ised phase III clinical trials [33]. In addition to its lack of effectiveness in phase III trials, toxicity in the form of hyperglycemias and hyperinsulinae-mias was also observed [34]. These disappointing results, which were re-ported in several figitumumab phase III trials, were mainly attributed to the inclusion of large groups of unselected patients in these studies [35]. The experience with figitumumab also highlights the importance of diagnostic techniques in the introduction of new biomarkers. Currently, companion diagnostics are a key component of individualised targeted therapy that will help ensure that only susceptible tumours are treated with a specific targeting agent [32].

Molecular profiling of cancer and patient stratification Due to the low costs and availability of the method, immunohistochemistry (IHC) has long been the main approach for evaluating target expression level on tumour biopsies. Fluorescence in situ hybridisation (FISH) is a more sen-sitive and reproducible technique with high concordance of acquired results but is much more laborious and expensive. Both techniques identify gene amplification either directly (through complimentary hybridisation) or indi-rectly (through detection of the pathologic protein products of these genes). One of the most successful examples of implementing biopsy-based molecu-lar profiling techniques in cancer therapy is the HercepTest. The expression of HER2 on a level of 3+ according to the HercepTest is predictive of breast cancer response to trastuzumab and lapatinib [36,37].

Invasiveness is the main drawback of biopsy-based methods. This limits the number of samples that can be obtained prior to and during therapy. In some cases, accessibility to metastases will be compromised due to their large numbers and dispersed locations. Non-representative sampling due to the heterogeneity of intratumoural expression [38,39] or discordance of ex-pression between the primary tumours and metastases [40-42] are additional limitations to be considered. In addition, the molecular profile of tumour samples determined earlier might not reflect the tumour status at the time of therapy. The main reason for this is clonal selection of malignant cells dur-ing the normal course of the disease or in response to previous treatments. [43,44]. Variations in antigen retrieval and differences in subjective interpre-tation of the results are additional complications associated with biopsy-based techniques [45,46]. There have been further improvements in biopsy-based diagnostic methods. CISH (compliment in situ hybridisation), SISH (silver in situ hybridisation), photo-activation based methods, assessment of mRNA overexpression using qRT-PCR and micro array along with MLPA (Multiplex Ligation-dependent Probe Amplification) are some promising examples [47].

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Response monitoring using anatomical imaging Anatomical imaging is the most commonly used technique for monitoring response to therapy in solid tumours. An evaluation criterion for measuring tumour progression in solid tumours in response to cancer therapy (RECIST) was published in 2000 and updated in 2009 [48,49]. RECIST relies on one-dimensional (non-volumetric) lesion measurement where the longest diame-ter (LD) of selected target lesions on an axial CT image is determined. The sum of the longest diameters (SLD) for all selected targets is used to evalu-ate the outcome of therapy.

In practice, using the RECIST criteria for monitoring anti-RTK treatment is suboptimal. These therapeutic agents are rather cytostatic than cytotoxic, and the tumour response might not be accompanied by tumour shrinkage [50]. For slowly growing tumours (CRC, NSCLC) response to treatment might not be recognisable. On the contrary, minor responses might translate into more profound tumour shrinkage in fast-growing tumours. Therefore, assessment of treatment outcome can be misleading, simply because a stable disease, according to the current criteria, can be achieved due to growth in-hibition or due to the natural slow growth rate of the tumour, even in the absence of any therapy. Therefore, evaluation of response rates by measur-ing size reduction is not ideal [51]. Choi and colleagues have developed a new criterion based on quantitative measurement of tumour density (in Hounsfield units) as a complementary parameter to tumour size [52]. Despite a number of refinements [53-55], the use of anatomic imaging methods for evaluating response to RTK-targeting drugs remains questionable. In addi-tion to challenges associated with determining the tumour growth rate, fun-damental limitations in response assessment may exist due to errors in lesion selection, lesion measurement or interobserver variability.

Obstacles associated with biopsy-based patient stratification and anatomi-cal imaging-therapy monitoring reveals the need for an effective alternative that can provide visualisation of target expression in all lesions during a sin-gle procedure.

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Radionuclide molecular imaging for personalising medicine

Radionuclide-based imaging techniques Radionuclide-based imaging techniques used in medicine can be divided into two categories: SPECT (Single Photon Emission Tomography) and PET (Positron Emission Tomography). SPECT relies on the detection of γ-quanta or high energy x-rays emitted by radionuclides e.g., 99mTc, 111In. PET is the technique for positron (β+) emitters e.g., 18F, 11C, 68Ga and is based on coincidence detection of annihilation photons. PET provides higher sensitivi-ty and spatial resolution than does SPECT and provides better quantification accuracy. Another advantage of PET over SPECT is the short half-life of β+ emitters, which minimises the radiation dose to the subject. On the other hand, SPECT is more readily available than PET. Incorporation of CT with both techniques in combined SPECT/CT or PET/CT gives anatomic “land-marks” to clinicians so they can accurately locate and identify the affected tissue.

Imaging metabolism and proliferation Today, the most commonly used tracer for clinical molecular imaging in oncology is 2-fluoro-2-deoxyglucose [18FDG]. 18FDG is a glucose analogue where fluorine-18 substitutes a hydroxyl group in position 2 of the glucose molecule. 18FDG-PET is an everyday expanding technique for both tumour detection and monitoring therapy response by measuring the metabolic activ-ity of cancer cells. Cancer cells that are slowly growing or dead (necrotic cells) in response to a certain treatment will be energetically inactive and hence will show a reduced uptake of the tracer. Because metabolic changes precede anatomical changes in several types of cancers, 18FDG-PET might provide early evidence of response compared with anatomical criteria. For example, in patients with GIST treated with imatinib mesylate, there was a close association between clinical outcome and the findings on 18FDG-PET but not the findings on computed tomography (CT) [18,56]. In addition, measurements of clinical benefit based on tumour size lag weeks and months

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behind the 18FDG-PET imaging results. Therefore, 18FDG-PET was actively evaluated for use as a sensitive and rapid indicator of response [57].

In addition to measuring alterations in glucose metabolism, measurement of changes in the DNA synthesis rate, which reflect proliferation, might also be used for response assessment. The thymidine analogues 3´-18F-fluoro-3´-deoxythymidine (18FLT) and 18F-1-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)5-methyluracil (18FMAU) might be superior to 18FDG for this purpose as they lack the unspecific uptake observed for the sugar ana-logue in several organs [58]. Early changes in tumour 18FLT uptake identi-fied the early response to a bevacizumab-irinotecan combination therapy in patients with glioma [59]. After being transported into the cell, FLT is phos-phorylated and acts as a thymidine analogue during DNA synthesis. Recent-ly, it was suggested that several factors other than cell proliferation could impact the sensitivity of 18F-FLT readouts for therapy monitoring. For in-stance, proliferating tumours with high thymidine levels do not take up a measurable level of the tracer [60].

Imaging of receptor tyrosine kinases Due to their complexity, imaging of the glucose metabolism and cell prolif-eration pathways for personalising medicine must be undertaken with cau-tion [61]. This makes it necessary to develop imaging probes targeting more cancer-specific molecular phenomena. Metabolic imaging of tumours with 18FDG cannot address many clinically important insights, such as screening the molecular profile of the tumour before starting treatment or understand-ing whether a specific pathway targeted by a drug has been altered. Measur-ing the drug pharmacokinetics during clinical trials, kinetic modelling and drug occupancy also extend beyond what could be learned from 18FDG. Al-ternatively, radionuclide molecular imaging of aberrantly expressed RTKs provides a highly sensitive, non-invasive screening tool for evaluating the molecular status of both primary tumours and all present metastatic lesions simultaneously.

The early work of Thomas Behr and colleagues confirmed the feasibility of using radiolabelled monoclonal antibodies for imaging HER2 prior to trastuzumab therapy [62]. They demonstrated that both the antitumour effi-cacy and toxicity of trastuzumab can be predicted with high accuracy based on radionuclide imaging prior to treatment. Of the 20 patients involved, 11 patients with tumour uptake of the tracer responded well to trastuzumab. Of the patients with no tumour uptake, only one patient did respond to the ther-apy. Another scintigraphy study demonstrated that the detection rate of sin-gle HER2-expressing tumour lesions was 45 %, and new tumour lesions were discovered in 13 of 15 patients using 111In-labelled trastuzumab [63]. Recently, trastuzumab has been labelled with the moderate-lived positron-

23

emitting radionuclide 64Cu and the long-lived 89Zr. This permits the use of more sensitive PET for visualisation of HER2 in metastatic breast cancer [64,65]. On the other hand, a group from Aarhus University hospital showed that 11C-labelled erlotinib can be used for the visualising of NSCLC lung tumours, including lymph nodes not identified with 18F-FDG [66]. Moreo-ver, this work also demonstrated that the target accessibility of the drug after systemic administration can be directly studied with its radiolabelled ana-logue. These and other results would substantiate the future use of radionu-clide-based imaging to individualise RTK-directed treatment.

Biochemical changes on a cellular level precede anatomical changes in response to therapy. For this reason, radionuclide molecular imaging (RMI) is a promising alternative to anatomic imaging for evaluating response to targeted therapy. It provides a noninvasive and direct method for in vivo visualisation and quantification of RTK expression, which can be repeatedly used. Of consideration is the work done by the Nijmegen group in assessing the changes in IGF-1R expression in response to therapy [67,68]. This group showed that it was possible to visualise membranous IGF-1R expression and target accessibility in vivo using 111In-figitumumab. This permitted the use of the receptor expression levels as a predictive biomarker for anti-IGF-1R therapy in Ewing’s sarcoma [67]. Niu and colleagues showed that it is feasi-ble to use 64Cu-DOTA-trastuzumab for in vivo monitoring of HER2 down-regulation by the HSP90 inhibitor 17-DMAG [69]. Holland and colleagues have demonstrated that treatment of mice bearing HER2-positive BT-474 breast cancer xenografts with the HSP90 inhibitor PU-H71 reduces the up-take of 89Zr-DFO-trastuzumab from 71±10 to 41±3 %ID/g at 48 h after in-jection [70]. In addition, reduction of 89Zr-trastuzumab in PET corresponded well to tumour reduction and downregulation of HER2 in gastric cancer models treated with afatinib, whereas 18F-FDG uptake remained constant [71]. Recently, trastuzumab-induced EGFR downregulation could be detect-ed noninvasively using the anti-EGFR monoclonal antibody panitumumab fragment 68Ga-PaniF(ab')2 [72].

However, it is important in this context to understand that antigen expres-sion is not the sole factor determining the success of personalised medicine when implementing RMI. For instance, several preclinical and clinical stud-ies have demonstrated that the correlation between tumour uptake of radio-tracer and target expression is not always direct. For example, the PET scans of 14 HER2-positive metastatic breast cancer patients using 89Zr-trastuzumab showed that approximately half of the patients showed no up-take of the tracer in certain tumour lesions. These specific lesions have been previously identified with conventional imaging [64]. Heskamp and col-leagues have also reported a discrepancy between IGF-1R expression in a metastatic breast cancer model and the radiolabelled antibody uptake after tamoxifen treatment. Despite the downregulation of membranous IGF-1R observed immunohistochemically, the uptake of 111In-R1507 remained con-

24

stant [68]. Similar variations have been observed previously in a different family of receptors (non-RTK), in particular the carbonic anhydrase receptor type IX (CAIX). Despite the immunohistochemically determined homoge-nous expression of CAIX, uptake of the mAb 131I-cG250 within the tumour was heterogeneous [73]. There could be different reasons underlying these observations, where some are radiotracer-related while others concern the antigen status. For instance, one can speculate that a discrepancy between tracer uptake and antigen expression in the previous examples is related to the suboptimal specificity and sensitivity of the tracer and hence that a more target-specific radiotracer would have a different outcome. Heskamp and colleagues have recently summarised the influence of drug type, localisation of the antigen, in vivo accessibility of the antigen, the enhanced permeability and retention (EPR) effect, receptor internalisation, tracer dose and timing of imaging on molecular imaging [74].

Imaging probes targeting RTK: current status Because they specifically bind to their targets, natural ligands (EGF, IGF, VEGF) and mAbs preferentially accumulate in tissues that overexpress these targets (tumours). It was reasonable to label these agents with radionuclides for the purpose of imaging RTKs. Selection of optimal radiolabelled imag-ing probes for visualisation of RTKs is a troublesome process [75]. For ex-ample, in vivo instability as well as physiological activity of radiolabelled natural ligands often resulted in suboptimal imaging outcomes. The use of radiolabelled TKIs, on the other hand, resulted in elevated hepatic uptake because of the lipophilic nature of these molecules. Although the potential of radiolabelled full-length IgG for both patient stratification and therapy moni-toring has been demonstrated, their use is associated with many apparent limitations, both in terms of sensitivity and specificity. Due to their size, antibodies demonstrate slow clearance from the body (several days) accom-panied with poor extravasation and diffusion into the extracellular space. In murine models, the tumour-to-blood ratio for the best full-length radiometal-labelled anti-HER2 monoclonal antibodies did not exceed 7. mAbs also ex-hibit a non-specific accumulation in tumours as a result of the abnormally permeable vasculature and inadequate lymphatic drainage of solid tumours, a phenomenon known as the enhanced permeability and retention (EPR) effect.

Advancements in biotechnology have made it possible to develop smaller enzymatically produced or engineered antibody fragments e.g., [Fab´]2, Fab´, diabodies, minibodies and nanobodies. Molecular display techniques (phage, ribosomal, yeast or bacterial surface display) also permitted the development of small size scaffold-based targeting agents e.g., affibody molecules and Designed Ankyrin Repeat Proteins (DARPins). A small targeting agent pro-

25

vides higher extravasation efficiency and better tumour penetration than do full-length mAbs. Together with rapid clearance from the body, these prop-erties increase the imaging contrast and consequently the sensitivity of RTK visualisation. Moreover, the time interval between injection and imaging will be reduced. The use of positron emitters with medium half-lives (com-patible with the smaller targeting agent biological half-life) such as 64Cu, 55Co, 76Br and 86Y, may further improve the sensitivity of imaging. The EPR effect was found to be effective for molecules with a molecular size of >45 kDa [76]. Antibody fragments (~55 kDa) are still considered to be too large to overcome this effect. Therefore, the use of even smaller targeting proteins, such as affibody molecules (~7 kDa), DARPins (~16 kDa) and nanobodies (~14 kDa), is important to obtain specific tumour targeting.

More details about radiolabelled probes targeting tyrosine-kinase recep-tors for personalised medicine can be found in two review articles published by our group [75,77]. Table 1 summarises some of these probes. In this ta-ble, imaging probes are classified according to whether they target the extra-cellular or the intracellular domain of RTKs. Radiotracers are also classified based on their nature: mAbs (monoclonal antibodies and their fragments), scaffold proteins (affibody, DARPins), natural peptide ligands and their de-rivatives (EGF, VEGF, IGF) and tyrosine kinase inhibitors and their ana-logues.

26

Table 1. Radiolabelled probes targeting tyrosine kinase receptors for personalised medicine [62-64, 67, 69, 78-107].

ǂ studied in humans.

RTK target-ed domain Class Targeting

agent Target examples

Extracellular domain

Monoclonal antibodies and

their derivatives

Monoclonal antibodies

HER2

111In-DTPA-trastuzumab ǂ 89Zr-DFO-trastuzumab ǂ 64Cu-DOTA-trastuzumab

EGFR

111In-Mab225 ǂ 89Zr-DFO-cetuximab

64Cu-DOTA-panitumumab

IGF-1R 111In- R1507

89Zr-DFO-R1507

Antibody fragments

HER2

111In-DOTA-DGHF (Fab’)2

68Ga-X-DGHF-(Fab’)2 111In-DTPA-trastzmb-Fab’

IGF-1R 111In-R1507-(Fab’)2 and

111In-R1507-Fab’

Nanobodies HER2 99mTc-2Rs15d EGFR 99mTc-7C12

Imaging agents based

on natural peptide

ligands of RTK

EGF EGFR 68Ga/111In-EGF

VEGF VEGFR 99mTc-VEGF121

64Cu-DOTA-VEGF121

IGF IGF-1R 111In-IGF-1(E3R)

De novo selected peptide ligands

Phage dis-play select-

ed short peptides

HER2 64Cu-NOTA-KCCYSL

EGFRvIII 4-[18F]fluorobenzoyl-FALGEA-NH2

Scaffold proteins

HER2 111In-DOTA-ZHER2:342 ǂ

99mTc-G3-PEG20 DARPin EGFR 111In-DOTA-ZEGFR:2377

IGF-1R 111In-DOTA-ZIGF1R:4551

PDGFRβ 111In-DOTA-ZPDGFRβ:09591

HER3 99mTc-ZHER3:08699

Intracellular domain

Radiolabelled TKI and

analogues

Radiolabelled TKIs EGFR TK [11C]-erlotinib ǂ

TKIs ana-logues

EGFR TK [11C]-PD153035 ǂ

(erlotinib analogue) VEGFR-2

TK [11C]-PAQ

(vandetanib analogue) Bcr-Abl

TK

18F-SKI696 (imatinib analogue)

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Affibody molecules

Background A promising approach for the development of high affinity, small-size imag-ing agents is the use of scaffold proteins [108]. In general, scaffold proteins are based on a structured framework of constant amino acids used to main-tain the tertiary structure, while a number of surface exposed residues have been randomised to produce a repertoire of binders [109,110]. Scaffold pro-teins have several advantages over antibodies and their fragments. Typically, scaffold proteins are small, highly soluble, rapidly folding and display high proteolytic, thermal and chemical stability. The rigid structure of scaffold proteins allows them to avoid entropic penalty and to preserve high affinity. Affibody molecules are the first and the most studied class of scaffold pro-teins in molecular imaging. These high affinity ligands are based on a 58-amino acid (6.5 kDa) trihelical scaffold derived from domain-B of the staph-ylococcal protein A [111,112]. Thirteen amino acids on helices 1 and 2 have been randomised, generating large, combinatorial libraries from which af-fibody variants binding the desired molecular targets could be selected (Fig. 2).

Randomisation of 13 selected positions on

helices 1 and 2

1x1013 Affibodylibrary members

Ig-binding domains of Protein A

E AD CB

Selection against specific target(HER2, EGFR…)

Z-domain

Figure 2. Selection of affibody molecules.

28

Affibody molecules can be produced recombinantly in E. coli or by solid-phase peptide synthesis. Affibody molecules contain no cysteines and there-fore introduction of a single cysteine during recombinant production makes it easy to site-specifically modify the scaffold (chelator, prosthetic group) using thiol-directed chemistry. The synthetic production permits the site-specific introduction of non-natural amino acids and chelators directly dur-ing production. Robustness, high affinity towards selected antigens and small size indicated that these molecules can be attractive candidates for tumour targeting applications [113]. Affibody molecules with subnanomolar affinity have been developed against several RTKs: HER2 [114], EGFR [100], IGF-1R [101], PDGFRβ [102], and HER3 [103].

The human epidermal growth factor receptor 2 (HER2) is amplified in approximately 18 % to 20 % of breast cancers and its status is also predictive for either resistance or sensitivity to several systemic therapies [115]. For this reason, the American Society of Clinical Oncology and College of American Pathologists have recommended that HER2 overexpression status should be determined in invasive breast cancer [115]. The affibody molecule targeting HER2 was the first and the most studied variant of this group of high affinity targeting scaffold proteins. The affinity of 22 pM has been achieved for the ZHER2:342 affibody molecule by affinity maturation [114]. Because of their small size, affibody molecules showed rapid clearance of unbound tracer, rapid extravasation and deep diffusion in tumours. The high affinity towards HER2 ensured high retention of the radiolabelled affibody molecule in the tumour. A tumour-to-blood ratio of approximately 200 has been determined in murine models bearing HER2-expressing xenografts a few hours after injection [116-118]. Importantly, the unspecific accumula-tion of radiolabelled affibody molecules in murine xenografts was very low, approximately 1-2 % of the specific accumulation [119]. Direct comparison of anti-HER2 affibody molecules and radiolabelled trastuzumab has been performed using different types of labels in xenograft models with different HER2 expression levels [120,121]. In both models, affibody molecules pro-vided an order of magnitude higher tumour-to-blood ratio than the antibody. Preclinical studies have demonstrated that affibody molecules can be used for the imaging of HER2 degradation in response to treatment with different HSP90 inhibitors [119,122]. Moreover, the feasibility of using affibody mol-ecules to monitor HER2-downregulation in response to trastuzumab therapy has been tested [123]. The authors concluded that (18F-FBEM)-ZHER2:342 is a potential PET-tracer that can predict response to trastuzumab in a HER2-positive human xenograft model. However, this conclusion was debated and it was noted that further intensive investigation is required before affibody molecules can be used to monitor response to trastuzumab [124,125]. A pilot clinical study has demonstrated feasibility of imaging of HER2-expressing breast cancer metastases using 111In- and 68Ga-labelled synthetic DOTA-ZHER2:342 affibody molecule (ABY-002) [97]. Recently, the first in-human

29

molecular imaging of HER2 expression in patients with metastatic breast cancer using the 111In-labelled second-generation anti-HER2 affibody mole-cule ABY-025 has been reported [98]. ABY-025 has an improved hydro-philicity and thermal stability, obtained by protein engineering of amino acids outside the HER2-binding region. In that study, seven patients with metastatic breast cancer and HER2-positive (n = 5) or -negative (n = 2) pri-mary tumours received an intravenous injection of 111In-ABY-025. Uptake of 111In-ABY-025 provided excellent visualisation of HER2-positive metas-tases. Immunohistochemical analysis of biopsies confirmed the HER2 ex-pression. Even in HER2-negative patients (n=2), visualisation of large le-sions with HercepTest scores of 0 (15,000–25,000 receptors) and +1 (80,000–110,000) was possible with weak signals. However, discrimination between tumours with high and low expression was possible. Collectively, these results indicate that 111In-ABY-025 can be used as a whole-body-oriented, noninvasive agent to discriminate between HER2-positive and HER2-negative metastases.

Labelling chemistry In addition to selection of optimal targeting proteins, optimisation of label-ling chemistry might improve the sensitivity of radionuclide-based molecu-lar targeting. Previous data suggest that the labelling chemistry has a pro-found influence on the tumour targeting properties of affibody molecules. Small changes in the physico-chemical properties of affibody molecules resulted in variation of the in vivo residualising vs. non residualising proper-ties of the tracer and its off-target interactions [126,127]. Difference in blood clearance, liver uptake, renal retention and route of excretion are some ex-amples of such effects. These effects were observed with different chelating moieties and even when the same chelator was used for labelling affibody molecules with different radionuclides [96]. It can be speculated that differ-ent radionuclide-chelator complexes can have different net charges, geome-try and lipophilicity. This can alter the overall interaction of affibody mole-cules with targeted receptors. Differences in charge may also influence bind-ing to plasma proteins and consequently the rate of excretion from the body. In addition, the type of radiocatabolites generated after intracellular degrada-tion may influence the retention properties in different organs.

As mentioned earlier, affibody molecules are designed not to include in-ternal cysteines. Introduction of a single cysteine into recombinantly pro-duced affibody molecules by genetic engineering enabled site-specific con-jugation of linkers and chelators using thiol-directed chemistry. Derivatives of affibody molecules containing a C-terminal cysteine have been developed [100,128]. These affibody molecules were site-specifically labelled using radiometals, such as 111In [100,128,130,133], 68Ga [117], 64Cu [129,131] and

30

57Co [132], and radiohalogens, such as 76Br, 131I and 18F [122,134-137]. Site specific-labelling provides a homogeneous product with high batch-to-batch reproducibility compared with random labelling where a heterogeneous mix-ture of conjugates containing a different number of chelators attached ran-domly at different positions is produced. Un-controlled labelling would gen-erate tracers with different biodistribution profiles and would render it diffi-cult to find the one with best targeting properties.

Macrocyclic chelators A chelator is needed to label proteins with radiometals. Many radionuclides have a metallic nature, e.g., 68Ga, 111In, 177Lu, and 64Cu. Chelators form com-plexes with metals through dative bonds. Complex formation between chela-tors and metals is a reversible process. Like any reversible reaction, the chelate stability is measured using dissociation constant KD. The dissociation constant can be expressed as concentration of reactants (free metal and free chelator) over products (metal-chelator complex) at equilibrium. In addition to thermodynamic stability, selection of an appropriate chelator for the label-ling of biomolecules with radiometals is also largely determined by its kinet-ic inertness. In blood, the concentration of naturally existing chelators (e.g., transferrin, ceuroplasmin and metal-binding enzymes) will far exceed the concentration of the radiolabelled protein. This will result in the dissociation of the radiometal from the metal-chelate complex, the binding of radiometal to natural chelators and consequently the possible accumulation of radioac-tivity in non-target tissues. Therefore, more inert chelates are required as they possess a slow dissociation rate i.e., they generally better retain the radiolabel in vivo. However, kinetically inert chelates also have a slow asso-ciation rate, and harsh conditions of labelling will be required, e.g., elevated temperature. The polyaminopolycarboxylate chelators are commonly used as bifunctional chelators in tracer development [138]. They can be divided into two classes: macrocyclic and acyclic chelators. Both classes form thermody-namically stable complexes with several radiometals. However, they differ in their kinetic inertness. Macrocyclic chelators (e.g., DOTA and NOTA) are more kinetically inert and have slow rates of dissociation, but elevated tem-peratures are required for complex formation with radiometals. For this rea-son, macrocyclic chelators are commonly used for labelling robust peptides that can tolerate high temperatures e.g., affibody molecules. Acyclic chela-tors, such as semi-rigid DTPA derivatives, on the other hand, are less inert and consequently more prone to dissociation in vivo. Their more rapid asso-ciation permits labelling under milder heating conditions, and they are there-fore preferable for heat-sensitive proteins e.g., mAbs.

31

Figure 3. Macrocyclic chelators.

Affibody molecules were labelled with several radiometals. For this, af-fibody molecules were coupled to different macrocyclic chelators with the aim of optimising targeting properties as a precondition for both efficient HER2 detection and peptide-based radionuclide therapy. It is important to mention that the formation of the most stable complex between different groups of metals and different chelators is determined by metal ionic radii, the size of the chelator and the coordination number of the metal vs. the che-lator denticity. The macrocyclic chelator DOTA was used to label affibody molecules with 111In, 68Ga, 64Cu and 57Co [100,117,129,135]. The use of DOTA provides stable complexing, and it is widely used in development of peptide-based radiopharmaceuticals [139]. However, DOTA is not always the chelator of choice. For example, DOTA is a suboptimal chelator for cop-per isotopes due to in vivo instability [140]. On the other hand, NOTA is a promising chelator for the labelling of targeting agents with a number of radiometals. NOTA is smaller than DOTA but forms more stable complexes with many three-valent radiometals. For example, the use of NOTA [141] or cross-bridged chelators [142] for labelling with radiocopper provided a more stable label than did DOTA. This translated into less radiocopper release in vivo and therefore lower liver associated radioactivity. Similarly, NOTA provided more stable complexes with gallium and indium than DOTA [143,144]. Several NOTA derivatives have been used for labelling of target-ing peptides with gallium isotopes [145-147] and 111In [145]. Biodistribution data indicated adequate in vivo stability of the conjugates. We evaluated the substitution of DOTA with a maleimido derivative of NOTA for site-specific labelling of affibody molecules [133]. A substantial reduction of accumulat-ed radioactivity was observed in the blood, bone and lungs. Moreover, the non-compromised tumour uptake and rapid blood clearance enabled the ac-quisition of higher contrast images a short time after injection. Later, the glutaric acid derivative of NOTA (NODAGA) was synthesised. NODAGA was evaluated for the labelling of several tumour-targeting peptides with

32

trivalent radiometals, such as 111In, 67Ga and 68Ga [145, 148-150], and the chelator provided high in vivo stability of the labels. Interestingly, substitu-tion of DOTA by NODAGA resulted in more rapid blood clearance of radi-oactivity for 68Ga-labelled RGD peptides, enabling higher tumour-to-organ ratios [150].

33

Optimisation of the targeting properties of affibody molecules for targeted radionuclide therapy

Targeted radionuclide therapy is a promising approach to overcoming the resistance experienced with the conventional targeted therapy. Full-length radiolabelled antibodies (150 kDa), which were the first vectors used, had limited success in the eradication of solid tumours due to the long blood circulation times, slow extravasation and low tumour penetration [151-153]. The long in vivo circulation time increases bone marrow radiation exposure, which may result in myelosuppression. The main goal of any therapeutic agent is to deliver the payload to the diseased tissue while sparing healthy tissues from damage (enhance the tumour absorbed dose and minimise tox-icity) [154]. Therefore, several alternative methods were developed to over-come the problems associated with conventional RIT. The main strategy is to reduce the size of the nuclide-bearing moiety. This might be achieved, for example, with the utilisation of smaller antibody fragments, scaffold proteins and short peptides [151,155]. Although a full-length antibody has the highest rate of tumour uptake, antibody fragments and smaller targeting agents achieve maximum uptake more rapidly. They also demonstrate more rapid elimination from blood and normal tissues. Collectively, these properties provide a high tumour-to-non tumour ratio [151]. Alternatively, pretargeting was developed. In this method, the targeting vector is injected first and then allowed to localise in the tumour and clear from blood and non-targeted tis-sues. Later the radionuclide-bearing moiety is injected. Because of its small size, the radionuclide-bearing moiety clears rapidly from the body but not the tumour where it reacts (with high affinity) with the targeting vector. This improves the tumour-to-non tumour ratio and consequently permits a high therapeutic margin [156,157].

Because of their rapid washout from non-target tissues, short residence time in the circulation and high affinity towards its target, affibody mole-cules are promising candidates for targeted radionuclide therapy applica-tions. For example, the anti-HER2 affibody molecule ZHER2:342 has demon-strated rapid clearance from the body but not from the tumour and has demonstrated high tumour retention [113]. In vitro cellular experiments showed that ZHER2:342 has a high affinity towards HER2 and slow cellular

34

internalisation. Only less than 30 % of the bound tracer was internalised by HER2-expressing cells after 24 h of incubation [158]. A slow internalisation rate will correspond to slower degradation and excretion of the radiolabelled affibody molecule from the tumour cells. These properties would ensure that an affibody molecule–based targeting agent will not be cleared from the body before delivering the payload to the target. Despite the improvement in in vivo properties of affibody molecules achieved through optimisation of the labelling chemistry, the high renal retention hampered their utilisation in targeted radionuclide therapy. The size of affibody molecules (7 kDa) is below the kidney cut-off (60 kDa), making them readily filtered through the glomerulus. Similar to many protein-based small-size drugs, filtered af-fibody molecules are prone to high reabsorption rates by the proximal con-voluted tubule cells. Affibody molecules labelled with radiometals such as 111In, 177Lu and 90Y, after internalisation and proteolytic degradation in the lysosomal compartment form hydrophilic, charged and bulky catabolites, which cannot diffuse through the lysosomal or cellular membrane. There-fore, radiometal-labelled affibody molecules have demonstrated high reten-tion of radioactivity in the kidneys i.e., residualising properties [159-161]. Alternatively, the use of radiohalogens such as 131I for labelling of affibody molecules provided a reasonably high tumour uptake but much lower renal retention of radioactivity [134,162]. Radiocatabolites of halogens are lipo-philic and can easily leak from the cells. Changing the label from a residual-ising to a non-residualising one, might overcome the high renal retention of radiolabelled-affibody molecules (Fig. 4). This might restrict the selection of therapeutic radionuclides to halogens. It is therefore more appropriate to reduce the high renal retention of radio-metal labelled affibody molecules, a strategy that will expand the selection panel.

Figure. 4. Comparative distribution of radioactivity after injection of affibody mole-cules labelled with residualising (111In) [161] and non-residualising (131I) [134] nu-clides in nude mice bearing HER2-expressing xenografts from two studies [163].

35

Optimisation of peptide-based chelators Technetium-99m (99mTc) (T½=6 h; Eγ=140.5 keV) is a generator-produced radionuclide. It is the most widely available and commonly used radionu-clide in nuclear medicine applications. As a metal, 99mTc requires chelator for attachment to a targeting protein. A variety of chelators has been pro-posed for 99mTc. It has been shown that a thiol-bearing moiety (mercaptoace-tyl or cysteine) together with adjacent amide nitrogens can form a stable chelator for 99mTc [164]. A series of experiments to label affibody molecules with 99mTc using peptide-based chelator chemistry were performed (Fig. 5).

Figure 5. Cysteine-containing peptide-based chelators on C-terminus (A). Mercap-toacetyl-containing peptide-based chelators on N-terminus (B). x1, x2 and x3 repre-sents different side groups of different amino acids. Red dots indicate the 13 resi-dues responsible for molecular recognition in affibody molecules.

Modifying the side-chains of the three adjacent amino acids comprising the SN3 chelator had a profound effect in the biodistribution profile of affibody molecules. This could be explained by alterations in charge and lipophilic properties of both the tracer and its degradation end-products. Initially, the 99mTc-labelling of affibody molecules was performed using the mercaptoace-tyl-glycyl-glycyl-glycyl (maGGG–) chelator coupled to the N-terminus of the anti-HER2 affibody molecule ZHER2:342 [165] (Fig. 5). Earlier, this chela-tor provided stable labelling of macromolecules with 99mTc and 188/186Re [166]. 99mTc-maGGG-ZHER2:342 was reasonably stable in vivo but demon-strated an elevated intestinal radioactivity due to hepatobiliary excretion. This was attributed to the high lipophilicity of the maGGG– chelator. Fur-ther experiments in mice demonstrated that reduction in hepatobilliary excre-tion correlated well with the introduction of more polar and hydrophilic ami-no acids. Substitution of all three glycines (maGGG–) in the chelator with polar serines (maSSS–) reduced the intestinal radioactivity three-fold (Fig. 6a) [167]. Substitution of glycines in maGGG– with glutamic-acid (charged and hydrophilic) residues in maEEE-ZHER:342 reduced the total gastrointesti-nal associated radioactivity by several fold (Fig. 6b) [168]. In contrast, renal

36

accumulated radioactivity of 99mTc-maEEE-ZHER:342 increased more than 10-fold. Further experiments (Fig. 6c) showed that a combination of serine and glutamic acids in mercaptoacetyl-containing chelators provided a radio-labelled conjugate, 99mTc-maESE-ZHER:342, with low hepatobiliary excretion and renal retention of radioactivity (Fig. 6c) [169].

Figure 6. Influence of composition of mercaptoacetyl-containing peptide-based chelators (N-terminus) on hepatic and renal uptake and the hepatobiliary excretion of 99mTc-labelled affibody molecules in mice. a. influence of serine; b. influence of glutamate; c. influence of order of serine and glutamate residues. [163]

Introduction of the naturally existing amino acid, cysteine, instead of mer-captoacetyl permitted recombinant production of the chelator containing affibody molecules. A peptide-based chelator with the common N3S format was obtained (Fig. 5). The tracer 99mTc-ZHER2:2395-Cys demonstrated excellent biodistribution except for an elevated kidney uptake [170]. To reduce the renal uptake, a –GSECG chelator (a “mirror” homologue of maESE–) was engineered on the C-terminus of anti-HER2 affibody molecule (99mTc-PEP05352) [171]. 99mTc-PEP05352 showed 3-fold lower renal retention of radioactivity in comparison to 99mTc-ZHER2:2395-Cys. For further optimisation of cysteine-containing peptide-based chelators, a series of recombinantly produced PEP05352 derivatives containing –GSEC, –GGGC, –GGSC, –GGEC, or –GGKC chelators at the C-terminus (designated as ZHER2:V1, ZHER2:V2, ZHER2:V3, ZHER2:V4 and ZHER2:V5, respectively) was produced [118]. These variants demonstrated low hepatobiliary excretion and rapid blood clearance. The tumour-to-blood ratio exceeded 180 as early as 4 h post-injection in mice bearing HER2-expressing xenografts. There was a consid-erable variation in the retained renal radioactivity. The variant with –GGGC

37

chelator 99mTc-ZHER2:V2 demonstrated the lowest kidney-associated radioac-tivity (6.4±0.6 %ID/g at 4 h p.i.) (Fig. 7). It was found that not only the composition of the peptide-based chelators can influence the uptake by exe-cratory organs but that the order of the amino acids in the chelator can also have an impact [172]. A series of anti-HER2 affibody molecules, containing –GSGC, –GEGC and –GKGC chelators have been prepared, characterised and compared with the previously studied 99mTc-labelled affibody molecules containing –GGSC, –GGEC and –GGKC chelators. In vivo data showed that uptake was very similar between the counterparts in the majority of organs, except for the kidneys where the major influence was obvious at early time points, i.e., 1 h p.i., but appreciably decreased by 4 h p.i.

liver kidney intestines*05

101520253035

6090

120-GGGC-GGSC-GGEC-GGKC-GSEC

Upt

ake,

% ID

/g

Figure 7. Influence of amino acid composition of cysteine-containing peptide-based chelators at the C-terminus on uptake of 99mTc-labelled affibody molecules in excre-tory organs of NMRI mice at 4 h p.i. [118]

Kidney reabsorption of radiolabelled affibody molecules: Understanding the mechanism As discussed in the previous section, altering the biodistribution of 99mTc-labelled affibody molecules was possible by engineering the amino acid composition of cysteine-containing peptide-based N3S chelators. This might permit labelling with the therapeutic radionuclides 186Re/188Re due to chemi-cal similarity of rhenium and technetium [173,174]. High-energy 188Re (T½=17.0 h; Eβ-max=2.1 MeV) or medium energy 186Re (T½=3.72 days; Eβ-max=1.08 MeV) are more suited for the eradication of bulky non-operable tumours and metastases. Eradication of residual or small lesions would re-quire short range β- or α-emitters such as 177Lu and 227Th. The difference in

38

the chemical nature of these radionuclides with 99mTc renders them inappro-priate to label affibody molecules through a peptide-based N3S chelator chemistry. Therefore, understanding the underlying mechanisms of kidney reabsorption and retention of radiolabelled affibody molecules would permit guided re-engineering of the scaffold to optimise the in vivo properties. This might also facilitate development of effective reabsorption blocking agents. Re-engineering of the affibody scaffold has been conducted on several occa-sions to optimise the targeting properties [163,175]. For example, the visual-isation of HER2-positive liver metastasis was not possible with the first-generation anti-HER2 affibody molecule, ABY-002 [97]. The protein engi-neering of this affibody molecule generated a more hydrophilic variant, ABY-025 [175]. This reduced the liver uptake, and the visualisation of liver metastasis in breast cancer patients was feasible [98].

The renal reabsorption of protein-based drugs was thoroughly investigat-ed. Megalin and cubilin, two scavenger receptors, co-localised in the proxi-mal tubules on the luminal side, were found to be responsible for the active reabsorption of many protein-based drugs [176,177]. After reabsorption, the radiolabelled targeting agent is degraded by means of proteolytic lysosomal enzymes. As discussed earlier, depending on their nature (lipophilic or hy-drophilic and charged or uncharged), radiocatabolites are either retained in the proximal tubule cells or diffuse to either blood or urine. It was demon-strated that megalin is the main mediator of renal accumulation of somato-statin analogues and a number of other small peptides [178,179]. Profound decrease of radioactivity accumulation in the kidneys of megalin-deficient mice was observed for several radiolabelled small peptides. This clearly indicates a direct involvement of the megalin\cubilin system in the reabsorp-tion process of these agents [177]. Recently, the involvement of megalin in renal reabsorption of radiolabelled anti-EGFR nanobodies (15 kDa) has been demonstrated [180]. Whether megalin/cubilin plays any role in the renal reabsorption of affibody molecules after glomerular filtration was not yet known, and further investigation was required. In this thesis, the mechanism of renal reabsorption of anti-HER2 affibody molecules was investigated.

filterated radiolabeled agent

endocytic PT receptors

Lysosomal enzymes

radiocatabolites

39

Figure 8. The mechanism of renal reabsorption of radiolabelled targeting agents.

40

The present investigation

Evaluation of anti-HER2 affibody molecules site-specifically labelled with 111In using a maleimido derivative of NODAGA (Paper I) Aim Earlier, affibody molecules were conjugated with different chelators such as, DTPA, DOTA and NOTA. These studies have shown that the type of chela-tor can have a profound effect on the off-target properties of radiometal la-belled affibody-based tracers. Even when the same conjugate is labelled with different radionuclides, it can have different properties. In paper I, we con-tinue to study the structure-properties relationship of the anti-HER2 affibody molecule ZHER2:2395 for the optimisation of imaging properties. For this pur-pose, ZHER2:2395 is site specifically coupled to the novel bifunctional malei-mido-derivatised macrocyclic chelator MMA-NODAGA at the C-terminus and labelled with the SPECT radionuclide 111In.

Method The maleimidoethylmonoamide NODAGA (MMA-NODAGA) was synthe-sised and conjugated to ZHER2:2395 affibody molecule having a C-terminal cysteine. The labelling efficiency and binding specificity to as well as inter-nalisation by HER2-expressing cells (SKOV-3 with high HER2 expression and DU-145 with low HER2 expression) of 111In-MMA-NODAGA-ZHER2:2395 was evaluated. Biodistribution of 111In-MMA-NODAGA-ZHER2:2395

and the previously studied variant 111In-MMA-DOTA-ZHER2:2395 was com-pared in nude male NMRI nu/nu mice bearing DU-145 prostate cancer xen-ografts.

Results and discussion The feasibility of conjugating NODAGA to an anti-HER2 affibody molecule (ZHER2:2395) using a thiol-directed chemistry has been validated. 111In-MMA-NODAGA-ZHER2:2395 showed a rapid internalisation rate in HER2-expressing cells compared with 111In-MMA-DOTA-ZHER2:2395 (Fig. 1).

41

Figure 1. Internalisation of 111In-MMA-NODAGA-ZHER2:2395 and 111In-MMA-DOTA-ZHER2:2395 by HER2-expressing DU-145 cell line.

Interestingly, 111In-NODAGA-ZHER2:2395 demonstrated more rapid clearance from the blood and lower uptake in normal organs compared with both DO-TA- and NOTA-conjugated variants (Fig. 2).

Figure 2. Targeting DU-145 xenografts in mice using 111In-MMA-NODAGA-ZHER2:2395 and 111In-DOTA- ZHER2:2395 4 h p.i. * data for intestines with content and carcass are presented as % of injected dose per whole sample

A similar phenomenon was observed earlier with the short peptide-based targeting agent RGD [150]. Studies of 68Ga-labelled RGD peptide have also shown that the use of NODAGA instead of DOTA increased clearance from blood and other non-specific compartments. The authors have speculated

42

that the increased clearance rate is most likely due to the reduced binding of NODAGA-conjugated peptide to blood plasma proteins. We can suggest a similar hypothesis for 111In-labelled affibody molecules. Although the bind-ing of 111In-MMA-NODAGA-ZHER2:2395 to blood proteins is so weak that it could not be detected in vitro, this might still slow down blood clearance.

The tumour uptake of 111In-MMA-NODAGA-ZHER2:2395 was lower in comparison with 111In-DOTA-ZHER2:2395 (4.73±0.79 %ID/g vs. 7.5±1.6 %ID/g) at 4 h p.i. (Fig. 2). Most likely, the main reason for the lower uptake of 111In-MMA-NODAGA-ZHER2:2395 in xenografts was a lower bioavailability of the conjugate due to rapid systemic clearance. Due to its rapid clearance and the lower uptake in unspecific tissues, 111In-MMA-NODAGA-ZHER2:2395

demonstrated higher tumour-to-organ ratios (Fig. 3). A high tumour-to-normal organ ratio is a precondition for high contrast imaging, especially in metastatic disease. Imaging at 4 h p.i. confirmed our biodistribution results.

Figure 3. Tumour-to-organ ratio of 111In-MMA-NODAGA-ZHER2:2395 and 111In-MMA-DOTA-ZHER2:2395 in mice bearing DU-145 xenografts 4 h .p.i.

Brief conclusion This study demonstrated that the incorporation of the novel bifunctional chelator maleimidoethylmonoamide-NODAGA enables the labelling of anti-HER2 affibody molecule with 111In. The incorporation of NODAGA as a macrocyclic chelator modified the biodistribution properties of affibody molecules and improved the tumour-to-blood ratio 5 times compared with the previously studied variants and enabled acquisition of high contrast im-ages.

43

Influence of 68Ga and 111In in combination with DOTA and NODAGA chelators on imaging properties of affibody molecules: a comparative evaluation (Paper II) Aim The use of positron emission tomography (PET) should increase sensitivity of HER2 imaging. The aim of this study was to compare maleimido deriva-tives of DOTA and NODAGA for site-specific labelling of a recombinant HER2-binding ZHER2:2395 affibody molecule with 68Ga. 111In-labelled variants are used as comparators.

Methods The macrocyclic chelators DOTA and NODAGA were site-specifically con-jugated to the ZHER2:2395 affibody molecule having a C-terminal cysteine and labelled with 68Ga and 111In. The in vivo and in vitro properties of 68Ga-NODAGA-ZHER2:2395,

68Ga-DOTA-ZHER2:2395, 111In-NODAGA-ZHER2:2395 and

111In-DOTA-ZHER2:2395 were studied in a comparative study.

Results and discussion This study demonstrates that it is possible to stably label NODAGA- con-taining anti-HER2 recombinant affibody molecule (ZHER2:2395) with 68Ga in high yield. Biodistribution studies in tumour-bearing mice demonstrated high renal retention of all conjugates following reabsorption. This is com-mon for radiometal labelled affibody molecules. Moreover, uptake of both 68Ga-labelled variants in the liver and spleen was significantly higher than 111In-labelled variants (Fig. 1). 68Ga-DOTA-ZHER2:2395 had the highest liver uptake compared with all other conjugates (3.2±0.3 and 3.06±0.12 at 1 and 2 h p.i.). Dicrostoforo and colleagues observed similar elevated hepatic uptake with 68Ga-DOTA-RGD and attributed this to the reduced stability of 68Ga-DOTA complexes in vivo [181]. This is unlikely to be the case in this work as the in vitro stability studies of all four tracers was comparable. In addi-tion, no similar effect was observed for the synthetic anti-HER2 affibody molecule (68Ga-ABY-002), where 68Ga-DOTA complex was located on the N-terminus [96]. This suggests that the allocation of the chelator-nuclide complex at different positions might influence the biodistribution pattern and the hepatic uptake of affibody molecules in tumour-bearing mice in particu-lar.

44

%IA

/g

liver

splee

n

tumor

kidney

0

5

10

15

20

100200300400

68Ga-NODAGA-Z239568Ga-DOTA-Z2395111In-NODAGA-Z2395111In-DOTA-Z2395

112

2

2

Figure 1 .Liver, spleen, tumour and kidney uptake of all four tracers in BALB/C nude mice bearing SKOV-3 xenografts 2 h p.i. (1) indicates significant difference between gallium-68 and indium-111 labelled conjugates, (2) indicates significant difference between 111In-NODAGA-ZHER2:2395 and

111In-DOTA-ZHER2:2395.

For 111In-labelled affibody molecules, 111In-NODAGA-ZHER2:2395 had lower uptake in liver, tumour and kidneys than did 111In-DOTA-ZHER2:2395 2 h p.i. (Fig. 1). This is in agreement with the results obtained in paper I. On the other hand, the overall tumour-to-organ ratio of 111In-DOTA-ZHER2:2395 was superior to 111In-NODAGA-ZHER2:2395. This is not in agreement with the re-sults of paper I. It is important to mention here that the short half-life of 68Ga (68 mins.) made it necessary to set the latest time point of the biodistribution study earlier than in paper I (2 h p.i. in paper II vs. 4 p.i. in paper I). Hence, the results of this study do not contradict the results of paper I as the time points and HER-2 expressing cell lines were different (DU-145 in paper I vs. SKOV-3 in paper II). In general, the overall tumour-to-normal-tissue ratios for all four conjugates are high enough to obtain a good quality image at a shorter time point (Fig. 2). However, 68Ga-NODAGA-ZHER2:2395 and 111In-DOTA-ZHER2:2395 demonstrated excellent targeting properties as illustrated in (Fig. 3). They also showed overall high tumour-to-normal-tissue ratios. The high tumour-to-liver (8±2 and 10.4±1), tumour-to-bone (36±15 and 45±14) and tumour-to-muscle (88±55 and 102±25) ratios obtained with 68Ga-NODAGA-ZHER2:2395 and 111In-DOTA-ZHER2:2395 would render these two tracers efficient in visualising HER2-expressing-metastases in these relevant organs.

45

Figure 2. Overall tumour-to-organ ratios of all four conjugates 1 and 2 h p.i.

Figure 3. Tumour-to-organ ratios of 68Ga-NODAGA-ZHER2:2395 and

111In-DOTA-ZHER2:2395 2 h p.i.

Brief conclusion This study demonstrated that both the type of nuclides and the type of chela-tors can alter the targeting properties of affibody molecules. Such effects were observed earlier for small size targeting agents but were unexpected for larger agents such as affibody molecules. The combination of 68Ga with NODAGA possesses favourable in vivo properties for affibody molecules. 68Ga-NODAGA-ZHER2:2395 is a potential candidate to be developed as a PET tracer for sensitive detection of HER2 expression.

46

In vivo and in vitro studies on renal uptake of radiolabelled affibody molecules: understanding the mechanism (Paper III) Aim Radiometal-labelled affibody molecules have a high rate of reabsorption and high retention by the kidneys after their excretion from circulation. This complicates the use of monomeric radiometal-labelled affibody molecules for radionuclide therapy. It is therefore important to understand the mecha-nism of renal reabsorption of anti-HER2 affibody molecules. Understanding this mechanism would facilitate the development of substances that can ef-fectively block this process and reduce the high kidney retention of radioac-tivity. This might permit the use of radio-metal labelled anti-HER2 affibody molecules in targeted radionuclide therapy.

Methods Renal accumulation of radiolabelled anti-HER2 affibody molecules was studied in a murine model using megalin knockout mice. The in vitro studies were conducted using opossum-derived proximal tubule (OK) cell lines.

Results and discussion It was shown previously that the kidney uptake of affibody molecules was not reduced with gelofusine, a well-known blocker of megalin (unpublished results). This indicated that megalin is not involved in the tubular reabsorp-tion of affibody molecules. Comparative micro-SPECT imaging between megalin-deficient and wild-type mice injected with111In-DOTA-ZHER2:2395 confirmed this finding (Fig. 1).

B.A.

Figure 1. Micro-SPECT images of megalin-deficient mice (A) versus wild-type mice (B) 4 h p.i. of 111In-DOTA-ZHER2:2395.

47

As shown in (Fig. 1), there was no difference in kidney-associated radioac-tivity between the wild-type and the megalin-knockout mice. After imaging, two groups of five mice were euthanised and the measured concentration of radioactivity in dissected kidneys demonstrated no significant difference between the two groups (214±26 %ID/g for wild-type mice vs. 202±16 %ID/g for megalin-knockout mice)

The ability of different affibody molecules (ZHER2:342, Ztaq:1154, Zinsulin:810 and ZEGFR:2377) and human albumin (megalin blocker) to inhibit the binding of 99mTc–ZHER2:2395-Cys to OK cells demonstrated that only ZHER2:342 had five orders of magnitude higher affinity (IC50= 1.46x10-9) to proximal tubule scavenger receptors in comparison to other affibody molecules and albumin (Fig. 2). This indicates that the interaction of anti-HER2 affibody molecules with proximal tubule receptors is binding site mediated.

99mTc-ZHER2:2395-Cys

ZHER2:342, ZEGFR:2377, Zinsulin:810, Ztaq:1154 and Albumin

Inhibitor IC50 concentration, MZHER:342 1.46 x 10-9

ZEGFR:2377 1.5-2 x 10-4

Zinsuline:810 2.5-3 x 10-4

Ztaq:1154 3.5-4 x 10-4

Albumine 5 x 10-4

0 M 9.7x10-4 M

Figure 2. Inhibition Concentrations (IC50) for different affibody molecules and al-bumin for binding of 99mTc–ZHER2:2395-Cys to Opossum-Derived Proximal Tubule Cells.

In agreement with in vivo studies, the results of this experiment (high albu-min IC50) suggested that the involvement of a megalin-mediated mechanism in the kidney reabsorption of anti-HER2 affibody molecules could be ex-cluded.

To determine whether proximal erbB-2 receptors (animal analogue of HER2) could be responsible for renal reabsorption of anti-HER2 affibody molecules, a comparison of the inhibition (IC50) of 99mTc–ZHER2:2395-Cys binding to OK and SKOV-3 (ovarian cancer with high HER2-expression)

48

was studied. The results show that the half maximal inhibitory concentration (IC50) on OK cells was 30-fold lower than that on SKOV-3 cells, where the uptake is HER-2-mediated (49.3 nM in SKOV-3 vs. 1.4 nM in OK cells). This difference suggests that another receptor, different from the targeted antigen HER2, is responsible for this extensive uptake of 99mTc–ZHER2:2395-Cys by tubular OK cells. Furthermore, the determination of the equilibrium dissociation constant, KD, indicated the presence of two types of receptors on the OK cells that bind the anti-HER2 affibody molecule 99mTc–ZHER2:2395-Cys, one with a high affinity of 0.8 nM (71500 receptors/cell) and the other with a lower affinity of approximately 9.2 nM (367000 receptor/cell) (Fig. 3).

050

0010

000

0.00

0.02

0.04

0.06

0.08

0.10

1000

00

2000

00

3000

00

Conc (pM)

pMol

/105 c

ells

KD1 = 0.8 nMBmax1 = 71 500 rec/cell

KD2 = 9.2 nMBmax2 = 367 000 rec/cell

Figure 3. Non-linear regression analysis for two-sites bind data from binding satura-tion experiment. Increasing concentrations of 99mTc-Z2395-C (x-axis) were added to dishes with OK cells.

Brief conclusion This study demonstrated that the renal reabsorption of anti-HER2 affibody molecules is mediated by an unknown receptor. The binding site of the af-fibody molecule is involved. Thus, efforts to overcome this phenomenon by reengineering the scaffold of affibody molecules cannot be successful. It would be more appropriate to optimise the labelling chemistry.

49

Selection of an optimal cysteine-containing peptide-based chelator for labelling of affibody molecules with 188Re (Paper IV) Aim Earlier, a series of studies involving labelling of affibody molecules with technetium-99m demonstrated that the targeting properties of labelled af-fibody molecules can be improved by selecting an optimal chelator. Techne-tium and rhenium are chemically similar elements. In this study, we evaluat-ed the influence of the composition of cysteine-containing C-terminal pep-tide-based chelators on the biodistribution and renal retention of 188Re-labelled anti-HER2 affibody molecules.

Methods The Affinity, specificity, retention by HER2 expressing cells and the biodis-tribution of eight rhenium-188 labelled affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) were compared with the parental affibody molecule ZHER2:2395 having a KVDC peptide chela-tor.

PolarNegatively

chargedPositively charged

Z2395 AENKFNKEMRNAYWEIALLPNLTNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQKVDC

ZV2 AENKFNKEMRNAYWEIALLPNLTNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQGGGC

ZV3 AENKFNKEMRNAYWEIALLPNLTNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQGGSC

ZV6 AENKFNKEMRNAYWEIALLPNLTNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQGSGC

ZV4 AENKFNKEMRNAYWEIALLPNLTNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQGGEC

ZV7 AENKFNKEMRNAYWEIALLPNLTNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQGEGC

ZV5 AENKFNKEMRNAYWEIALLPNLTNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQGGKC

ZV8 AENKFNKEMRNAYWEIALLPNLTNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQGKGC

non-polar non-charged

Figure 1. Alignment of the eight studied 188Re-labelled variants. (K) lysine, (V) valine, (G) glycine, (S) serine, (E) Glutamate and (C) cysteine.

50

Results and discussion The affibody molecules having a cysteine-containing peptide-based chelator at their C-terminus can be labelled with 188Re in acidic conditions with high yield (>95 % for all variants except ZHER2:2395 for which labelling yield was 89.3±1.2 %). All constructs retained low picomolar affinity to HER2-expressing cells after labelling. The composition of the peptide-based chela-tors had an apparent influence on the cellular retention of the 188Re-labelled conjugates after binding to HER2-expressing SKOV-3 cells (Fig. 2.).

Figure 2. Influence of the amino acid composition and their position in the cysteine-containing chelators on the cellular retention of 188Re-labelled affibody molecules by SKOV-3 cells.

Cellular retention was highest for the variants with lysine-containing chela-tors [188Re-ZHER2:2395 (–KVDC), 188Re-ZHER2:V5 (–GGKC) and 188Re-ZHER2:V5

(–GKGC)]. The –GGXC variants 188Re-ZHER2:V3 (–GGSC), 188Re-ZHER2:V4 (–GGEC) and 188Re-ZHER2:V5 (–GGKC) provided better retention of radioactivi-ty than their –GXGC counterparts, 188Re-ZHER2:V6 (–GSGC), 188Re-ZHER2:V7 (–GEGC) and 188Re-ZHER2:V8 (–GKGC). This pattern was in excellent agree-ment with our earlier data concerning cellular processing of 99mTc-labelled variants [172]. As the affinities of binding to HER2-expressing cells were very similar for all the variants (KD values ranging between 4 and 51 pM), the difference in cellular retention was most likely due to differences in the cellular processing of radiocatabolites after internalisation and lysosomal degradation. Retention of 188Re-ZHER2:V2 (–GGGC) was low, but the biologi-cal half-life was T1/2= 19 h, which would permit efficient tumour targeting.

The biodistribution of all 188Re-labelled affibody molecules was, in gen-eral, comparable with the main observed difference found in the uptake and retention of radioactivity in excretory organs (liver, kidneys) and in the lungs.

51

Figure 3. Influence of the –GXYC peptide-based chelator on the liver uptake of 188Re-labelled affibody molecules 1 and 4 h p.i.

The liver uptake of the eight 188Re-labelled variants is shown in (Fig. 3). The lysine-containing variants (ZHER2:2395, ZHER2:V5 and ZHER2:V8) showed the high-est uptake in the liver 1 h p.i. and retention 4 h p.i. compared with the re-maining variants. However, the hepatobiliary excretion of these variants remained low. Earlier, we observed a similar relationship between positive charge and radioactivity concentration in the liver. For example, elevated hepatic uptake was also observed with a histidine-containing tag substituted with lysine residues for labelling with [99mTc-(CO)3]

+ irrespective of the tag position (C- or N- terminus) [182]. To explain the retention of 188Re labelled constructs containing lysine (positively charged), we can speculate that these variants act as good substrates for the uptake but not the excretion transport molecules in hepatocytes. The –GXGC variants (ZHER2:V6 and ZHER2:V7) demonstrated rapid clearance between 1 and 4 h p.i., but their –GGXC coun-terparts (ZHER2:V3 and ZHER2:V4) did not have a similar effect. ZHER2:V2 (–GGGC) showed the lowest initial uptake and the lowest hepatic retention of radioactivity.

Figure 4. Influence of the –GXYC peptide-based chelator on the kidney uptake of 188Re-labelled affibody molecules 1 and 4 h p.i.

52

The renal uptake of 188Re-ZHER2:V2 was 2.5-6-fold lower than the uptake of other conjugates at 1 h p.i (Fig. 4). At 4 h p.i., the renal uptake of 188Re-ZHER2:V2 was 40-50-fold lower than the uptake of constructs with lysine-containing chelators (188Re-ZHER2:2395,

188Re-ZHER2:V5 and 188Re-ZHER2:V8), indi-cating less retention of the 188Re-ZHER2:V2 catabolites by the proximal tubule cells. The glutamate-containing variants (ZHER2:V4 and ZHER2:V7) showed a 6-fold decrease between 1 and 4 h p.i but were still higher than 188Re-ZHER2:V2. This was confirmed with imaging in normal mice injected with either 188Re- ZHER2:2395, ZHER2:V4 or ZHER2:V2. The visualisation of the kidneys was possible only with ZHER2:2395 and ZHER2:V4 but not ZHER2:V2 at 4 h p.i.(Fig. 5).

Figure 5. Imaging of mice injected with 188Re–labelled, ZHER2:2395 (–KVDC), ZHER2:V2 (–GGGC) and ZHER2:V4 (–GGEC) at 4 h p.i.

Brief conclusion The –GGGC chelator located on the C-terminus of the HER2 binding af-fibody molecule provides a low retention of radioactivity in the kidneys. 188Re-ZHER2:V2 (–GGGC) is a promising candidate for further development as a radiolabelled therapeutic agent against HER2-expressing tumours.

188Re-‐ZHER2:2395 188Re-‐ZHER2:V4 188Re-‐ZHER2:V2

K

B

(-‐KVDC) (-‐GGEC) (-‐GGGC)

53

188Re-ZHER2:V2, a promising affibody-based targeting agent against HER2-expressing tumors: preclinical assessment (Paper V). Aim The aim of this study was to evaluate 188Re-ZHER2:V2 as a potential candidate for radionuclide therapy of HER2-expressing tumours.

Methods The anti-HER2 ZHER2:V2 affibody molecule was labelled with 188Re using a gluconate-containing kit. Targeting properties and biodistribution were stud-ied in mice bearing ovarian carcinoma SKOV-3 xenografts with a high ex-pression of HER2. Absorbed doses in tumours and normal tissues in mice were calculated using the anatomically realistic murine Moby phantom. Ab-sorbed doses in humans were estimated using OLINDA/EXM 1.0.

Results and discussion 188Re-ZHER2:V2 provided efficient specific targeting of HER2-expressing xen-ografts. The results of the biokinetics study (20 min-48 h p.i.) revealed a rapid clearance of radioactivity from blood. Twenty minutes after injection, the kidney-accumulated radioactivity was at its maximum followed by rapid decrease. The tumour-associated radioactivity at 4 h p.i. was several fold higher than the activity in other organs and showed higher retention with time (T1/2= 15 h). This was in agreement with the results obtained in paper IV (in vitro). The area under curve (AUC) for the tumour exceeded the AUC for blood by 47 fold, for bone by 70 fold, and for the kidneys by 2.8 fold (Fig. 1).

Figure 1. Uptake of 188Re-ZHER2:V2 in blood, kidney, tumour and bone over time in NMRI nu/nu mice. The areas under the curve (AUC) in these organs are presented in the attached table.

54

In addition to emitting β-particles, 188Re also emits a 155 keV (15 %) gamma photon, which can be used for imaging the biodistribution of 188Re-labelled targeting agents using SPECT. The imaging of tumour bearing mice using 188Re-ZHER2:V2, 1 and 4 h p.i., confirmed the results of the biodistribution study and showed that tumour xenografts were the only sites with prominent accumulation of radioactivity 4 h p.i (Fig. 2).

Figure 2. Imaging of HER2 expression in SKOV-3 xenografts in NMRI nu/nu mice using 188Re-ZHER2-GGGC (188Re-ZHER2:V2) 1 and 4 h p.i.

In the future, imaging might permit specific dosimetry calculations for each patient individually, i.e., patient-specific dosimetry prior to and during tar-geted radionuclide therapy.

The results of absorbed dose evaluation in mice using the Moby phantom are presented in Fig. 3.

Abso

rbed

dos

e G

y/M

Bq

Liver

Large

intesti

ne

Small i

ntestine

Red marro

wLu

ngs

KidneyTumor

0.0

0.1

0.2

0.3

0.4

0.5Self-doseTotal-dose

Figure 3. Calculated absorbed dose in (Gy/MBq) for 188Re-ZHER2:V2 in mice using murine Moby phantom.

55

Self-doses were in agreement with the AUC data. However, the total ab-sorbed dose (self-dose + cross-dose) in several organs was much higher. The most important observation is the difference between the self- and total-dose absorbed in the bone marrow. The maximum range of 188Re beta-particles in tissue is approximately 10.4 mm. This is comparable to the mouse dimen-sions and explains the elevated cross-dose to red-marrow and other mouse organs. According to the model, the tumour is the main source of cross-irradiation to different organs because of the high uptake and retention of 188Re-ZHER2:V2. Therefore, delivery of therapeutically meaningful absorbed doses to tumours in mice would be associated with the delivery of lethal absorbed doses to the radiosensitive bone marrow.

Due to the larger size of the human body, the influence of the cross-dose to bone marrow is expected to be much smaller than in mice. The results of the estimated absorbed doses (mGy/MBq) of 188Re-ZHER2:V2 in humans using Olinda/EXM 1.0 are presented in Table 1.

Table 1. Calculated absorbed dose (mGy/MBq) of 188Re-ZHER2:V2 in humans using Olinda/EXM 1.0.

Organ Absorbed dose 188Re-ZHER2:V2

Liver 0.029 LLI Wall 0.022

Small intestine 0.016Red marrow 0.010

Lungs 0.011Kidney 0.189Tumour 0.65

Total body 0.009

In clinical practice, a maximum bone marrow absorbed dose of 2 Gy and kidney absorbed dose of 23 Gy is generally accepted in planning radionu-clide therapy. Dosimetry calculations in humans suggest that 188Re-ZHER2:V2

may provide an absorbed dose to tumours of 79 Gy while keeping the ab-sorbed dose to kidneys below 23 Gy and the absorbed dose to bone marrow below 1.2 Gy. The short half-life of 188Re would permit the irradiation of tumours with a high-dose rate. Moreover, fast kinetics of 188Re-ZHER2:V2 along with the in-site availability of a 188W/188Re generator would together facilitate the fractionation of therapy. Both high-dose rate and fractionation may be critical for the success of targeted radionuclide therapy by facilitat-ing the reoxygenation of radioresistant quiescent hypoxic cells. Fractionation can also provide a more uniform radiation dose distribution for bulky, poorly vascularised tumours; this cannot be achieved with single administration.

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Brief conclusion 188Re-ZHER2:V2, a promising targeteding agent for radionuclide therapy against HER2-expressing tumours, has been developed. The study demonstrated that an optimal labelling chemistry permits the efficient modification of biodis-tribution of affibody molecules. This study also revealed that future human radiotherapy studies may be feasible.

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Concluding remarks

This thesis was dedicated to studying the structure-properties relationship of anti-HER2 affibody molecules. The coupling of different chelators and la-belling with different radionuclides may have a profound effect on the in vitro and in vivo properties of these targeting agents. This is important in-formation for the development of affibody molecules for radionuclide mo-lecular imaging. It would be reasonable to speculate that the results of this work can be extrapolated to other affibody molecules targeting different molecular targets (e.g., IGF-1R, PDGFR-β, CAIX and HER3) and to other anti-HER2-targeting-scaffold-protein-based probes e.g., ADAPTs or DARPins.

The chances of radiolabelled anti-HER2 affibody molecules being used in targeted radionuclide therapy largely depends on understanding the mecha-nism of the renal reabsorption of these targeting agents. The current thesis studied this phenomenon in murine and real kidney cell models. Efforts to optimise the cysteine containing peptide based chelator for labelling of af-fibody molecule with 188Re generated the promising therapeutic targeting agent 188Re-ZHER2-V2.

The major conclusions from this research are as follows: • The coupling of the novel macrocyclic chelator MMA-NODAGA to the

anti-HER2 affibody molecule ZHER2:2395 was possible and this provided a conjugate with an affinity of 66 pM. NODAGA-ZHER2:2395 was success-fully and stably labelled with indium-111 with high yield (>95 %).

• The clearance of 111In-NODAGA-ZHER2:2395 from normal tissues was more rapid than the clearance of 111In-DOTA-ZHER2:2395, providing higher tumour-to-organ ratios. High-contrast images of HER2-expressing xen-ografts were acquired shortly (4 h) after injection.

• The comparative evaluation of anti-HER2 affibody molecules labelled with 68Ga and 111In using maleimido derivatives of DOTA and NODAGA suggested that 111In-labelled affibody molecules cannot be used to exactly predict in vivo properties of their 68Ga-labelled ana-logues. Similarly, the substitution of therapeutic radiometals (e.g., 90Y) with imaging surrogates (e.g., 111In) for dosimetry estimations might re-sult in severe errors in dosimetry estimations.

• The substitution of DOTA in 68Ga-DOTA-ZHER2:2395 with NODAGA in 68Ga-NODAGA-ZHER2:2395 decreased the liver and spleen uptake. This suggests that affibody molecules labelled with the same radiometal using

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different macrocyclic chelators may have different properties. Reducing the liver uptake is an important property for a HER2 radiotracer as liver is one of the main metastatic sights for several HER2-expressing tu-mours.

• The megalin/cubilin scavenger receptors play no role in the renal reab-sorption of anti-HER2 affibody molecules. The process is rather mediat-ed by an unknown receptor. Optimising the labelling chemistry might be a more efficient strategy than blocking the proximal-tubule receptors.

• An optimal labelling strategy may allow for the optimisation of biodis-tribution and targeting properties of 188Re-labelled affibody molecules having cysteine-containing, peptide-based chelators on the C-terminus.

• Labelling with 188Re showed high stability with no release over time. This is important for targeted radionuclide therapy.

• The introduction of lysine into cysteine-containing, peptide-based chela-tors provided variants with higher uptake and retention in execratory or-gans.

• Variants where glycine is located between the substituted amino acid and cysteine (–GXGC) showed more rapid clearance from excretory or-gans than their (–GGXC) counterparts.

• The variant with –GGGC (ZHER2:V2) provided non-residualising proper-ties in the kidneys. The in vitro inhibition of the lysosomal activity in tubular kidney cells improved the retention of 188Re-ZHER2:V2. Lower re-tention of 188Re in kidney may be attributed to non-residualising char-acter of the 188Re-GGGC label.

• 188Re-ZHER2:V2 is a promising candidate for targeted radionuclide therapy of HER2-expressing tumours.

• The tumour-associated radioactivity of 188Re-ZHER2:V2 was several fold higher than the activity in other organs (especially the kidneys and bone marrow) and showed higher retention with time.

• SPECT images showed that tumour xenografts were the only sites with prominent accumulation of radioactivity as early as 4 h p.i. In the future, imaging the biodistribution of 188Re-ZHER2:V2 would permit for patient-specific dosimetry.

• Dosimetry results indicate that 188Re-ZHER2-V2 is a promising therapeutic agent for the eradication of bulky non-operable HER2 expressing tu-mours.

In conclusion, this thesis shows that the labelling strategy is of absolute im-portance when designing radionuclide-based, tumour-targeting agents. This understanding resulted in the design of promising affibody-based targeting agents for both radionuclide-based imaging and therapy. We believe that the lessons learned from the optimisation of anti-HER2 affibody molecules can be of importance for the development of other scaffold protein-based target-ing agents.

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Ongoing and future studies

Several approaches must be taken to address the challenges of cancer. Clas-sical approaches such as surgery and conventional therapy continue to be important, but new solutions are always needed. The detection of aberrantly expressed molecular targets in cancer cells prior to the initiation of targeted therapy and the loading of targeting agents with cytotoxic effector molecules are two promising strategies.

Affibody molecules have previously been proven to be attractive for radi-onuclide molecular imaging applications. The anti-HER2 affibody molecule was the most studied variant in this series of small, high affinity targeting agents. In this thesis, we continued to optimise the targeting properties of anti-HER2 affibody molecules labelled with SPECT and PET radionuclides. The information obtained from papers I and II can help in optimising the targeting properties of newly generated anti-RTK affibody molecules. Alt-hough the structure-properties relationship is specific for each specific case, it will still be useful to have ZHER2:2395 as a reference model. Future studies should evaluate the influence of the radiolabelling of affibody molecules targeting IGF-1R, VEGFR2, HER3, and PDGFRβ using different macrocy-clic chelators on both the on-target and off-target interactions of these mole-cules. The levels of expression of IGF-1R, VEGFR2, HER3 and PDGFRβ are low in comparison with those of HER2. Therefore, the careful optimisa-tion of labelling chemistry to obtain radiotracers with favourable imaging properties is a must. In this case, results from the HER2 experience is of significance. Favourable imaging properties would permit the sensitive de-tection of these molecular targets and can provide reliable tumour bi-omarkers that can be repeatedly measured in several types of cancer, e.g., liver cancer, renal cancer and NSCLC.

Despite their excellent targeting properties, successful use of radiometal-labelled affibody molecules in targeted radionuclide therapy was not possi-ble due to the high renal reabsorption and retention of radionuclides. The findings presented in papers IV and V showed that 188Re-ZHER2:V2 is a prom-ising candidate for targeted radionuclide therapy of HER2-expressing tu-mours with low renal retention. Dosimetry estimations indicate that the re-quired activity to deliver a 79 Gy dose to the tumour (maximum dose with-out exceeding the kidneys and bone marrow dose limits) is 121 GBq. Up-scaling experiments suggest that a specific activity of 17.5 GBq/mg is achievable. A conservative estimation of the maximum amount of protein

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that could be injected in humans without self-blocking is 2.4 mg. This means that up to 42 GBq activity can be injected per patient using 188Re-ZHER2:V2. The injection of the 121 GBq (delivering a 79 Gy absorbed dose to the tu-mour) would be possible by dose fractionation, i.e., three successive frac-tions. The generator production of 188Re and the rapid clearance of 188Re-ZHER2:V2 would permit such a therapeutic regimen. In the future, full GMP production and performance of human dosimetry and toxicity studies are crucial steps for the introduction of 188Re-ZHER2:V2 in full-scale clinical trials.

188Re emits high-energy beta particles and hence can only be used for the eradication of bulky non-operable tumours. The chemistry of 188Re cannot be expanded to label affibodies with short range β- or α-emitters such as 177Lu and 227Th that can be used for residual and small lesions. Pretargeting is an attractive and promising strategy to permit labelling with therapeutic radio-metals. Pretargeting has been developed for coupling radionuclides to target-ing vectors after their clearance from non-targeted tissues (kidneys, blood), but not the tumour [151]. This improves the tumour-to-non-tumour ratio and consequently permits a high therapeutic margin. We are developing a pretar-geting technique using anti-HER2 affibody molecules and a bioorthogonal chemistry (Diels-Alder reaction) (Fig. 1) [183]. Bioorthogonal chemistry-based pretargeting has been validated previously for antibodies [184,185]. Briefly, a trans-cyclooctene conjugated non-radiolabelled mAb (CC49-TCO) was preinjected, given time to localise in the tumour surface and clear from the blood and normal tissues. One day later, the radiolabelled hapten (111In-DOTA-tetrazine) was injected. 111In-DOTA-tetrazine quickly cleared from body compartments and localised in the tumour, where the TCO-CC49 is retained. Importantly, radiolabelled tetrazine has low uptake in the kid-neys. The use of full-length antibodies may have two limitations. First, the high rate of internalisation by cancer cells would result in a reduced availa-bility of primary targeting agents on the cells surface. Second, the rate of clearance from the blood is slow, which would mean longer waiting times between the first and second injections. Incomplete clearance can result in the binding of the secondary agent to the primary one in the blood compart-ment. Affibody molecules with their slow internalisation and rapid blood clearance may represent good substitutes for antibodies. Preliminary in vitro studies using the first generation of affibody-TCO conjugates and 111In/68Ga-labelled DOTA-tetrazine have demonstrated the feasibility of specific pre-targeting of HER2-expressing cancer cell-lines.

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Figure 1. Illustrative representation of the Diels-Alder pretargeting using affibody molecules.

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Acknowledgements

I would like to thank all those who supported me to reach this day. Vladimir Thank you for everything since the first day you accepted me as a project student in your group. Thank you for all the things you taught me in and out of research and for all the nice discussions we always had about science and politics. Thank you for sharing your infinite experience with me. Anna, Thank you for your endless support and the long hours you spent with me in the cell lab when I was a fresh project student. Not to forget all what you taught me regarding mice experiments and your support in trimming long texts and for the tastiest Lax and salads you prepared during our group dinners. Marika, Thank you for your nice inspiring discussions and for the enlight-ening ideas you always brought. My group mates Hadis, Well I think you fall far behind just being a group mate, indeed you have been my lovely, crazy sister. Remembering all of the ups and downs we have been through together simply touches me from inside “Mamali likes you so much” Joanna, Thank you my friend for all the hot discussions we had starting with science passing by medicine and ending in women rights, politics and religions. You´ll make a super oncologist in the near future. Javad, Thank you for your kindness and nice discussion and for all the cookies you shared with us. Jennie, You were really cheering us all and so supportive. Zohreh, I remember the long hours we spent in BMC studying when we were doing our masters and the “You do the physics, I do the chemistry deal” . Bogdan, The kind and super intelligent guy, although I supervised you dur-ing your project I learned from you more than you did from me . Maria, Thanks for the nice discussions in Lyon and for being mitt endast koleague som pratade svenska med mig då och då . All BMS, Bo, Kiki, Helene, Hanna, Jonas, John, Kale, Jos, Andris, Ann-Sofie, Sara A, Diana, Sara HS, Amelie, Anja, Jorgen, Lars, Christl, thank you for your help, the nice activities we did together and collabora-tions.

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All PPP, Mats, Olof, Ram, Ole, Sergio, Veronika and the others thank you for tolerating me ringing the bell every now and then, and letting me in to your department. I think I should´ve asked for an access card it would have been much easier ;-). Akademiska Sjukhuset Aseel, Madina, Matias, Ulrike Garske, Anders Sundin. Thank you all for help and support. Our collaborators in KTH, Stefan, Amelie, Törbjörn, Helena, Daniel, Ca-milla, Anna-perols and all the others, thank you for the nice work we did together. Out of BMS Omer, I hate you my best friend for all of the torture I had in the gym. Thanks for being there when I needed you. Suheir, Shokran 3ala kol shi. You have played an important role in produc-ing this thesis by your support, smile and “fatayer”. You are the craziest person on Earth and probably the whole galaxy. Wish you the best in your studies and life. أنقزينم وا ما م وال طلع أنقزوا ? إتRosalia and Aaron, Akiko and Ernils, Scarlet and Christian: Ni är den bästa par I världen, Tack för era stöd och allt mina kära vänner. Maria-Teresa: Tack för dem tider när vi umgick med varandra och till Eli-sabeth vars sötta ord ringer i mitt öra hittills. All the Williams and meetup friends. Hanan and Ammar: thank you and still waiting for the Mansaf. The Saudi Club in Stockholm especially Zayed, Mishari, Rami, Saad, and all the others. Mario, Roberta, Vicky, Ivon you guys rock! Out of Sweden My friends in Yemen: My school friends and the diplomatic zone gang: A.aziz, Youssef, M.Jabr, Taha, M. Najeeb, A.Hamid, Ahmed Altashi, A. Alhaimi, Usama, Tareq S, Waseem, Nashwan (R.I.P), Mutaz, Raidan, A. Elaghil, M. Jamal, Mayad, Waddah. Time runs so fast folks. Univeristy friends: A. Attaya, Obada, Hattami and Mustafa. US friends: Algailani, Hussam, Amani. My family in Sudan: Omer Dad thank you for teaching me everything. Despite you were busy, I remember those days when you came after work

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and revised with me my lessons, your sudden visits to school which did not always end in a nice way ;-). I learned from you how to be a hardworking person and aiming for success and most important the basics of pharmacy. Layla my spoiled sweet mom thank you for all the sociological discussions you had with me and all the ideas you seeded in my mind. I remember your words “education is the only thing that is worth and what makes your identi-ty is your education”. Moaied: My brother, thank you for all the childhood times with the teenage mutant ninja turtles epidemic we had (by the way I guess we should watch the new one together), thanks for video games time we spent together which always ended with a fight and being banned from playing. Thanks for always trusting in me. Ruba My lovely daughter not only my sister, I still remember the first day you arrived home as a chubby baby wrapped with a diaper, today you are a pharmacist, I wish to see you the most successful pharmacist in the planet. To the soul of my grandfather and uncle who died two years ago and my aunt who died this year. Warm regards to my bigger family who always wanted me to bring back home the 7th PhD degree in the family.

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References

1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646-74.

2. Lemmon MA, Schlessinger J. Cell signaling by receptor tyrosine kinas-es. Cell 2010;141:1117-34.

3. Lin SY et al. Nuclear localization of EGF receptor and its potential new role as a transcription factor. Nat Cell Biol 2001;3:802-8.

4. Grassot J et al. RTKdb: database of Receptor Tyrosine Kinase. Nucleic Acids Res. 2003;31:353-8.

5. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2000; 103:211-25.

6. McCubrey JA et al. Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim Biophys Acta 2007;1773:1263-84.

7. Sharma PS et al. Receptor tyrosine kinase inhibitors as potent weapons in war against cancers. Curr Pharm Des. 2009;15:758-76.

8. Wong KK. Recent developments in anti-cancer agents targeting the Ras/Raf/ MEK/ERK pathway. Recent Pat Anticancer Drug Discov 2009; 4:28-35.

9. Hatzivassiliou G et al. ERK inhibition overcomes acquired resistance to MEK inhibitors. Mol Cancer Ther. 2012;11:1143-54.

10. Hudis CA. Trastuzumab--mechanism of action and use in clinical prac-tice. N Engl J Med. 2007;357:39-51.

11. Gualberto A et al. Emerging role of insulin-like growth factor receptor inhibitors in oncology: early clinical trial results and future directions. Oncogene. 2009;28:3009-21.

12. Strosberg JR et al. A multi-institutional, phase II open-label study of ganitumab (AMG 479) in advanced carcinoid and pancreatic neuroendo-crine tumors. Endocr Relat Cancer. 2013;20:383-90.

13. Pappo AS et al. R1507, a monoclonal antibody to the insulin-like growth factor 1 receptor, in patients with recurrent or refractory Ewing sarcoma family of tumors: results of a phase II Sarcoma Alliance for Research through Collaboration study. J Clin Oncol. 2011;29:4541-7.

14. Zhu AX et al. A phase II and biomarker study of ramucirumab, a human monoclonal antibody targeting the VEGF receptor-2, as first-line mono-therapy in patients with advanced hepatocellular cancer. Clin Cancer Res. 2013;19:6614-23.

66

15. Fuchs CS et al. Ramucirumab monotherapy for previously treated ad-vanced gastric or gastro-oesophageal junction adenocarcinoma (RE-GARD): an international, randomised, multicentre, placebo-controlled, phase 3 trial. Lancet. 2014;383:31-9.

16. Johnson LN. Protein kinase inhibitors: contributions from structure to clinical compounds. Q Rev Biophys. 2009;42:1-40.

17. Sawyers CL. Opportunities and challenges in the development of kinase inhibitor therapy for cancer. Genes Dev. 2003;17:2998-3010.

18. Demetri GD et al. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med. 2002;347:472-80

19. Geyer CE et al. Lapatinib plus capecitabine for HER2-positive advanced breast cancer. N Engl J Med 2006;355:2733–43.

20. FDA approved drug products data base www.accessdata.fda.gov. 21. Pohlmann PR et al.Resistance to Trastuzumab in Breast Cancer. Clin

Cancer Res. 2009;15:7479-7491. 22. Holohan C et al. Cancer drug resistance: an evolving paradigm. Nat Rev

Cancer. 2013;13:714-26. 23. Rosenzweig SA et al. Acquired resistance to drugs targeting receptor

tyrosine kinases. Biochem Pharmacol. 2012;83:1041-8. 24. Wu AM et al. Arming antibodies: prospects and challenges for immuno-

conjugates. Nat Biotechnol. 2005;23:1137-46. 25. Carter P. Improving the efficacy of antibody-based cancer therapies. Nat

Rev Cancer 2001;1:118-29. 26. Verma S et al. Trastuzumab emtansine for HER2-positive advanced

breast cancer. N Engl J Med. 2012;367:1783-91. 27. The MARIANNE clinical trial (NCT01120184) 28. Gonzalez de Castro D et al. Personalized cancer medicine: molecular

diagnostics, predictive biomarkers, and drug resistance. Clin Pharmacol Ther. 2013;93:252-9.

29. Burke HB et al. The American Joint Committee on Cancer. Criteria for prognostic factors and for an enhanced prognostic system. Cancer. 1993; 72:3131-5.

30. Biomarkers Definitions Working Group. Biomarkers and surrogate end-points: preferred definitions and conceptual framework. Clin Pharmacol Ther. 2001;69:89-95.

31. Stehle F et al. Towards defining biomarkers indicating resistances to targeted therapies. Biochim Biophys Acta. 2014 May;1844(5):909-916.

32. Bates SE et al. Drug development: portals of discovery. Clin Cancer Res. 2012;18:23-32.

33. Pollak M. The insulin and insulin-like growth factor receptor family in neoplasia: an update. Nat Rev Cancer. 2012;12:159-69.

67

34. Jassem J et al. Randomized, open label, phase III trial of figitumumab in combination with paclitaxel and carboplatin versus paclitaxel and car-boplatin in patients with non-small cell lung cancer (NSCLC). J Clin Oncol 2010;28:7500.

35. Pollak M. The insulin receptor/insulin-like growth factor receptor family as a therapeutic target in oncology. Clin Cancer Res. 2012;18:40-50.

36. Pegram MD et al. Results of two open-label, multicenter phase II studies of docetaxel, platinum salts, and trastuzumab in HER2-positive ad-vanced breast cancer. J Natl Cancer Inst. 2004;96:759-69.

37. Di Leo A et al. Phase III, double-blind, randomized study comparing lapatinib plus paclitaxel with placebo plus paclitaxel as first-line treat-ment for metastatic breast cancer. J Clin Oncol. 2008;26:5544-52

38. Dei Tos AP et al. Assessing epidermal growth factor receptor expression in tumours: what is the value of current test methods? Eur J Cancer 2005;41:1383-92.

39. Apple SK et al. Comparison of fluorescent in situ hybridization HER-2/neu results on core needle biopsy and excisional biopsy in primary breast cancer. Mod. Pathol 2009;22:1151-9.

40. Koda M et al. Expression of the insulin-like growth factor-I receptor in primary breast cancer and lymph node metastases: correlations with es-trogen receptors alpha and beta. Horm Metab Res 2003;35:794-801.

41. Scartozzi M et al. Epidermal growth factor receptor (EGFR) status in primary colorectal tumors does not correlate with EGFR expression in related metastatic sites: implications for treatment with EGFR-targeted monoclonal antibodies. J Clin Oncol 2004;22:4772-8.

42. Fehm T et al. ERalpha-status of disseminated tumour cells in bone mar-row of primary breast cancer patients. Breast Cancer Res 2008;10:R76.

43. Rasbridge SA et al. The effects of chemotherapy on morphology, cellu-lar proliferation, apoptosis and oncoprotein expression in primary breast carcinoma. Br J Cancer 1994;70:335-41.

44. Choong LY et al. Progressive loss of epidermal growth factor receptor in a subpopulation of breast cancers: implications in target-directed thera-peutics. Mol Cancer Ther 2007;6:2828-42.

45. Bartlett JM et al. Evaluating HER2 amplification and overexpression in breast cancer. J Pathol 2001;195:422-8.

46. Press MF et al. Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials. Clin Cancer Res.2005;11:6598-6607.

47. Moelans CB et al. Current technologies for HER2 testing in breast can-cer.Crit Rev Oncol Hematol. 2011;80:380-92.

68

48. Therasse P et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst. 2000;92:205-16.

49. Eisenhauer EA et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009;45:228-47.

50. Contractor KB et al. Monitoring predominantly cytostatic treatment re-sponse with 18F-FDG PET. J Nucl Med 2009; 50 Suppl 1: 97S-105S.

51. Weber WA. Assessing tumor response to therapy. J Nucl Med 2009;50 Suppl 1:1S-10S.

52. Choi H et al. Correlation of computed tomography and positron emission tomography in patients with metastatic gastrointestinal stromal tumor treated at a single institution with imatinib mesylate: proposal of new computed tomography response criteria. J Clin Oncol 2007;25:1753-9.

53. Mozley PD et al. Measurement of Tumor Volumes Improves RECIST-Based Response Assessments in Advanced Lung Cancer. Transl Oncol 2012;5:19-25.

54. Nishino M et al. Personalized tumor response assessment in the era of molecular medicine: cancer-specific and therapy-specific response crite-ria to complement pitfalls of RECIST. AJR Am J Roentgenol 2012;198: 737-45.

55. Smith AD et al. Morphology, Attenuation, Size, and Structure (MASS) criteria: assessing response and predicting clinical outcome in metastatic renal cell carcinoma on antiangiogenic targeted therapy. AJR Am J Roentgenol 2010;194:1470-8.

56. Van den Abbeele AD. The lessons of GIST--PET and PET/CT: a new paradigm for imaging. Oncologist. 2008;13 Suppl 2:8-13.

57. Hillner BE et al. Impact of positron emission tomography/computed tomography and positron emission tomography (PET) alone on expected management of patients with cancer: initial results from the National Oncologic PET Registry. J Clin Oncol 2008;26:2155-61.

58. Dunphy MP et al. Radiopharmaceuticals in preclinical and clinical de-velopment for monitoring of therapy with PET. J Nucl Med. 2009; 50 Suppl 1:106S-21S.

59. Chen W et al. Predicting treatment response of malignant gliomas to bevacizumab and irinotecan by imaging proliferation with [18F] fluoro-thymidine positron emission tomography: a pilot study. J Clin Oncol 2007;25:4714-21.

60. Zhang CC et al. [(18)F]FLT-PET imaging does not always "light up" proliferating tumor cells. Clin Cancer Res 2012;18:1303-12.

61. Peterson TE et al. Molecular imaging: 18F-FDG PET and a whole lot more. J Nucl Med Technol. 2009;37:151-61.

62. Behr TM et al. Trastuzumab and breast cancer. N Engl J Med 2001 27; 345:995-6.

69

63. Perik PJ et al. Indium-111-labeled trastuzumab scintigraphy in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer. J Clin Oncol. 2006;24:2276-82.

64. Dijkers EC et al. Biodistribution of 89Zr-trastuzumab and PET imaging of HER2-positive lesions in patients with metastatic breast cancer. Clin Pharmacol Ther. 2010;87:586-92.

65. Mortimer JE et al. Functional imaging of human epidermal growth factor receptor 2-positive metastatic breast cancer using (64)Cu-DOTA-trastuzumab PET. J Nucl Med. 2014;55:23-9.

66. Memon AA et al. PET imaging of patients with non-small cell lung can-cer employing an EGF receptor targeting drug as tracer. Br J Cancer. 2011;105:1850-5.

67. Fleuren ED et al. Predicting IGF-1R therapy response in bone sarcomas: immuno-SPECT imaging with radiolabeled R1507. Clin Cancer Res 2011;17;7693-703.

68. Heskamp S et al. Dynamics of IGF-1R Expression During Endocrine Breast Cancer Treatment. Mol Imaging Biol. 2014;16:529-37.

69. Niu G et al. Monitoring therapeutic response of human ovarian cancer to 17-DMAG by noninvasive PET imaging with (64)Cu-DOTA-trastuzumab. Eur J Nucl Med Mol Imaging. 2009;36:1510-9.

70. Holland JP et al. Measuring the pharmacodynamic effects of a novel Hsp90 inhibitor on HER2/neu expression in mice using Zr-DFO-trastuzumab. PLoS One. 2010 ;5:e8859.

71. Janjigian YY et al. Monitoring afatinib treatment in HER2-positive gas-tric cancer with 18F-FDG and 89Zr-trastuzumab PET. J Nucl Med. 2013;54:936-43.

72. Ma T et al. Molecular Imaging Reveals Trastuzumab-Induced Epidermal Growth Factor Receptor Downregulation In Vivo. J Nucl Med. 2014;55:1002-1007. [Epub ahead of print]

73. Steffens MG et al. Immunohistochemical analysis of intratumoral heter-ogeneity of [131I]cG250 antibody uptake in primary renal cell carcino-mas. Br J Cancer. 1998;78:1208-13.

74. Heskamp S et al. Radionuclide imaging of drug delivery for patient se-lection in targeted therapy. Expert Opin Drug Deliv. 2014;11:175-85.

75. Tolmachev V et al. Radiolabelled receptor-tyrosine-kinase targeting drugs for patient stratification and monitoring of therapy response: pro-spects and pitfalls. Lancet Oncol. 2010;11:992-1000.

76. Wester HJ et al. Molecular targeting with peptides or peptide-polymer conjugates: just a question of size? J Nucl Med. 2005;46:1940-5.

77. Altai M et al. Radiolabeled Probes Targeting Tyrosine-Kinase Receptors For Personalized Medicine. Curr Pharm Des. 2014;20:2275-92

70

78. McLarty K et al. Associations between the uptake of 111In-DTPA-trastuzumab, HER2 density and response to trastuzumab (Herceptin) in athymic mice bearing subcutaneous human tumour xenografts. Eur J Nucl Med Mol Imaging.2009;36:81-93.

79. Gaykema SB et al. Zirconium-89-trastuzumab positron emission tomog-raphy as a tool to solve a clinical dilemma in a patient with breast can-cer. J Clin Oncol 2012;30:e74-5.

80. Divgi CR et al. Phase I and imaging trial of indium 111-labeled anti-epidermal growth factor receptor monoclonal antibody 225 in patients with squamous cell lung carcinoma. J Natl Cancer Inst. 1991;83:97-104.

81. Aerts HJ et al. Disparity between in vivo EGFR expression and 89Zr-labeled cetuximab uptake assessed with PET. J Nucl Med 2009;50:123-31.

82. Niu G et al. PET of EGFR antibody distribution in head and neck squa-mous cell carcinoma models. J Nucl Med.2009;50:1116-23.

83. Heskamp S et al. ImmunoSPECT and immunoPET of IGF-1R expres-sion with the radiolabeled antibody R1507 in a triple-negative breast cancer model. J Nucl Med 2010;51:1565-72.

84. Smith-Jones PM et al. Imaging the pharmacodynamics of HER2 degra-dation in response to Hsp90 inhibitors. Nat Biotechnol 2004; 22: 701-6.

85. Smith-Jones PM et al. Early tumor response to Hsp90 therapy using HER2 PET: comparison with 18F-FDG PET. J Nucl Med 2006;47:793-6.

86. Heskamp et al. Optimization of IGF-1R SPECT/CT imaging using 111In-labeled F(ab')2 and Fab fragments of the monoclonal antibody R1507. Mol Pharm. 2012;9:2314-21.

87. Vaneycken I et al. Preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancer. FASEB J 2011;25:2433-46.

88. Gainkam LO et al. Correlation between epidermal growth factor recep-tor-specific nanobody uptake and tumor burden: a tool for noninvasive monitoring of tumor response to therapy. Mol Imaging Biol. 2011;13: 940-8.

89. Velikyan I et al. Preparation and evaluation of (68)Ga-DOTA-hEGF for visualization of EGFR expression in malignant tumors. J Nucl Med 2005;46:1881-8.

90. Reilly RM et al. A comparison of EGF and MAb 528 labeled with 111In for imaging human breast cancer. J Nucl Med 2000;41:903-11.

91. Backer MV et al. Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes. Nat Med. 2007;13: 504-9.

92. Cai W et al. PET of vascular endothelial growth factor receptor expres-sion. J Nucl Med 2006;47:2048-56.

71

93. Cornelissen B et al. The level of insulin growth factor-1 receptor expres-sion is directly correlated with the tumor uptake of (111)In-IGF-1(E3R) in vivo and the clonogenic survival of breast cancer cells exposed in vitro to trastuzumab (Herceptin). Nucl Med Biol. 2008;35:645-53.

94. Kumar SR et al. In vitro and in vivo evaluation of ⁶⁴Cu-radiolabeled KCCYSL peptides for targeting epidermal growth factor receptor-2 in breast carcinomas. Cancer Biother Radiopharm. 2010;25:693-703.

95. Denholt CL et al. Evaluation of 4-[18F]fluorobenzoyl-FALGEA-NH2 as a positron emission tomography tracer for epidermal growth factor re-ceptor mutation variant III imaging in cancer. Nucl Med Biol 2011;38: 509-15.

96. Tolmachev V et al. A HER2-binding Affibody molecule labelled with 68Ga for PET imaging: direct in vivo comparison with the 111In-labelled analogue. Eur J Nucl Med Mol Imaging 2010;37:1356-67

97. Baum RP et al. Molecular imaging of HER2-expressing malignant tu-mors in breast cancer patients using synthetic 111In- or 68Ga-labeled af-fibody molecules. J Nucl Med 2010;51:892-7.

98. Sörensen J et al. First-in-Human Molecular Imaging of HER2 Expres-sion in Breast Cancer Metastases Using the 111In-ABY-025 Affibody Molecule. J Nucl Med. 2014;55:730-5.

99. Zahnd C et al. Efficient tumor targeting with high-affinity designed ankyrin repeat proteins: effects of affinity and molecular size. Cancer Res. 2010;70:1595-605.

100. Tolmachev V et al. Imaging of EGFR expression in murine xenografts using site-specifically labelled anti-EGFR 111In-DOTA-Z EGFR:2377 Affibody molecule: aspect of the injected tracer amount. Eur J Nucl Med Mol Imaging. 2010;37:613-22.

101. Tolmachev V, et al. Imaging of insulinlike growth factor type 1 recep-tor in prostate cancer xenografts using the affibody molecule 111In-DOTA-ZIGF1R:4551. J Nucl Med. 2012;53:90-7.

102. Tolmachev V et al. Imaging of platelet-derived growth factor receptor β expression in glioblastoma xenografts using affibody molecule 111In-DOTA-Z09591. J Nucl Med. 2014;55:294-300.

103. Orlova A et al. Imaging of HER3-expressing xenografts in mice using a 99mTc(CO) 3-HEHEHE-Z HER3:08699 affibody molecule. Eur J Nucl Med Mol Imaging. 2014;41:1450-9.

104. Memon AA et al. Positron emission tomography (PET) imaging with [11C]-labeled erlotinib: a micro-PET study on mice with lung tumor xenografts. Cancer Res. 2009;69:873-8.

105. Meng X et al. Molecular imaging with 11C-PD153035 PET/CT pre-dicts survival in non-small cell lung cancer treated with EGFR-TKI: a pilot study. J Nucl Med. 2011;52:1573-9.

72

106. Samén E et al. Visualization of angiogenesis during cancer develop-ment in the polyoma middle T breast cancer model: molecular imaging with (R)-[11C]PAQ. EJNMMI Res. 2014;4:17.

107. Glekas AP et al. In vivo imaging of Bcr-Abl overexpressing tumors with a radiolabeled imatinib analog as an imaging surrogate for imatinib. J Nucl Med. 2011;52:1301-7.

108. Miao Z et al. Protein scaffold-based molecular probes for cancer mo-lecular imaging. Amino Acids. 2011 ;41:1037-47.

109. Nygren PA, Skerra A. Binding proteins from alternative scaffolds. J Immunol Methods. 2004;290:3-28.

110. Binz HK, Amstutz P, Plückthun A. Engineering novel binding proteins from nonimmunoglobulin domains. Nat Biotechnol 2005;23:1257-68.

111. Nygren PA et al. Alternative binding proteins: affibody binding pro-teins developed from a small three-helix bundle scaffold. FEBS J. 2008; 275:2668-76.

112. Löfblom J et al. Affibody molecules: engineered proteins for therapeu-tic, diagnostic and biotechnological applications. FEBS Lett. 2010;584: 2670-80.

113. Ahlgren S et al. Radionuclide molecular imaging using Affibody mole-cules. Curr Pharm Biotechnol. 2010;11:581-9.

114. Orlova A et al. Tumor imaging using a picomolar affinity HER2 bind-ing affibody molecule. Cancer Res 2006;66:4339-48.

115. Wolff AC et al. American Society of Clinical Oncology; College of American Pathologists. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epi-dermal growth factor receptor 2 testing in breast cancer. J Clin Oncol. 2007;25:118-45.

116. Orlova A et al. Evaluation of [(111/114m)In]CHX-A''-DTPA-ZHER2:342, an affibody ligand coniugate for targeting of HER2-expressing malignant tumors. Q J Nucl Med Mol Imaging. 2007;51: 314-23.

117. Kramer-Marek G et al. 68Ga-DOTA-affibody molecule for in vivo assessment of HER2/neu expression with PET. Eur J Nucl Med Mol Imaging. 2011;38:1967-76.

118. Wållberg H et al. Molecular design and optimization of 99mTc-labeled recombinant affibody molecules improves their biodistribution and im-aging properties. J Nucl Med 2011;52:461-9.

119. Orlova A et al. Synthetic affibody molecules: a novel class of affinity ligands for molecular imaging of HER2-expressing malignant tumors. Cancer Res 2007;67:2178-86.

120. Orlova A et al. On the selection of a tracer for PET imaging of HER2-expressing tumors: direct comparison of a 124I-labeled affibody mole-cule and trastuzumab in a murine xenograft model. J Nucl Med 2009; 50:417-25.

73

121. Malmberg J et al. Comparative biodistribution of imaging agents for in vivo molecular profiling of disseminated prostate cancer in mice bear-ing prostate cancer xenografts: focus on 111In- and 125I-labeled anti-HER2 humanized monoclonal trastuzumab and ABY-025 affibody. Nucl Med Biol 2011;38:1093-102.

122. Kramer-Marek G et al. [18F]FBEM-Z(HER2:342)-Affibody molecule-a new molecular tracer for in vivo monitoring of HER2 expression by positron emission tomography. Eur J Nucl Med Mol Imaging 2008;35:1008-18.

123. Kramer-Marek G et al. Potential of PET to predict the response to trastuzumab treatment in an ErbB2-positive human xenograft tumor model. J Nucl Med. 2012;53:629–637.

124. Tagliabue E et al. PET prediction of response to trastuzumab in ErbB2-positive human xenograft model. J Nucl Med. 2012;53:1654-5; author reply 1655-6.

125. Capala J et al. Reply: PET prediction of response to trastuzumab in ErbB2-positive human xenograft model. J Nucl Med. 2012 Sep 4.

126. Orlova A et al. Update: affibody molecules for molecular imaging and therapy for cancer. Cancer Biother Radiopharm. 2007;22:573-84.

127. Tolmachev, V et al. Influence of labeling methods on biodistribution and imaging properties of radiolabelled peptides for visualisation of molecular therapeutic targets. Curr. Med. Chem.2010;17;2636–2655.

128. Ahlgren S et al. Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant Affibody molecules. Bioconjug Chem 2008;19:235-243.

129. Cheng Z et al. 64Cu-labeled affibody molecules for imaging of HER2 expressing tumors. Mol Imaging Biol. 2010;12:316-24.

130. Tolmachev V et al. Evaluation of a maleimido derivative of CHX-A'' DTPA for site-specific labeling of affibody molecules. Bioconjug Chem 2008;19:1579-1587.

131. Miao Z et al. Small-animal PET imaging of human epidermal growth factor receptor positive tumor with a 64Cu labeled affibody protein. Bi-oconjug Chem. 2010;21:947-54.

132. Wållberg H et al. Evaluation of the radiocobalt-labeled [MMA-DOTA-Cys61]-Z HER2:2395(-Cys) affibody molecule for targeting of HER2-expressing tumors. Mol Imaging Biol. 2010;12:54-62

133. Tolmachev V et al. Evaluation of a Maleimido Derivative of NOTA for Site-Specific Labeling of Affibody Molecules. Bioconjug Chem 2011; 22:894-902.

134. Tolmachev V et al. Influence of valency and labelling chemistry on in vivo targeting using radioiodinated HER2-binding Affibody molecules. Eur J Nucl Med Mol Imaging. 2009;36:692-701.

74

135. Cheng Z et al. Small-animal PET imaging of human epidermal growth factor receptor type 2 expression with site-specific 18F-labeled protein scaffold molecules. J Nucl Med. 2008;49:804-13.

136. Mume E et al. Evaluation of ((4-hydroxyphenyl)ethyl)maleimide for site-specific radiobromination of anti-HER2 affibody. Bioconjug Chem. 2005;16:1547-55.

137. Heskamp S et al. Imaging of human epidermal growth factor receptor type 2 expression with 18F-labeled affibody molecule ZHER2:2395 in a mouse model for ovarian cancer. J Nucl Med. 2012;53:146-53.

138. Wadas TJ, Wong EH, Weisman GR, Anderson CJ. Coordinating radi-ometals of copper, gallium, indium, yttrium, and zirconium for PET and SPECT imaging of disease. Chem Rev. 2010;110:2858-902.

139. De Leon-Rodríguez et al. The synthesis and chelation chemistry of DOTA-peptide conjugates. Bioconjugate Chem. 2008;19:391–402.

140. Anderson C. J et al. Cross-bridged macrocyclic chelators for stable complexation of copper radionuclides for PET imaging. Q. J. Nucl. Med. Mol. Imaging. 2008;52:185–192.

141. Prasanphanich A. F. et al. [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] tar-geting vector for positron-emission tomography imaging of gastrin-releasing peptide receptor-expressing tissues. Proc. Natl. Acad. Sci. U.S.A. 2007;104:12462–12467.

142. Garrison J. C. et al. In vivo evaluation and small-animal PET/CT of a prostate cancer mouse model using 64Cu bombesin analogs: side-by-side comparison of the CB-TE2A and DOTA chelation systems. J. Nucl. Med. 2007;48:1327–1337.

143. Clarke E et al. Stabilities of the Fe(III), Ga(III) and In(III) chelates of N,N’,N’’-triazacyclononanetriacetic acid. Inorg. Chim. Acta. 1991;181:273–280.

144. Clarke E.T. et al. Stabilities of trivalent metal ion complexes of the tetraacetate derivatives of 12-, 13-, and 14-membered tetraazamacrocy-cles. Inorg. Chim. Acta 1992;190: 37–46.

145. Eisenwiener KP et al. NODAGATOC, a new chelator-coupled somato-statin analogue labeled with [67/68Ga] and [111In] for SPECT, PET, and targeted therapeutic applications of somatostatin receptor (hsst2) expressing tumors. Bioconjug Chem. 2002;13:530-41.

146. Jeong, J.M et al. Preparation of a promising angiogenesis PET imaging agent: 68Ga-labeled c(RGDyK)-isothiocyanatobenzyl-1,4,7-triazacyclononane-1,4,7-triaceticacid and feasibility studies in mice. J. Nucl. Med. 2008;49:830–836.

147. Liu, Z et al. 68Ga-labeled cyclic RGD dimers with Gly3 and PEG4 linkers: promising agents for tumor integrin alphavbeta3 PET imaging. Eur. J. Nucl. Med. Mol. Imaging 2009;36:947–957.

75

148. Velikyan I et al. Convenient preparation of 68Ga-based PET-radiopharmaceuticals at room temperature. Bioconjug Chem. 2008;19:569-73.

149. Velikyan I et al. The importance of high specific radioactivity in the performance of 68Ga-labeled peptide. Nucl Med Biol. 2008;35:529-36.

150. Knetsch PA et al. [68Ga]NODAGA-RGD for imaging αvβ3 integrin expression. Eur J Nucl Med Mol Imaging. 2011;38:1303-12.

151. Sharkey RM, Goldenberg DM. Perspectives on cancer therapy with radiolabeled monoclonal antibodies. J Nucl Med 2005;46(Suppl 1):115S–27

152. Jurcic JG et al. Radioimmunotherapy of hematological cancer: prob-lems and progress. Clin Cancer Res 1995;1:1439–1446.

153. Schlom J et al. Monoclonal antibodies: they’re more and less than you think in Molecular Foundations of Oncology, Brode, Ed., Williams and Wilkens pp. 1991;95:134.

154. Knox SJ et al. Clinical radioimmunotherapy. Semin Radiat Oncol 2000;10:73–93.

155. Kenanova V et al. Tailoring antibodies for radionuclide delivery. Ex-pert Opin Drug Deliv. 2006;3:53-70.

156. Goldenberg DM et al. Cancer imaging and therapy with bispecific anti-body pretargeting. Update Cancer Ther 2007;2:19–31.

157. Frampas E et al. Improvement of radioimmunotherapy using pretarget-ing. Front Oncol. 2013;3:159.

158. Wållberg H et al.Slow internalization of anti-HER2 synthetic affibody monomer 111In-DOTA-ZHER2:342-pep2: implications for develop-ment of labeled tracers. Cancer Biother Radiopharm. 2008;23:435-42.

159. Tolmachev V et al. Radionuclide therapy of HER2-positive microxen-ografts using a 177Lu-labeled HER2-specific Affibody molecule. Can-cer Res 2007;67:2773–82.

160. Fortin MA et al. Labelling chemistry and characterization of [90Y/177Lu]-DOTAZHER2:342–3 Affibody molecule, a candidate agent for locoregional treatment of urinary bladder carcinoma. Int J Mol Med 2007;19:285–91.

161. Ahlgren S et al. Targeting of HER2-expressing tumors using 111In-ABY-025, a second-generation Affibody molecule with a fundamental-ly reengineered scaffold. J Nucl Med 2010;51:1131–1138.

162. Orlova A et al. Comparative in vivo evaluation of technetium and io-dine labels on an anti-HER2 affibody for single-photon imaging of HER2 expression in tumors. J Nucl Med. 2006;47:512-9.

163. Feldwisch J, Tolmachev V. Engineering of affibody molecules for ther-apy and diagnostics. Methods Mol Biol. 2012;899:103-26.

164. Schwartz A et al. A novel approach to Tc-99m labeled monoclonal antibodies. J Nucl Med 1987;28:721.

76

165. Engfeldt T et al. Imaging of HER2-expressing tumours using a synthet-ic Affibody molecule containing the 99mTc-chelating mercaptoacetyl-glycyl-glycyl-glycyl (MAG3) sequence. Eur J Nucl Med Mol Imaging 2007;34:722–733.

166. Wang Y et al. An improved synthesis of NHS-MAG3 for conjugation and radiolabeling of biomolecules with 99mTc at room temperature. Nat Protoc 2007;2:972–978

167. Engfeldt T et al. 99mTc-chelator engineering to improve tumour target-ing properties of a HER2-specific Affibody molecule. Eur J Nucl Med Mol Imaging 2007;34:1843–1853.

168. Tran T et al. 99mTc-maEEE-ZHER2:342, an Affibody molecule-based tracer for the detection of HER2 expression in malignant tumors. Bio-conjug Chem 2007;18:1956–1964

169. Ekblad T et al. Development and preclinical characterisation of 99mTc-labelled Affibody molecules with reduced renal uptake. Eur J Nucl Med Mol Imaging 2008;35:2245–2255.

170. Ahlgren S et al. Targeting of HER2-expressing tumors with a site-specifically 99mTc-labeled recombinant affibody molecule, ZHER2:2395, with C-terminally engineered cysteine. J Nucl Med. 2009;50:781-9.

171. Tran TA et al. Design, synthesis and biological evaluation of a multi-functional HER2-specific Affibody molecule for molecular imaging. Eur J Nucl Med Mol Imaging 2009;36:1864–1873.

172. Altai M et al. Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribu-tion of 99mTc-labeled recombinant Affibody molecules. Amino Acids. 2012;42:1975-85.

173. Liu G, Hnatowich DJ. Labeling biomolecules with radiorhenium: a review of the bifunctional chelators. Anticancer Agents Med Chem. 2007;7:367-77.

174. Orlova A et al. (186)Re-maSGS-Z (HER2:342), a potential Affibody conjugate for systemic therapy of HER2-expressing tumours. Eur J Nucl Med Mol Imaging. 2010;37:260-9.

175. Feldwisch J et al. Design of an optimized scaffold for affibody mole-cules. J Mol Biol. 2010;398:232-47.

176. Verroust PJ et al. Megalin and cubilin – the story of two multipurpose receptors unfold. Nephrol Dial Transplant 17:1867-1871, 2002.

177. Vegt E et al. Renal uptake of different radiolabelled peptides is mediat-ed by megalin: SPECT and biodistribution studies in megalin-deficient mice. Eur J Nucl Med Mol Imaging 38:623–632, 2011.

178. de Jong M et al. Megalin Is Essential for Renal Proximal Tubule Reab-sorption of 111In-DTPA-Octreotide. J Nucl Med 2005;46:1696-1700.

77

179. Vegt E et al. Renal toxicity of radiolabeled peptides and antibody frag-ments: mechanisms, impact on radionuclide therapy, and strategies for prevention. J Nucl Med 2010;51:1049-1058.

180. Gainkam LO et al. Localization, mechanism and reduction of renal retention of technetium-99m labeled epidermal growth factor receptor-specific nanobody in mice. Contrast Media Mol Imaging 2011;6:85.

181. Decristoforo C et al. 68Ga- and 111Inlabelled DOTA-RGD peptides for imaging of alphavbeta3 integrin expression. Eur J Nucl Med Mol Imag-ing 2008;35:1507–15.

182. Hofström C et al. HAHAHA, HEHEHE, HIHIHI, or HKHKHK: Influ-ence of Position and Composition of Histidine Containing Tags on Bio-distribution of [(99m)Tc(CO)3](+)-Labeled Affibody Molecules. J Med Chem. 2013;56:4966-74.

183. Knight JC, Cornelissen B. Bioorthogonal chemistry: implications for pretargeted nuclear (PET/SPECT) imaging and therapy. Am J Nucl Med Mol Imaging. 2014;4:96-113.

184. Rossin R et al. In vivo chemistry for pretargeted tumor imaging in live mice. Angew Chem Int Ed Engl. 2010;49:3375-8.

185. Rossin Ret al. Diels-Alder reaction for tumor pretargeting: in vivo chemistry can boost tumor radiation dose compared with directly la-beled antibody. J Nucl Med. 2013;54:1989-95.

Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1013

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